 |
   |
           |
|
 | |
|
Biochemistry Cell
Biology Computational
Biology Developmental
Biology Ecology Evolution Genetics Microbiology Molecular
Biology Plant
Biology Science
Education Structural
Biology |
Kang, Y., T. Taldone, C.C. Clement, S.W. Fewell, J. Aguirre, J.L. Brodsky, and G. Chiosis (2008) Design of a fluorescence polarization assay platform for the study of human Hsp70. Bioorg. Med. Chem. Lett. 18:3749-3751 The 70kDa heat shock proteins (Hsp70) are molecular chaperones that assist in folding of newly synthesized polypeptides, refolding or denaturation of misfolded proteins, and translocation of proteins across biological membranes. In addition, Hsp70 play regulatory roles in signal transduction, cell cycle, and apoptosis. Here, we present a novel assay platform based on fluorescence polarization that is suitable for investigating the yet elusive molecular mechanics of human Hsp70 allosteric regulation.
Goeckeler, J.L., A.P. Petruso, J. Aguirre, C.C. Clement, G. Chiosis, and J.L. Brodsky (2008) The yeast Hsp110, Sse1p, exhibits high-affinity peptide binding. FEBS Lett. 582:2393-2396 Hsp110s are divergent relatives of Hsp70 chaperones that hydrolyze ATP. Hsp110s serve as Hsp70 nucleotide exchange factors and act directly to maintain polypeptide solubility. To date, the impact of peptide binding on Hsp110 ATPase activity is unknown and an Hsp110/peptide affinity has not been measured. We now report on a peptide that binds to the yeast Hsp110, Sse1p, with a K(D) of approximately 2nM. Surprisingly, the binding of this peptide fails to stimulate Sse1p ATP hydrolysis. Moreover, an Hsp70-binding peptide is unable to associate with Sse1p, suggesting that Hsp70s and Hsp110s possess partially distinct peptide recognition motifs.
Nakatsukasa, K., and J.L. Brodsky (2008) The recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum. Traffic 17:1614-1626 Secretory and membrane proteins that fail to fold in the endoplasmic reticulum (ER) are retained and may be sorted for ER-associated degradation (ERAD). During ERAD, ER-associated components such as molecular chaperones and lectins recognize folding intermediates and specific oligosaccharyl modifications on ERAD substrates. Substrates selected for ERAD are then targeted for ubiquitin- and proteasome-mediated degradation. Because the catalytic steps of the ubiquitin-proteasome system reside in the cytoplasm, soluble ERAD substrates that reside in the ER lumen must be retrotranslocated back to the cytoplasm prior to degradation. In contrast, it has been less clear how polytopic, integral membrane substrates are delivered to enzymes required for ubiquitin conjugation and to the proteasome. In this review, we discuss recent studies addressing how ERAD substrates are recognized, ubiquitinated and delivered to the proteasome and then survey current views of how soluble and integral membrane substrates may be retrotranslocated. Tonsor, S.J., C. Scott, I. Boumaza, T.R. Liss, J.L. Brodsky, and E. Vierling (2008) Heat shock protein 101 effects in A. thaliana: genetic variation, fitness and pleiotropy in controlled temperature conditions. Mol. Ecol. 17:1614-1626 The Hsp100/ClpB heat shock protein family is ancient and required for high temperature survival, but natural variation in expression and its phenotypic effects is unexplored in plants. In controlled environment experiments, we examined the effects of variation in the Arabidopsis cytosolic AtHsp101 (hereafter Hsp101). Ten wild-collected ecotypes differed in Hsp101 expression responses across a 22 to 40 degrees C gradient. Genotypes from low latitudes expressed the least Hsp101. We tested fitness and pleiotropic consequences of varying Hsp101 expression in 'control' vs. mild thermal stress treatments (15/25 degrees C D/N vs. 15/25 degrees D/N plus 3 h at 35 degrees C 3 days/week). Comparing wild type and null mutants, wt Columbia (Col) produced approximately 33% more fruits compared to its Hsp101 homozygous null mutant. There was no difference between Landsberg erecta null mutant NIL (Ler) and wt Ler; wt Ler showed very low Hsp101 expression. In an assay of six genotypes, fecundity was a saturating function of Hsp101 content, in both experimental treatments. Thus, in addition to its essential role in acquired thermal tolerance, Hsp101 provides a substantial fitness benefit under normal growth conditions. Knocking out Hsp101 decreased fruit production, days to germination and days to bolting, total dry mass, and number of inflorescences; it increased transpiration rate and allocation to root mass. Root : total mass ratio decayed exponentially with Hsp101 content. This study shows that Hsp101 expression is evolvable in natural populations. Our results further suggest that Hsp101 is primarily an emergency high-temperature tolerance mechanism, since expression levels are lower in low-latitude populations from warmer climates. Hsp101 expression appears to carry an important trade-off in reduced root growth. This trade-off may select for suppressed expression under chronically high temperatures.
Nakatsukasa, K., G. Huyer, S. Michaelis, and J.L. Brodsky (2008) Dissecting the ER-Associated Degradation of a Misfolded Polytopic Membrane Protein. Cell 132:101-112 It remains unclear how misfolded membrane proteins are selected and destroyed during endoplasmic reticulum-associated degradation (ERAD). For example, chaperones are thought to solubilize aggregation-prone motifs, and some data suggest that these proteins are degraded at the ER. To better define how membrane proteins are destroyed, the ERAD of Ste6p( *), a 12 transmembrane protein, was reconstituted. We found that specific Hsp70/40s act before ubiquitination and facilitate Ste6p( *) association with an E3 ubiquitin ligase, suggesting an active role for chaperones. Furthermore, polyubiquitination was a prerequisite for retrotranslocation, which required the Cdc48 complex and ATP. Surprisingly, the substrate was soluble, and extraction was independent of a ubiquitin chain extension enzyme (Ufd2p). However, Ufd2p increased the degree of ubiquitination and facilitated degradation. These data indicate that polytopic membrane proteins can be extracted from the ER, and define the point of action of chaperones and the requirement for Ufd2p during membrane protein quality control.
Buck, T.M., C.M. Wright, and J.L. Brodsky (2007) The activities and function of molecular chaperones in the endoplasmic reticulum. Semin. Cell Dev. Biol. 18:751-761 Most proteins in the secretory pathway are translated, folded, and subjected to quality control at the endoplasmic reticulum (ER). These processes must be flexible enough to process diverse protein conformations, yet specific enough to recognize when a protein should be degraded. Molecular chaperones are responsible for this decision making process. ER associated chaperones assist in polypeptide translocation, protein folding, and ER associated degradation (ERAD). Nevertheless, we are only beginning to understand how chaperones function, how they are recruited to specific substrates and assist in folding/degradation, and how unique chaperone classes make quality control "decisions".
Wright, C.M., R.J. Chovatiya, N.E. Jameson, D.M. Turner, G. Zhu, S. Werner, D.M. Huryn, J.M. Pipas, B.W. Day, P. Wipf, and J.L. Brodsky (2007) Pyrimidinone-peptoid hybrid molecules with distinct effects on molecular chaperone function and cell proliferation. Bioorg. Med. Chem. 16:3291-3301 The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, including breast cancer. Hsp70 ATP hydrolysis is relatively weak but is stimulated by J domain-containing proteins. We identified pyrimidinone-peptoid hybrid molecules that inhibit cell proliferation with greater potency than previously described Hsp70 modulators. In many cases, anti-proliferative activity correlated with inhibition of J domain stimulation of Hsp70.
Feng, D., X. Zhao, C. Soromani, J. Toikkanen, K. Romisch, S.S. Vembar, J.L. Brodsky, S. Keranen, and J. Jantti (2007) The trans-membrane domain is sufficient for Sbh1p function, its association with the Sec61 complex and its interaction with Rtn1p. J. Biol. Chem. 282:30618-30628 The Sec61 protein translocation complex in the endoplasmic reticulum (ER) membrane is composed of three subunits: The a-subunit, called Sec61p in yeast, is a multi-spanning membrane protein that forms the protein conducting channel. The functions of the smaller, carboxy-terminally tail-anchored ss subunit Sbh1p, its close homologue Sbh2p, and the subunit Sss1p are not well understood. Here we show that co-translational protein translocation into the ER is reduced in sbh1 sbh2 cells, whereas there is a limited reduction of post-translational tranlocation and no effect on export of a mutant form of alfa-factor precursor for ER-associated degradation in the cytosol. The translocation defect and the temperature-sensitive growth phenotype of sbh1 sbh2 cells were rescued by expression of the trans-membrane domain of Sbh1p alone and the Sbh1p trans-membrane domain was sufficient for coimmunoprecipitation with Sec61p and Sss1p. Furthermore, we show that Sbh1p co-precipitates with the ER trans-membrane protein Rtn1p. Sbh1p-Rtn1p complexes appear not to contain Sss1p and Sec61p. Our results define the trans-membrane domain as the minimal functional domain of the Sec61ss homologue Sbh1p in ER translocation, identify a novel interaction partner for Shb1p and imply that Sbh1p has additional functions that are not directly linked to protein translocation in association with the Sec61-complex.
Brodsky, J.L., and C.M. Scott (2007) Tipping the delicate balance: Defining how proteasome maturation affects the degradation of a substrate for autophagy and endoplasmic reticulum associated degradation (ERAD). Autophagy 3:623-625 An increasing body of data links endoplasmic reticulum (ER) function to autophagy. Not surprisingly, then, some aberrant proteins in the ER can be destroyed either via ER associated degradation (ERAD), which is proteasome-mediated, or via autophagy. One such substrate is the "Z" variant of the alpha-1 protease inhibitor (A1Pi), variably known as A1Pi-Z or AT-Z ("anti-trypsin, Z variant"). The wild type protein is primarily synthesized in the liver and is secreted. In contrast, AT-Z, like other ERAD substrates, is retro-translocated from the ER and delivered to the proteasome. However, AT-Z can form high molecular weight polymers that are degraded via autophagy, and cells that accumulate AT-Z polymers ultimately succumb, which leads to liver disease. Therefore, identifying genes that have an impact AT-Z turnover represents an active area of research. To this end, a yeast expression system for AT-Z has proven valuable. For example, a recent study using this system indicates that the activity of a proteasome assembly chaperone (PAC) is critical for maximal AT-Z turnover, which suggests a new role for PACs. Because PACs are conserved, it will be critical to analyze whether these dedicated chaperones are implicated in other diseases associated with ERAD and autophagy.
Hrizo, S.L., V. Gusarova, D.M. Habiel, J.L. Goeckeler, E.A. Fisher, and J.L. Brodsky (2007) The Hsp110 molecular chaperone stabilizes apolipoprotein B from endoplasmic reticulum associated degradation (ERAD). J. Biol. Chem. 282:32665-32675 Apolipoprotein B (apoB) is the most abundant protein in low-density lipoproteins and plays key roles in cholesterol homeostasis. The co-translational degradation of apoB is controlled by fatty acid levels in the ER and is mediated by the proteasome. To define the mechanism of apoB degradation, we employed a cell-free system in which proteasome-dependent degradation is recapitulated with yeast cytosol, and we developed an apoB yeast expression system. We discovered that a yeast Hsp110, Sse1p, associates with and stabilizes apoB, which contrasts with data indicating that Hsp70 and Hsp90 facilitate apoB degradation. However, the Ssb chaperones have no effect on apoB turnover. To determine whether our results are relevant in mammalian cells, Hsp110 was over-expressed in hepatocytes and enhanced apoB secretion was observed. This study indicates that chaperones within distinct complexes can play unique roles during ER associated degradation (ERAD), establishes a role for Sse1/Hsp110 in ERAD, and identifies Hsp110 as a target to lower cholesterol.
Sparvero, L.J., S. Patz, J.L. Brodsky, and C.M. Coughlan (2007) Proteomic analysis of the amyloid precursor protein fragment C99: expression in yeast. Anal. Biochem. 370:162-170 The accumulation and aggregation of fragments of amyloid precursor protein (APP) are central to the development of Alzheimer's disease. The production of the small fragment C99 is thought to form the rate-limiting step in the APP processing pathway, which can lead to the production of the toxic Abeta peptide. It has also been suggested that the proteasome contributes to APP catabolism. While the identities and aggregation propensities of many APP fragments have been studied in vitro, the sequences, structures, and cellular sources of fragments generated in vivo remains poorly elucidated. To better identify the specific APP fragments generated in vivo and to elucidate the role of the proteasome in APP processing, we developed a C99 yeast expression system. Using Zip Tip immunocapture, a specific anti-Abeta antiserum (6E10), and matrix-assisted laser desorption ionization- time of flight mass spectrometry, we identified over one dozen APP-generated peptide fragments in wild-type yeast (PRE1PRE2) and over three dozen unique fragments in proteasome mutant cells (pre1- 1pre2-1) expressing C99. Based on the identities of the immunocaptured species, we propose that defects in proteasome function are compensated by other proteases and that the combination of techniques described here will be invaluable to further delineate the APP processing pathway in vivo.
Kashlan, O.B., G.M. Mueller, M.Z. Qamar, P.A. Poland, A. Ahner, R.C. Rubenstein, R.P. Hughey, J.L. Brodsky, and T.R. Kleyman (2007) Small heat shock protein alpha A-crystallin regulates epithelial sodium channel expression. J. Biol. Chem. 282:28149-28156 Integral membrane proteins are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). After being translocated or inserted into the ER, they fold and undergo post-translational modifications. Within the ER, proteins are also subjected to quality control checkpoints during which misfolded proteins may be degraded by proteasomes via a process known as ER associated degradation (ERAD). Molecular chaperones, including the small heat shock protein {a}A-crystallin, have recently been shown to play a role in this process. We have now found that aA-crystallin is expressed in cultured mouse collecting duct cells, where apical Na+ transport is mediated by epithelial Na+ channels (ENaC). ENaC-mediated Na+ currents in Xenopus oocytes were reduced by co-expression of {a}A-crystallin. This reduction in ENaC activity reflected a decrease in the number of channels expressed at the cell surface. Furthermore, we observed that the rate of ENaC delivery to the cell surface of Xenopus oocytes was significantly reduced by co-expression of {a}A-crystallin, whereas the rate of channel retrieval remained unchanged. We also observed that {a}A-crystallin and ENaC coimmunoprecipitate. These data are consistent with the hypothesis that small heat shock proteins recognize ENaC subunits at ER quality control checkpoints and can target ENaC subunits for ERAD.
Scott, C.M., K.B. Kruse, B.Z. Schmidt, D.H. Perlmutter, A.A. McCracken, and J.L. Brodsky (2007) ADD66, a gene involved in the endoplasmic reticulum associated degradation (ERAD) of alpha-1-antitrypsin-Z in yeast, facilitates proteasome activity and assembly. Mol. Biol. Cell 18:3776-3787 Monitoring Editor: Peter Walter Antitrypsin Deficiency is a primary cause of juvenile liver disease and arises from expression of the "Z" variant of the alpha-1 protease inhibitor (A1Pi). Whereas A1Pi is secreted from the liver, A1PiZ is retro-translocated from the endoplasmic reticulum (ER) and degraded by the proteasome, an event that may offset liver damage. To better define the mechanism of A1PiZ degradation, a yeast expression system was developed and a gene, ADD66, was identified that facilitates A1PiZ turn-over (Palmer et al., J. Cell. Sci. 116, 2361-2373, 2003). We report here that ADD66 encodes an approximately 30 kDa soluble, cytosolic protein and that the chymotrypsin-like activity of the proteasome is reduced in add66Delta mutants. This reduction in activity may arise from the accumulation of 20S proteasome assembly intermediates or from qualitative differences in assembled proteasomes. Add66p also appears to be a proteasome substrate. Consistent with its role in ER associated degradation (ERAD), synthetic interactions are observed between the genes encoding Add66p and Ire1p, a transducer of the unfolded protein response, and yeast deleted for both ADD66 and/or IRE1 accumulate polyubiquitinated proteins. These data identify Add66p as a proteasome assembly chaperone (PAC) and provide the first link between PAC activity and ERAD.
Rodina, A., M. Vilenchik, K. Moulick, J. Aguirre, J. Kim, A. Chiang, J. Litz, C.C. Clement, Y. Kang, Y. She, N. Wu, S. Felts, P. Wipf, J. Massague, X. Jiang, J.L. Brodsky, G.W. Krystal, and G. Chiosis (2007) Selective compounds define Hsp90 as a major inhibitor of apoptosis in small-cell lung cancer. Nat. Chem. Biol. 3:498-507 The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome-channeled. These results provide important evidence for a transformation-specific interplay between chaperones in regulating apoptosis in malignant cells.
Wahlman, J., G.N. Demartino, W.R. Skach, N.J. Bulleid, J.L. Brodsky, and A.E. Johnson (2007) Real-Time fluorescence detection of ERAD substrate retrotranslocation in a mammalian in vitro system. Cell 129:943-955 Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or "retrotranslocated" into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encapsulated with selected lumenal factors inside mammalian microsomes. After mixing microsomes with fluorescence-quenching agents and selected cytosolic proteins, the rate of substrate efflux was monitored continuously in real time by the decrease in fluorescence intensity as cytosolic quenchers contacted dye-labeled substrates. The retrotranslocation kinetics of nonglycosylated pro-alpha factor were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase or all cytosolic proteins with only PA700, the 19S regulatory particle of the 26S proteasome. Retrotranslocation was blocked by antibodies against a putative retrotranslocation channel protein, derlin-1, but not Sec61alph
Gusarova, V., J. Seo, M.L. Sullivan, S.C. Watkins, J.L. Brodsky, and E.A. Fisher (2007) Golgi-associated maturation of very low density lipoproteins involves conformational changes of apolipoprotein B, but is not dependent on apolipoprotein E. J. Biol. Chem. 282:19453-19462 The major protein component in secreted very low density lipoproteins (VLDL); is apolipoprotein B (apoB), and it is established that these particles can reach sizes approaching 100 nm. We previously employed a cell-free system to investigate the nature of the vesicles in which this large cargo exits the endoplasmic reticulum (ER). We found that apoB-containing lipoproteins exited the ER as dense lipid-protein complexes regardless of the final sizes of the particles, and that further expansion occurred via post-ER lipidation. Here, we focus on maturation in the Golgi apparatus. In three separate approaches, we found that VLDL maturation (as assessed by changes in buoyant density) is associated with conformational changes in apoB. In addition, as the size of VLDL expands, apoprotein E (apoE) concentrates in a sub-class of Golgi microsomes or Golgi-derived vesicles that co-migrate with apoBcontaining microsomes or vesicles, respectively. A relationship between apoB and apoE was further confirmed by colocalization studies by immunoelectron microscopy. These combined results are consistent with previous suggestions that apoE is required for VLDL maturation. To our surprise, however, we observed robust secretion of mature VLDL either when apoE synthesis is inhibited in rat hepatoma cells or in apoE-/-mouse primary hepatocytes. We conclude that VLDL maturation in the Golgi involves apoB conformational changes and that the expansion of the lipoprotein does not require apoE; rather, the increase in VLDL surface area favors apoE binding.
Brodsky, J.L. (2007) The protective and destructive roles played by molecular chaperones during ERAD (endoplasmic-reticulum-associated degradation). Biochem. J. 404:353-363 Over one-third of all newly synthesized polypeptides in eukaryotes interact with or insert into the membrane or the lumenal space of the ER (endoplasmic reticulum), an event that is essential for the subsequent folding, post-translational modification, assembly and targeting of these proteins. Consequently, the ER houses a large number of factors that catalyse protein maturation, but, in the event that maturation is aborted or inefficient, the resulting aberrant proteins may be selected for ERAD (ER-associated degradation). Many of the factors that augment protein biogenesis in the ER and that mediate ERAD substrate selection are molecular chaperones, some of which are heat- and/or stress-inducible and are thus known as Hsps (heat-shock proteins). But, regardless of whether they are constitutively expressed or are inducible, it has been assumed that all molecular chaperones function identically. As presented in this review, this assumption may be false. Instead, a growing body of evidence suggests that a chaperone might be involved in either folding or degrading a given substrate that transits through the ER. A deeper appreciation of this fact is critical because (i) the destruction of some ERAD substrates results in specific diseases, and (ii) altered ERAD efficiency might predispose individuals to metabolic disorders. Moreover, a growing number of chaperone-modulating drugs are being developed to treat maladies that arise from the synthesis of a unique mutant protein; therefore it is critical to understand how altering the activity of a single chaperone will affect the quality control of other nascent proteins that enter the ER.
Perlmutter, D.H., J.L. Brodsky, W.F. Balistreri, and B.C. Trapnell (2007) Molecular pathogenesis of alpha-1-antitrypsin deficiency-associated liver disease: A meeting review. Hepatology 45:1313-1323 In recent years, we have witnessed several important paradigm shifts in understanding the molecular basis of liver disease in alpha-1-antitrypsin (AT) deficiency. These shifts have become possible as a result of a number of advances in research on the cell biology of aggregation-prone mutant proteins and in research on the pathobiological mechanisms of liver disease in general. Late-breaking research in these areas was the subject of an AASLD/Alpha-1 Foundation Single Topic Conference in Atlanta, Georgia, on January 26 to 28, 2006. The conference was titled "Alpha-1-Antitrypsin Deficiency and Other Liver Diseases Caused by Aggregated Proteins." Investigators from all over the world, representing a broad array of scientific disciplines and perspectives, discussed the pathobiology of AT deficiency, mechanisms of cell injury in diseases associated with aggregation-prone proteins, pathways by which cells respond to protein aggregation and mislocalization, and mechanisms of liver injury in general and in diseases related to AT deficiency. A session of the meeting was devoted to novel therapeutic strategies being developed for AT deficiency as well as to strategies either in development or already being applied to the class of diseases associated with mutant proteins. (HEPATOLOGY 2007;45:1313-1323.).
Spring, J.H., S.R. Robichaux, N. Kaufmann, and J.L. Brodsky (2007) Localization of a Drosophila DRIP-like aquaporin in the malpighian tubules of the house cricket, Acheta domesticus. Comp. Biochem. Phys. A 148:92-100 Malpighian tubules (Mt) are the primary excretory and osmoregulatory organs of insects, capable of rapidly transporting extraordinary volumes of fluid when stimulated by diuretic factors. In the house cricket, Acheta domesticus, the Mt are composed of three morphologically distinct regions (proximal, mid, and distal). Unlike the dipteran Mt, which have both primary and stellate cells, each region of the Acheta Mt consists of a morphologically uniform cell type. The mid and distal regions are both secretory in function and increase secretion rate in response to dibutyryl cAMP (cAMP). Achetakinin-2, while acting synergistically with cAMP on the mid-Mt, inhibits secretion by the distal Mt, and the effects can be reversed by cAMP. Using an antibody to the water-specific Drosophila aquaporin (DRIP), we demonstrated that DRIP-like immunoreactivity was found in both the distal and mid-Mt. The distribution of the aquaporin altered in response to stimulation and was consistent with the secretory data. The regulation of secretion in Acheta Mt is quite different from that of Drosophila, with both cation and anion/water transport occurring in the same cells. This is the first demonstration of the presence of an insect aquaporin, namely DRIP, in the Mt of an order other than the Diptera.
Jelenska, J., N. Yao, B.A. Vinatzer, C.M. Wright, J.L. Brodsky, and J.T. Greenberg (2007) A J domain virulence effector of Pseudomonas syringae remodels host chloroplasts and suppresses defenses. Curr. Biol. 17:499-508 BACKGROUND: The plant pathogen Pseudomonas syringae injects 20-40 different proteins called effectors into host plant cells, yet the functions and sites of action of these effectors in promoting pathogenesis are largely unknown. Plants in turn defend themselves against P. syringae by activating the salicylic acid (SA)-mediated signaling pathway. The P. syringae-specific HopI1 effector has a putative chloroplast-targeting sequence and a J domain. J domains function by activating 70 kDa heat-shock proteins (Hsp70). RESULTS: HopI1 is a ubiquitous P. syringae virulence effector that acts inside plant cells. When expressed in plants, HopI1 localizes to chloroplasts, the site of SA synthesis. HopI1 causes chloroplast thylakoid structure remodeling and suppresses SA accumulation. HopI1's C terminus has bona fide J domain activity that is necessary for HopI1-mediated virulence and thylakoid remodeling. Furthermore, HopI1-expressing plants have increased heat tolerance, establishing that HopI1 can engage the plant stress-response machinery. CONCLUSIONS: These results strongly suggest that chloroplast Hsp70 is targeted by the P. syringae HopI1 effector to promote bacterial virulence by suppressing plant defenses. The targeting of Hsp70 function through J domain proteins is known to occur in a mammalian virus, SV40. However, this is the first example of a bacterial pathogen exploiting a J domain protein to promote pathogenesis through alterations of chloroplast structure and function.
Wright, C.M., S.W. Fewell, M.L. Sullivan, J.M. Pipas, S.C. Watkins, and J.L. Brodsky (2007) The hsp40 molecular chaperone, Ydj1p, along with the protein kinase C pathway, impact cell wall integrity in the yeast Saccharomyces cerevisiae. Genetics 175:1649-1664 Molecular chaperones, such as Hsp40, regulate cellular processes by aiding in the folding, localization, and activation of multi-protein machines. To identify new targets of chaperone action we performed a multi-copy suppressor screen for genes that improved the slow growth defect of yeast lacking the YDJ1 chromosomal locus and expressing a defective Hsp40 chimera. Among the genes identified were MID2, which regulates cell wall integrity, and PKC1, which encodes protein kinase C and is linked to cell wall biogenesis. We found that ydj1Delta yeast exhibit phenotypes consistent with cell wall defects and these phenotypes were improved by Mid2p or Pkc1p over-expression or by over-expression of activated down-stream components in the PKC pathway. Yeast containing a thermosensitive allele in the gene encoding Hsp90 also exhibited cell wall defects, and Mid2p or Pkc1p over-expression improved the growth of these cells at elevated temperatures. To determine the physiological basis for suppression of the ydj1Delta growth defect, wild type and ydj1Delta yeast were examined by electron microscopy and we found that Mid2p over-expression thickened the mutant's cell wall. Together, these data provide the first direct link between cytoplasmic chaperone function and cell wall integrity, and suggest that chaperones orchestrate the complex biogenesis of this structure.
Mandal, A.K., P. Lee, J.A. Chen, N. Nillegoda, A. Heller, S. Distasio, H. Oen, J. Victor, D.M. Nair, J.L. Brodsky, and A.J. Caplan (2007) Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation. J. Cell. Biol. 176:319-328 Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases ( approximately 50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis.
Ahner, A., K. Nakatsukasa, H. Zhang, R.A. Frizzell, and J.L. Brodsky (2007) Small Heat Shock Proteins Select DF508-CFTR for ER Associated Degradation (ERAD). Mol. Biol. Cell 18:806-814 Secreted proteins that fail to achieve their native conformations, such as CFTR (Cystic Fibrosis Transmembrane conductance Regulator) and particularly the DeltaF508-CFTR variant can be selected for ER-associated degradation (ERAD) by molecular chaperones. Because the message corresponding to HSP26, which encodes a small heat shock protein (sHsp) in yeast was up-regulated in response to CFTR expression, we examined the impact of sHsps on ERAD. First, we observed that CFTR was completely stabilized in cells lacking two partially redundant sHsps, Hsp26p and Hsp42p. Interestingly, the ERAD of a soluble and a related integral membrane protein were unaffected in yeast deleted for the genes encoding these sHsps, and CFTR polyubiquitination was also unaltered, suggesting that Hsp26p/Hsp42p are not essential for polyubiquitination. Next, we discovered that DeltaF508-CFTR degradation was enhanced when a mammalian sHsp, alphaA-crystallin, was overexpressed in HEK293 cells, but wild type CFTR biogenesis was unchanged. Because alphaA-crystallin interacted preferentially with DeltaF508-CFTR and because purified alphaA-crystallin suppressed the aggregation of CFTR's first nucleotide-binding domain, we suggest that sHsps maintain the solubility of DeltaF508-CFTR during the ERAD of this polypeptide.
Brodsky, J.L., and G. Chiosis (2006) Hsp70 molecular chaperones: emerging roles in human disease and identification of small molecule modulators. Curr. Top. Med. Chem. 6:1215-1225 Molecular chaperones are best known for their ability to aid in the solubilization of mis-folded proteins, and as a result play essential roles in protein folding, degradation, and transport. However, many molecular chaperones also play essential roles in signal transduction cascades. For example, Hsp70 molecular chaperones are a highly conserved, abundant class of chaperones that are found in every species and in nearly every cellular compartment in eukaryotes. In addition to their well-established roles in facilitating protein folding and in the targeting of proteins to organelles and to proteolytic machines, Hsp70s are anti-apoptotic and inhibition of Hsp70 function in some cases is sufficient to induce tumor cell death. Hsp70 function is also vital for the replication of viruses. Based on these data, small molecule Hsp70 modulators might, in principle, be used for the treatment of specific cancers, infections, and protein conformational diseases. In this review, we summarize the structural and functional characteristics of Hsp70 chaperones, and then discuss their roles in cellular physiology. Finally, we will review the recent discovery of small molecules that alter Hsp70 expression and function. Kruse, K.B., J.L. Brodsky, and A.A. McCracken (2006) Autophagy: An ER Protein Quality Control Process. Autophagy 2:135-137 Protein quality control processes active in the endoplasmic reticulum (ER), including ER-associated protein degradation (ERAD) and the unfolded protein response (UPR), prevent the cytotoxic effects that can result from the accumulation of misfolded proteins. Characterization of a yeast mutant deficient in ERAD, a proteasome-dependent degradation pathway, revealed the employment of two overflow pathways from the ER to the vacuole when ERAD was compromised. One removes the soluble misfolded protein via the biosynthetic pathway and the second clears aggregated proteins via autophagy. Previously, autophagy had been implicated in the clearance of cytoplasmic aggresomes, but was not known to play a direct role in ER protein quality control. These findings provide insight into the molecular mechanisms that result in the gain-of-function liver disease associated with both alpha1-deficiency and hypofibrinogenemia (abnormally low levels of plasma fibrinogen, which is required for blood clotting), and emphasize the need for a more complete understanding of the molecular mechanisms of autophagy and its relationship to protein quality control.
Kruse, K.B., J.L. Brodsky, and A.A. McCracken (2006) Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble z variant of human a-1 proteinase inhibitor (A1PiZ) and another for aggregates of A1PiZ. Mol. Biol. Cell 17:203-212 The Z variant of human alpha-1 proteinase inhibitor (A1PiZ) is a substrate for endoplasmic reticulum-associated protein degradation (ERAD). To identify genes required for the degradation of this protein, A1PiZ degradation-deficient (add) yeast mutants were isolated. The defect in one of these mutants, add3, was complemented by VPS30/ATG6, a gene that encodes a component of two phosphatidylinositol 3-kinase (PtdIns 3-kinase) complexes: complex I is required for autophagy, whereas complex II is required for the carboxypeptidase Y (CPY)-to-vacuole pathway. We found that upon overexpression of A1PiZ, both PtdIns 3-kinase complexes were required for delivery of the excess A1PiZ to the vacuole. When the CPY-to-vacuole pathway was compromised, A1PiZ was secreted; however, disruption of autophagy led to an increase in aggregated A1PiZ rather than secretion. These results suggest that excess soluble A1PiZ transits the secretion pathway to the trans-Golgi network and is selectively targeted to the vacuole via the CPY-to-vacuole sorting pathway, but excess A1PiZ that forms aggregates in the endoplasmic reticulum is targeted to the vacuole via autophagy. These findings illustrate the complex nature of protein quality control in the secretion pathway and reveal multiple sites that recognize and sort both soluble and aggregated forms of aberrant or misfolded proteins.
Zhang, H., B.Z. Schmidt, F. Sun, S.B. Condliffe, M.B. Butterworth, R.T. Youker, J.L. Brodsky, M. Aridor, and R.A. Frizzell (2006) Cysteine string protein monitors late steps in CFTR biogenesis. J. Biol. Chem. 281:11312-11321 We examined the role of the cysteine string protein (Csp) in CFTR biogenesis in relation to another J-domain protein, Hdj-2, a recognized CFTR co-chaperone. Increased expression of Csp produced a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR. Exogenous expression of Hdj-2 also increased CFTR band B, but unlike Csp, Hdj-2 increased band C as well. The Csp-induced block of CFTR maturation required Hsp70, since a J-domain mutant (H43Q) that interferes with Csp's ability to stimulate Hsp70 ATPase activity relieved the Csp-induced block of CFTR maturation. Nevertheless, Csp H43Q still increased immature CFTR. Csp-induced band B CFTR was found adjacent to the nucleus, co-localizing with calnexin, and it remained detergent soluble. These data indicate that Csp did not block CFTR maturation by promoting the aggregation or degradation of immature CFTR. Csp knockdown by RNAi produced a 5-fold increase in mature CFTR and augmented cAMP-stimulated CFTR currents. Thus, the production of mature CFTR is inversely related to Csp expression level. Both Csp and Hdj-2 associated with the CFTR R domain in vitro, and Hdj-2 binding was displaced by Csp, suggesting common interaction sites. Combined expression of Csp and Hdj-2 mimicked the effect of Csp alone: a block of CFTR maturation. But together, Csp and Hdj-2 produced additive increases in CFTR band B, and this did not depend on their interactions with Hsp70, consistent with direct chaperone actions of these proteins. Like Hdj-2, Csp reduced the aggregation of NBD1 in vitro in the absence of Hsp70. Our data suggest that both Csp and Hdj-2 facilitate the biosynthesis of immature CFTR, acting as direct CFTR chaperones, but in addition, Csp is positioned later in the CFTR biogenesis cascade where it regulates the production of mature CFTR by limiting its exit from the ER.
Karlgren, S., N. Pettersson, B. Nordlander, J.C. Mathai, J.L. Brodsky, M.L. Zeidel, R.M. Bill, and S. Hohmann (2005) Conditional osmotic stress in yeast: a system to study transport through aquaglyceroporins and osmostress signaling. J. Biol. Chem. 280:7186-7193 The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1Delta gpd2Delta mutant both to produce glycerol and to adapt to high osmolarity to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1Delta gpd2Delta mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-Delta1 of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-Delta1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-Delta1 showed similarly low permeability for water. The growth defect on polyols in the gpd1Delta gpd2Delta mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx, causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1Delta gpd2Delta mutants were treated with glycerol, xylitol, or sorbitol, the osmosensing HOG pathway was activated, and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system.
Chiosis, G., and J.L. Brodsky (2005) Small molecule microarrays: from proteins to mammalian cells - are we there yet? Trends Biotechnol. 23:271-274 A recent publication by Stockwell and colleagues documents a leap forward toward the continued development of small molecule microarray (SMM) technology. By creating microarrays of small molecules impregnated in a biodegradable polymer, the authors have, for the first time, shown that SMMs can be used in a cell-based format. This technological improvement opens the door for using SMMs to perform high-throughput screens in mammalian cells.
Nishikawa, S., J.L. Brodsky, and K. Nakatsukasa (2005) Roles of molecular chaperones in endoplasmic reticulum (ER) quality control and ER-Associated degradation (ERAD). J. Biochem. 137:551-555 Secreted proteins are synthesized at the endoplasmic reticulum (ER), and a quality control mechanism in the ER is essential to maintain secretory pathway homeostasis. Newly synthesized soluble and integral membrane secreted proteins fold into their native conformations with the aid of ER molecular chaperones before they are transported to post-ER compartments. However, terminally mis-folded proteins may be retained in the ER and degraded by a process called ER-associated degradation (ERAD). Recent studies using yeast have shown that molecular chaperones both in the ER and in the cytosol play key roles during the ERAD of mis-folded proteins. One important role for chaperones during ERAD is to prevent substrate protein aggregation. Substrate selection is another important role for molecular chaperones during ERAD.
Kaufmann, N., J.C. Mathai, W.G. Hill, J.A. Dow, M.L. Zeidel, and J.L. Brodsky (2005) Developmental expression and biophysical characterization of a Drosophila melanogaster aquaporin. Am. J. Physiol. Cell. Ph. 289:397-407 Aquaporins (AQP) accelerate the movement of water and other solutes across biological membranes, yet the molecular mechanisms of each AQP[[rad]]s transport function and the diverse physiological roles played by AQP family members are still being defined. We therefore characterized an AQP in a model organism, Drosophila melanogaster, which is amenable to genetic manipulation and developmental analysis. To further understand mechanisms of Malpighian tubule-facilitated water transport, we identified 7 putative AQPs in the Drosophila genome (dAQPs) and found that one of these, previously named DRIP, has the highest sequence similarity to those vertebrate AQPs which exhibit the fastest rates of water transport. In situ mRNA analyses showed that DRIP is expressed in both embryonic and adult Malpighian tubules, as well as in other tissues in which fluid transport is essential. In addition, the pattern of DRIP expression was dynamic. To define DRIP-mediated water transport, the protein was expressed in Xenopus oocytes and in yeast secretory vesicles and significantly elevated rates of water transport correlated with DRIP expression. Moreover, the activation energy for water transport in DRIP-expressing secretory vesicles was 4.9 kcal/mole. This low value is characteristic of AQP-mediated water transport whereas the value in control vesicles was 16.4 kcal/mole. In contrast, glycerol, urea, ammonia, and proton transport were unaffected by DRIP expression, suggesting that DRIP is a highly selective water-specific channel. This result is consistent with the homology between DRIP and mammalian water-specific AQPs. Together, these data establish Drosophila as a new model system to investigate AQP function.
Brodsky, J.L. (2005) An in vitro assay for the selective endoplasmic reticulum associated degradation of an unglycosylated secreted protein. Methods 35:354-359 The endoplasmic reticulum (ER) represents the first compartment into which nascent secreted proteins traffic, and not coincidentally the ER lumen houses a high concentration of factors that facilitate protein folding, such as molecular chaperones. To off-set the potentially lethal consequences of mis-folded secreted protein accumulation, aberrant proteins may be selected for degradation via a process known as ER associated degradation (ERAD). After their selection ERAD substrates are retro-translocated back to the cytoplasm and then degraded by the 26S proteasome. Key features of the selection, retro-translocation, and degradation steps that constitute the ERAD pathway were elucidated through the development of an in vitro ERAD assay. In this assay the fates of two yeast proteins can be distinguished after their translocation, or import into ER-derived microsomes. Whereas a wild type, glycosylated protein ("GpalphaF") is stable, a non-glycosylated version of the same protein ("palphaF") is rapidly degraded when microsomes containing radiolabeled forms of these substrates are incubated in cytosol and ATP. The purpose of this chapter is first to discuss the experimental findings from the use of the in vitro assay, and then to describe the assay in detail. Finally, future potential uses of the in vitro system are illustrated.
Ahner, A., F.M. Whyte, and J.L. Brodsky (2005) Distinct but overlapping functions of Hsp70, Hsp90, and an Hsp70 nucleotide exchange factor during protein biogenesis in yeast. Arch. Biochem. Biophys. 435:32-41 Hsp70 and Hsp90 molecular chaperones play essential roles in protein expression and maturation, and while catalyzing protein folding they can "decide" to target mis-folded substrates for degradation. In this report, we show for the first time distinct but partially overlapping requirements for Hsp90, Hsp70, and an Hsp70 nucleotide exchange factor (NEF) at different steps during the biogenesis of a model substrate, firefly luciferase (FFLux), in yeast. By examining the inducible expression of FFLux in wild type cells and in specific yeast mutants, we find that the Fes1p NEF is required for efficient FFLux folding, whereas the Hsp70, Ssa1p, is required for both protein folding and stability, and to maintain maximal FFLux mRNA levels. In contrast, Hsp90 function was primarily necessary to express the FFLux-encoding gene from an inducible promoter. Together, these data indicate previously unknown roles for these proteins and point to the complexity with which chaperones and cochaperones function in the cell.
Alder, N.N., Y. Shen, J.L. Brodsky, L.M. Hendershot, and A.E. Johnson (2005) The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum. J. Cell Biol. 168:389-399 The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain-binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding-induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain-binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates.
Shomura, Y., Z. Dragovic, H.C. Chang, N. Tzvetkov, J.C. Young, J.L. Brodsky, V. Guerriero, F.U. Hartl, and A. Bracher (2005) Regulation of Hsp70 function by HspBP1; structural analysis reveals an alternate mechanism for Hsp70 nucleotide exchange. Mol. Cell 17:367-379 HspBP1 belongs to a family of eukaryotic proteins recently identified as nucleotide exchange factors for Hsp70. We show that the S. cerevisiae ortholog of HspBP1, Fes1p, is required for efficient protein folding in the cytosol at 37 C. The crystal structure of HspBP1, alone and complexed with part of the Hsp70 ATPase domain, reveals a mechanism for its function distinct from that of BAG-1 or GrpE, previously characterized nucleotide exchange factors of Hsp70. HspBP1 has a curved, all alpha-helical fold containing four armadillo-like repeats unlike the other nucleotide exchange factors. The concave face of HspBP1 embraces lobe II of the ATPase domain, and a steric conflict displaces lobe I, reducing the affinity for nucleotide. In contrast, BAG-1 and GrpE trigger a conserved conformational change in lobe II of the ATPase domain. Thus, nucleotide exchange on eukaryotic Hsp70 occurs through two distinct mechanisms.
Fewell, S.W., C.M. Smith, M.A. Lyon, T.P. Dumitrescu, P. Wipf, B.W. Day, and J.L. Brodsky (2004) Small molecule modulators of endogenous and co-chaperone-stimulated Hsp70 ATPase activity. J. Biol. Chem. 279:51131-51140 The molecular chaperone and cytoprotective activities of the Hsp70 and Hsp40 chaperones represent therapeutic targets for human diseases such as cancer and those that arise from defects in protein folding; however, very few Hsp70 and no Hsp40 modulators have been described. Using an assay for ATP hydrolysis, we identified and screened small molecules with structural similarity to 15-deoxyspergualin and NSC 630668-R/1 for their effects on endogenous and Hsp40-stimulated Hsp70 ATPase activity. Several of these compounds modulated Hsp70 ATPase activity, consistent with the action of NSC 630668-R/1 observed previously (Fewell, S. W., Day, B. W., and Brodsky, J. L. (2001) J. Biol. Chem. 276, 910-914). In contrast, three compounds inhibited the ability of Hsp40 to stimulate Hsp70 ATPase activity but did not affect the endogenous activity of Hsp70. Two of these agents also compromised the Hsp70 /Hsp40-mediated post-translational translocation of a secreted pre-protein in vitro. Together, these data indicate the potential for continued screening of small molecule Hsp70 effectors and that specific modulators of Hsp70-Hsp40 interaction can be obtained, potentially for future therapeutic use.
Huyer, G., W.F. Piluek, Z. Fansler, S.G. Kreft, M. Hochstrasser, J.L. Brodsky, and S. Michaelis (2004) Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein. J. Biol. Chem. 279:38369-38378 The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane are monitored by ER quality control. Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. Whereas luminal and integral membrane ERAD substrates both require the proteasome for their degradation, the ER quality control machinery for these two classes of proteins likely differs because of their distinct topologies. Here we establish the requirements for the ERAD of Ste6p*, a multispanning membrane protein with a cytosolic mutation, and compare them with those for mutant form of carboxypeptidase Y (CPY*), a soluble luminal protein. We show that turnover of Ste6p* is dependent on the ubiquitin-protein isopeptide ligase Doa10p and is largely independent of the ubiquitin-protein isopeptide ligase Hrd1p/Der3p, whereas the opposite is true for CPY*. Furthermore, the cytosolic Hsp70 chaperone Ssa1p and the Hsp40 co-chaperones Ydj1p and Hlj1p are important in ERAD of Ste6p*, whereas the ER luminal chaperone Kar2p is dispensable, again opposite their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to require the Sec61p translocon pore but, like CPY*, could depend on the Sec61p homologue Ssh1p. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of Ste6p* employs distinct machinery from that of the soluble luminal substrate CPY* and that Ste6p* is a valuable model substrate to dissect the cellular machinery required for the ERAD of multispanning membrane proteins with a cytosolic mutation.
Youker, R.T., P. Walsh, T. Beilharz, T. Lithgow, and J.L. Brodsky (2004) Distinct roles for the Hsp40 and Hsp90 molecular chaperones during cystic fibrosis transmembrane conductance regulator degradation in yeast. Mol. Biol. Cell. 15:4787-4797 Monitoring Editor: Reid Gilmore Aberrant secreted proteins can be destroyed by ER associated protein degradation (ERAD), and a prominent, medically-relevant ERAD substrate is CFTR. To better define the chaperone requirements during CFTR maturation the protein was expressed in yeast. Because Hsp70 function impacts CFTR biogenesis in yeast and mammals we first sought ER-associated Hsp40 cochaperones involved in CFTR maturation. Ydj1p and Hlj1p enhanced Hsp70 ATP hydrolysis but CFTR degradation was slowed only in yeast mutated for both YDJ1 and HLJ1, suggesting functional redundancy. In contrast, CFTR degradation was accelerated in an Hsp90 mutant strain, suggesting that Hsp90 preserves CFTR in a folded state, and consistent with this hypothesis Hsp90 maintained the solubility of an aggregation-prone domain (NBD1) in CFTR. Soluble ERAD substrate degradation was unaffected in the Hsp90 or the Ydj1p/Hlj1p mutants, and surprisingly CFTR degradation was unaffected in yeast mutated for Hsp90 cochaperones. These results indicate that Hsp90, but not the Hsp90 complex, maintains CFTR structural integrity, whereas Ydj1p/Hlj1p catalyze CFTR degradation.
Ahner, A., and J.L. Brodsky (2004) Checkpoints in ER-associated degradation: excuse me, which way to the proteasome? Trends Cell Biol. 14:474-478 The failure of secreted proteins to fold results in their retrotranslocation from the endoplasmic reticulum (ER) and degradation by the proteasome in a process called 'ER-associated degradation' (ERAD). Two recent studies indicate that ERAD substrates are targeted to different pathways depending on the topology of the substrate and the subcellular location of the misfolded domain. Brodsky, J.L., V. Gusarova, and E.A. Fisher (2004) Vesicular trafficking of hepatic apolipoprotein B100 and its maturation to very low-density lipoprotein particles: Studies from cells and cell-free systems. Trends Cardiovas. Med. 14:127-132 A cell-free system was established to study the process by which apolipoprotein (apo)B100-containing lipoproteins exit the endoplasmic reticulum (ER). ApoB was found in COPII vesicles with physical properties distinct from those containing other secreted proteins. When lipid synthesis in rat hepatoma cells was stimulated by fatty acid addition, fully lipidated apoB-lipoproteins of very low-density lipoprotein density were absent from the vesicles, but instead formed in a post-ER compartment. These data suggest that the COPII machinery in cells of hepatic and intestinal origin has evolved to sequester secreted cargoes with unique properties compared with those in other tissues, and that final lipidation occurs after a protein quality-control checkpoint is passed in the ER. Lee, R.J., C.W. Liu, C. Harty, A.A. McCracken, M. Latterich, K. Romisch, G.N. DeMartino, P.J. Thomas, and J.L. Brodsky (2004) Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein. EMBO J. 23:2206-2215 Aberrant polypeptides in the endoplasmic reticulum (ER) are retro-translocated to the cytoplasm and degraded by the 26S proteasome via ER-associated degradation (ERAD). To begin to resolve the requirements for the retro-translocation and degradation steps during ERAD, a cell-free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER-derived microsomes during the ERAD of a model, soluble polypeptide. As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system. Proficient ERAD was observed if wild-type cytosol was substituted with either purified yeast or mammalian proteasomes. Moreover, addition of only the 19S cap of the proteasome catalyzed ATP-dependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle.
Coughlan, C.M., J.L. Walker, J.C. Cochran, K.D. Wittrup, and J.L. Brodsky (2004) Degradation of mutated bovine pancreatic trypsin inhibitor (BPTI) in the yeast vacuole suggests post-endoplasmic reticulum protein quality control. J. Biol. Chem 279:15289-15297 The rate-limiting step in protein secretion is folding, which occurs in the ER lumen, and almost all secreted proteins contain disulfide bonds that form in the ER and stabilize the native state. Secreted proteins unable to fold may aggregate or they may be subject to ER associated protein degradation (ERAD). To examine the fate of aberrant forms of a well-characterized, disulfide-bonded secreted protein, we expressed bovine pancreatic trypsin inhibitor (BPTI) in yeast. BPTI is a single domain, 58 amino acid polypeptide containing three disulfide bonds, and yeast cells secrete the wild type protein. In contrast, the Y35L mutant, which folds rapidly but is unstable, remains soluble and is not secreted. Surprisingly, the proteolysis of Y35L is unaffected in yeast containing mutations in genes encoding factors required for ERAD and is stable if artificially retained in the ER. Rather, Y35L is diverted from the Golgi to the vacuole and degraded. Because only the mutant protein is quantitatively proteolyzed these data suggest that a post-ER quality control check-point diverts unstable proteins to the vacuole for degradation.
Shaner, L., A. Trott, J.L. Goeckeler, J.L. Brodsky, and K.A. Morano (2004) The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related Hsp70 family. J. Biol. Chem. 279:21992-22001 The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1 yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1 and lethality of sse1 sse2 cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme carboxyl-terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 carboxyl-terminal residues not included in the PBD were active. Remarkably, the two domains of Sse1 when expressed in trans functionally complement the sse1 growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill-defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.
Gusarova, V., J.L. Brodsky, and E.A. Fisher (2003) Apolipoprotein B100 exit from the endoplasmic reticulum (ER) Is COPII-dependent,and its lipidation to very low density lipoprotein occurs post-ER. J. Biol. Chem. 278:48051-48058 Hepatic apolipoprotein B100 (apoB100) associates with lipids to form dense lipoprotein particles in the endoplasmic reticulum (ER) and is further lipidated to very low density lipoproteins (VLDL). Because the VLDL diameter can exceed 200 nm, classical ER-derived vesicles may be unable to accommodate VLDLs. Using hepatic membranes and cytosol to reconstitute ER budding, apoB100-containing vesicles sedimented distinct from those harboring more typical cargo but contained Sec23. Moreover, ER exit of apoB was inhibited by dominant-negative Sar1. Budding required Sar1 regardless of whether oleic acid (OA) was added to stimulate apoB lipidation; therefore, either large apoB100-lipoproteins reside in secretory vesicles, or full lipidation occurs post-ER. Using membranes from cells incubated in the presence or absence of OA, we determined that apoB100-lipoproteins in ER vesicles had not become lipidated to VLDLs. VLDL particles resided in the Golgi, but not the ER, after fractionation of OA-treated cells. We conclude that apoB100-lipoproteins exit the ER in COPII vesicles, but under conditions favorable for VLDL formation final lipid loading occurs post-ER.
McCracken, A.A., and J.L. Brodsky (2003) Evolving questions and paradigm shifts in ER associated degradation (ERAD). BioEssays 25:868-877 ER-associated degradation (ERAD) is a component of the protein quality control system, ensuring that aberrant polypeptides cannot transit through the secretory pathway. This is accomplished by a complex sequence of events in which unwanted proteins are selected in the ER and exported to the cytosol for degradation by the proteasome. Given that protein quality control can be essential for cell survival, it is not surprising that ERAD is linked to numerous disease states. Here we review the molecular mechanisms of ERAD, its role in metabolic regulation and biomedical implications, and the unanswered questions regarding this process. Kabani, M., J.M. Beckerich, and J.L. Brodsky (2003) The yeast Sls1 and Fes1 proteins define a new family of Hsp70 nucleotide exchange factors. Curr. Genomics 4:463-473 Hsp70 molecular chaperones play a variety of functions in every organism, cell type and compartment, and their activities have been implicated in a number of disease states. Hsp70 activity relies on ATP binding and hydrolysis by the N-terminal domain, which modulates peptide binding and release by the C-terminal domain. In Escherichia coli, the Hsp70 family member DnaK is regulated by DnaJ, which stimulates its ATPase activity, and by GrpE, a nucleotide exchange factor that promotes ADP release. In eukaryotic cells, Hsp70 regulation is far more complex and many families of positive and negative regulators have been characterized, such as Bag-1 that was first identified as an anti-apoptotic Bcl2-binding protein. Whereas eukaryotic DnaJ homologs have extensively been studied, GrpE homolog are found only in the mitochondria or chloroplasts. In fact, until the discovery of Bag-1 in mammalian cells, nucleotide exchange factors were presumed absent from the eukaryotic cytosol and organelles that comprise the secretory pathway, such as the endoplasmic reticulum (ER). However, members of a novel class of nucleotide exchange factors in the ER and in the cytoplasm have recently been identified that act on the ER lumen and cytoplasmic Hsp70 proteins, respectively. Although first uncovered in yeast and named Sls1p and Fes1p, we report here that the class of Hsp70 nucleotide exchange factors defined by Sls1p and Fes1p is conserved from yeast to humans. Kabani, M., S.S. Kelley, M.W. Morrow, D.L. Montgomery, R. Sivendran, M.D. Rose, L.M. Gierasch, and J.L. Brodsky (2003) Dependence of endoplasmic reticulum associated degradation (ERAD) on the peptide binding domain and concentration of BiP. Mol. Biol. Cell 14:3437-3448 ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD. Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al. J. Biol. Chem. 274, 3453-3460). The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate. Peptide-stimulated ATPase activity was also reduced, suggesting that the interdomain coupling in Kar2-1p is partially compromised. In contrast, Hsp40 cochaperone-activation of Kar2-1p's ATPase activity was unaffected. Consistent with UPR induction in kar2-1 yeast, an ERAD substrate aggregated in microsomes prepared from this strain but not from wild-type yeast. Overexpression of wild-type BiP increased substrate solubility in microsomes obtained from the mutant, but the ERAD defect was exacerbated, suggesting that simply retaining ERAD substrates in a soluble, retro-translocation-competent conformation is insufficient to support polypeptide transit to the cytoplasm.
Coughlan, C.M., and J.L. Brodsky (2003) Yeast as a model system to investigate protein conformational diseases. Methods Mol. Biol. 232:77-89 The yeast Saccharomyces cerevisiae has long served as a model euakaryote by virtue of the plethora of tools with which it can be manipulated genetically. Importantly, genetic dissections of yeast physiology have led serendipitously to significant advances in our understanding of several human diseases, most notably cancer, via the seminal studies performed by Hartwell and colleagues (1) on the regulation of the cell cycle. More recently, however, the genetic and biochemical tools available in yeast have been co-opted for the purpose of directly examining the molecular basis and to aid in the treatment of several human diseases. First, yeast has served as a "bio-factory" for the over-expression and purification of insulin and granulocyte-macrophage colony-stimulating factor (GM-CSF) and for the production of the antigen for the hepatitis B vaccine. Second, large-scale screening methods have been used to identify novel pharmacological targets produced in yeast or, via the two-hybrid screen, to obtain protein partners of medically-relevant gene-products. Finally, the heterologous expression of proteins in yeast that lead to human disease has been used to uncover physiological responses to these proteins; yeast also encode homologues of several disease-causing proteins. In particular, the expression of specific proteins in yeast that fail to adopt their proper conformations and/or whose conformation lead to a pathological state has helped us to understand how "conformational diseases" (2) arise, and how eukaryotic cells respond to aberrant polypeptides. Palmer, E.A., K.B. Kruse, S.W. Fewell, S.M. Buchanan, J.L. Brodsky, and A.A. McCracken (2003) Differential requirements of novel A1PiZ degradation deficient (ADD) genes in ER-associated protein degradation. J. Cell Sci. 116:2361-2373 In the eukaryotic cell, a protein quality control process termed endoplasmic reticulum-associated degradation (ERAD) rids the ER of aberrant proteins and unassembled components of protein complexes that fail to reach a transport-competent state. To identify novel genes required for ERAD, we devised a rapid immunoassay to screen yeast lacking uncharacterized open reading frames that were known targets of the unfolded protein response (UPR), a cellular response that is induced when aberrant proteins accumulate in the ER. Six genes required for the efficient degradation of the Z variant of the alpha1-proteinase inhibitor (A1PiZ), a known substrate for ERAD, were identified, and analysis of other ERAD substrates in the six A1PiZ-degradation-deficient (add) mutants suggested diverse requirements for the Add proteins in ERAD. Finally, we report on bioinformatic analyses of the new Add proteins, which will lead to testable models to elucidate their activities.
Sullivan, M.L., R.T. Youker, S.C. Watkins, and J.L. Brodsky (2003) Localization of the BiP Molecular Chaperone with Respect to Endoplasmic Reticulum Foci Containing the Cystic Fibrosis Transmembrane Conductance Regulator in Yeast. J. Histochem. Cytochem. 51:545-548 Almost all secreted proteins pass through the endoplasmic reticulum (ER), an organelle that is equipped to tolerate and/or degrade misfolded proteins. We report here that yeast expressing the cystic fibrosis transmembrane conductance regulator (CFTR) concentrate the protein at defined sites in the ER membrane that are not necessarily enriched for the ER molecular chaperone BiP. We propose that these sites are Russell bodies, an ER subcompartment in which misfolded proteins are stored and can be targeted for degradation.
Kabani, M., C. McLellan, D.A. Raynes, V. Guerriero, and J.L. Brodsky (2002) HspBP1, a homologue of the yeast Fes1 and Sls1 proteins, is an Hsc70 nucleotide exchange factor. FEBS Lett. 531:339-342 The yeast FES1 and SLS1 genes encode conserved nucleotide exchange factors that act on the cytoplasmic and endoplasmic reticulum luminal Hsp70s,Ssa1p and BiP, respectively. We report here that mammalian HspBP1 is homologous to Fes1p and that HspBP1 promotes nucleotide dissociation from both Ssa1p and mammalian Hsc70. In contrast, Fes1p inefficiently strips nucleotide from mammalian Hsc70, and unlike HspBP1 does not inhibit chaperone-mediated protein refolding in vitro. Together, our data indicate that HspBP1 is a member of this new class of nucleotide exchange factors that exhibit varying degrees of compartment and species specificity. Goeckeler, J.L., A. Stephens, P. Lee, A.J. Caplan, and J.L. Brodsky (2002) Overexpression of Yeast Hsp110 Homolog Sse1p Suppresses ydj1-151 Thermosensitivity and Restores Hsp90-dependent Activity. Mol. Biol. Cell 13:2760-2770 The Saccharomyces cerevisiae heat-shock protein Hsp40, Ydj1p, is involved in a variety of cellular activities that control polypeptide fate, such as folding and translocation across intracellular membranes. To elucidate the mechanism of Ydj1p action, and to identify functional partners, we screened for multicopy suppressors of the temperature-sensitive ydj1-151 mutant and identified a yeast Hsp110, SSE1. Overexpression of Sse1p also suppressed the folding defect of v-Src kinase in the ydj1-151 mutant and partially reversed the alpha-factor translocation defect. SSE1-dependent suppression of ydj1-151 thermosensitivity required the wild-type ATP-binding domain of Sse1p. However, the Sse1p mutants maintained heat-denatured firefly luciferase in a folding-competent state in vitro and restored human androgen receptor folding in sse1 mutant cells. Because the folding of both v-Src kinase and human androgen receptor in yeast requires the Hsp90 complex, these data suggest that Ydj1p and Sse1p are interacting cochaperones in the Hsp90 complex and facilitate Hsp90-dependent activity.
Kabani, M., J.M. Beckerich, and J.L. Brodsky (2002) Nucleotide exchange factor for the yeast hsp70 molecular chaperone Ssa1p. Mol. Cell. Biol. 22:4677-4689 We report on the identification of Fes1p (yBR101cp) as a cytosolic homologue of Sls1p, an endoplasmic reticulum (ER) protein previously shown to act as a nucleotide exchange factor for yeast BiP (M. Kabani, J.-M. Beckerich, and C. Gaillardin, Mol. Cell. Biol. 20:6923-6934, 2000). We found that Fes1p associates preferentially to the ADP-bound form of the cytosolic Hsp70 molecular chaperone Ssa1p and promotes nucleotide release. Fes1p activity was shown to be compartment and species specific since Sls1p and Escherichia coli GrpE could not substitute for Fes1p. Surprisingly, whereas Sls1p stimulated the ATPase activity of BiP in cooperation with luminal J proteins, Fes1p was shown to inhibit the Ydj1p-mediated activation of Ssa1p ATPase activity in steady-state and single-turnover assays. Disruption of FES1 in several wild-type backgrounds conferred a strong thermosensitive phenotype but partially rescued ydj1-151 thermosensitivity. The Dfes1 strain was proficient for posttranslational protein translocation, as well as for the ER-associated degradation of two substrates. However, the Dfes1 mutant showed increased cycloheximide sensitivity and a general translational defect, suggesting that Fes1p acts during protein translation, a process in which Ssa1p and Ydj1p are known to be involved. In support of this hypothesis, Fes1p was found to be associated with ribosomes.
Fewell, S.W., D.M. Markle, and J.L. Brodsky (2002) The carboxy terminus of simian virus 40 large T antigen is required to disrupt the yeast cell cycle. J. Virol. 76:4621-4624 Wild-type and J domain mutant simian virus 40 large T antigens alter the cell cycle and bud morphology of Saccharomyces cerevisiae. In contrast, yeast cells expressing mutant T antigen lacking the carboxy-terminal 150 aa exhibit normal morphology, indicating that this region of T antigen is required for cell cycle disruption.
Zhang, Y., S. Michaelis, and J.L. Brodsky (2002) CFTR expression and ER-associated degradation in yeast. Methods Mol. Med. 70:257-265 Fewell, S.W., J.M. Pipas, and J.L. Brodsky (2002) Mutagenesis of a functional chimeric gene in yeast identifies mutations in the simian virus 40 large T antigen J domain. Proc. Natl. Acad. Sci., USA 99:2002-2007 Simian virus 40 large T antigen contains an amino terminal J domain that catalyzes T antigen-mediated viral DNA replication and cellular transformation. To dissect the role of the J domain in these processes, we exploited the genetic tools available only in the yeast Saccharomyces cerevisiae to isolate 14 loss-of-function point mutations in the T antigen J domain. This screen also identified mutations that, when engineered into simian virus 40, resulted in T antigen mutants that were defective for the ability to support viral growth, to transform mammalian cells in culture, to dissociate the p130-E2F4 transcription factor complex, and to stimulate ATP hydrolysis by hsc70, a hallmark of J domain-containing molecular chaperones. These data correlate the chaperone activity of the T antigen J domain with its roles in viral infection and cellular transformation and support a model by which the viral J domain recruits the cytoplasmic hsc70 molecular chaperone in the host to rearrange multiprotein complexes implicated in replication and transformation. More generally, this study presents the use of a yeast screen to identify loss-of-function mutations in a mammalian virus and can serve as a widely applicable method to uncover domain functions of mammalian proteins for which there are yeast homologues with selectable mutant phenotypes.
Fewell, S.W., K.J. Travers, J.S. Weissman, and J.L. Brodsky (2001) The action of molecular chaperones in the early secretory pathway. Annu. Rev. Genet. 35:149-191 The endoplasmic reticulum (ER) serves as a way-station during the biogenesis of nearly all secreted proteins, and associated with or housed within the ER are factors required to catalyze their import into the ER and facilitate their folding. To ensure that only properly folded proteins are secreted and to temper the effects of cellular stress, the ER can target aberrant proteins for degradation and/or adapt to the accumulation of misfolded proteins. Molecular chaperones play critical roles in each of these phenomena. Morrow, M.W., and J.L. Brodsky (2001) Yeast ribosomes bind to highly purified reconstituted Sec61p complex and to mammalian p180. Traffic 2:705-716 To determine whether the yeast Sec61p translocation pore is a high-affinity ribosome receptor in the endoplasmic reticulum,we isolated the Sec61p complex using an improved protocol in which contaminants found previously to be associated with the complex are absent. The purified complex, which contains Sec61p with an amino terminal hexahistidine tag, was active since it rescued a sec61-3 post-translational translocation defect in a reconstituted system. Co-reconstitution of the Sec61p and Sec63p complexes into liposomes failed to support post-translational translocation, suggesting that Sec62p is required for this process. By Scatchard analysis, the purified Sec61p complex bound to yeast ribosomes when reconstituted into liposomes with a KD of 5.6 nM, a value similar to the KD obtained when ribosome binding to total microsomal protein was measured (2.7 nM). In addition, a mammalian protein, p180, which has been proposed to be a ribosome receptor, was expressed in yeast, and endoplasmic reticulum-derived microsomes isolated from this strain exhibited approximately 2.3-fold greater binding to yeast ribosomes. Despite this increase in ribosome binding, neither co- nor post-translational translocation was compromised in vivo. In sum, our data suggest that the Sec61p complex is a ribosome receptor in the yeast endoplasmic reticulum membrane. Brodsky, J.L. (2001) Chaperoning the maturation of the cystic fibrosis transmembrane conductance regulator. Am. J. Physiol. Lung C 281:L39-42
Nishikawa, S.I., S.W. Fewell, Y. Kato, J.L. Brodsky, and T. Endo (2001) Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation. J. Cell Biol. 153:1061-1070 Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-alpha- factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.
Gusarova, V., A.J. Caplan, J.L. Brodsky, and E.A. Fisher (2001) Apoprotein B degradation is promoted by the molecular chaperones hsp90 and hsp70. J. Biol. Chem. 276:24891-24900 Apoprotein B (apoB) is the major protein of hepatic-derived atherogenic lipoproteins. The net production of apoB can be regulated by pre-secretory degradation mediated by the ubiquitin-proteasome pathway and cytosolic hsp70. To further explore the mechanisms of apoB degradation, we have established a cell-free system in which degradation can be faithfully recapitulated. Human apoB48 synthesized in vitro was translocated into microsomes, glycosylated, and ubiquitinylated. Subsequent incubation with rat hepatic cytosol lead to proteasome-mediated degradation. To explore whether hsp90 is required for apoB degradation, geldanamycin (GA) was added during the degradation assay. GA increased the recovery of microsomal apoB48 approximately 3-fold and disrupted the interaction between hsp90 and apoB48. Confirming the hsp90 effect in the cell-free system, we also found that transfection of hsp90 cDNA into rat hepatoma cells enhanced apoB48 degradation. Finally, apoB48 degradation was reconstituted in vitro using cytosol prepared from wild type yeast. Notably, degradation was attenuated when apoB48-containing microsomes were incubated with cytosol supplemented with GA, or with cytosol prepared from yeast strains with mutations in the homologues of mammalian hsp70 and hsp90. Overall, our data suggest that hsp90 facilitates the interaction between ER-associated apoB and components of the proteasomal pathway, perhaps in cooperation with hsp70
Zhang, Y., G. Nijbroek, M.L. Sullivan, A.A. McCracken, S.C. Watkins, S. Michaelis, and J.L. Brodsky (2001) Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. Mol. Biol. Cell 12:1303-1314 Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD). To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast. We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover. Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.
Fewell, S.W., B.W. Day, and J.L. Brodsky (2001) Identification of an inhibitor of hsc70-mediated protein translocation and ATP hydrolysis. J. Biol. Chem. 276:910-914 Members of the hsc70 family of molecular chaperones are critical players in the folding and quality control of cellular proteins. Because several human diseases arise from defects in protein folding, the activity of hsc70 chaperones is a potential therapeutic target for these disorders. Using a known hsc70-modulator, 15-deoxyspergualin, as a seed, we identified a novel inhibitor of hsc70 activity. This compound, R/1, inhibits the endogenous and DnaJ-stimulated ATPase activity of hsc70 by 48% and 51%, respectively, and blocks the hsc70-mediated translocation of a preprotein into yeast ER-derived microsomal vesicles. Biochemical studies demonstrate that R/1 most likely exerts these effects by altering the oligomeric state of hsc70.
McCracken, A.A., and J.L. Brodsky (2000) A molecular portrait of the response to unfolded proteins. Genome Biol. 1:1013.1-1013.3 Using DNA microarrays, 381 genes have been found to be induced in response to unfolded proteins. The identity of the previously characterized 208 of these, and further experiments, have revealed new details on the scope of the unfolded protein response and its connection to the degradation of proteins at the endoplasmic reticulum.
McClellan, A.J., and J.L. Brodsky (2000) Mutation of the ATP-binding pocket of SSA1 indicates that a functional interaction between Ssa1p and Ydj1p is required for post-translational translocation into the yeast endoplasmic reticulum. Genetics 156:501-512 The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.
Sullivan, C.S., J.D. Tremblay, S.W. Fewell, J.A. Lewis, J.L. Brodsky, and J.M. Pipas (2000) Species-specific elements in the large T-antigen J domain are required for cellular transformation and DNA replication by simian virus 40. Mol. Cell. Biol. 20:5749-5757 The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo.
McCracken, A.A., E.D. Werner, M.J. Powell, and J.L. Brodsky (2000) Differential fates of invertase mutants in the yeast endoplasmic reticulum. Yeast 16:49-55 A number of proteins have been identified as substrates for endoplasmic reticulum (ER)-associated protein degradation (ERAD) and we describe here a new model substrate with which to study this process. Two secretion-defective forms of yeast invertase that accumulated in the ER to greatly different levels were examined: Suc2-538p levels were low, while Suc2-533p was present in high amounts. Because Suc2-533p and Suc2-538p mRNA levels were comparable, we examined whether Suc2-538p was targeted for degradation. Both mutant polypeptide levels were unaffected in a yeast strain deficient in vacuolar protease activity and, additionally, we showed that Suc2-538p was stabilized in ERAD-deficient strains, demonstrating that Suc2-538p was a substrate for ERAD. Coury, L.A., M.L. Zeidel, and J.L. Brodsky (1999) Use of yeast sec6 mutant for purification of vesicles containing recombinant membrane proteins. Methods Enzymol. 306:169-186 Brodsky, J.L., and A.A. McCracken (1999) ER protein quality control and proteasome-mediated protein degradation. Semin. Cell Dev. Biol. 10:507-513 A variety of mutant polypeptides that are associated with human disease are targeted for degradation by an endoplasmic reticulum (ER) quality control system. In addition, physiological signals and viral gene products can target the degradation of several ER resident proteins and secreted proteins passing through the ER. Although the mechanism of protein quality control and the site of degradation were obscure, recent data indicate that degradation requires the cytosolic proteasome. Biochemical and genetic analyses have indicated that both lumenal and integral membrane proteins are selected for proteolysis and exported to the cytosol by a process that in several cases requires ER associated molecular chaperones. Coury, L.A., M. Hiller, J.C. Mathai, E.W. Jones, M.L. Zeidel, and J.L. Brodsky (1999) Water transport across yeast vacuolar and plasma membrane-targeted secretory vesicles occurs by passive diffusion. J. Bacteriol. 181:4437-4440 To determine whether solute transport across yeast membranes was facilitated, we measured the water and solute permeations of vacuole-derived and late secretory vesicles in Saccharomyces cerevisiae; all permeations were consistent with passive diffusive flow. We also overexpressed Fps1p, the putative glycerol facilitator in S. cerevisiae, in secretory vesicles but observed no effect on water, glycerol, formamide, or urea permeations. However, spheroplasts prepared from the strain overexpressing Fps1p showed enhanced glycerol uptake, suggesting that Fps1p becomes active only upon insertion in the plasma membrane.
Trimbur, G.M., J.L. Goeckler, J.L. Brodsky, and C.J. Walsh (1999) Cloning, sequencing, and nucleolar targeting of the basal-body-binding nucleolar protein BN46/51. J. Cell Sci. 112:1159-1168 BN46/51 is an acidic protein found in the granular component of the nucleolus of the amebo-flagellate Naegleria gruberi. When Naegleria amebae differentiate into swimming flagellates, BN46/51 is found associated with the basal body complex at the base of the flagella. In order to determine the factors responsible for targeting BN46/51 to a specific subnucleolar region, cDNAs coding for both subunits were isolated and sequenced. Two clones, JG4.1 and JG12.1 representing the 46 kDa and 51 kDa subunits, respectively, were investigated in detail. JG12.1 encoded a polypeptide of 263 amino acids with a predicted size of 30.1 kDa that co-migrated with the 51 kDa subunit of BN46/51 when expressed in yeast. JG4.1 encoded a polypeptide of 249 amino acids with a predicted size of 28.8 kDa that co-migrated with the 46 kDa subunit of BN46/51. JG4.1 was identical to JG12.1 except for the addition of an aspartic acid between positions 94 and 95 of the JG12.1 sequence and the absence of 45 amino acids beginning at position 113. The predicted amino acid sequences were not closely related to any previously reported. However, the sequences did have 26-31% identity to a group of FKPBs (FK506 binding proteins) but lacked the peptidyl-prolyl cis-trans isomerase domain of the FKBPs. Both subunits contained two KKE and three KKX repeats found in other nucleolar proteins and in some microtubule binding proteins. Using 'Far Western' blots of nucleolar proteins, BN46/51 bound to polypeptides of 44 kDa and 74 kDa. The 44 kDa component was identified as the Naegleria homologue of fibrillarin. BN46/51 bound specifically to the nucleoli of fixed mammalian cells, cells which lack a BN46/51 related polypeptide. When the JG4.1 and JG12.1 cDNAs were expressed in yeast, each subunit was independently targeted to the yeast nucleolus. We conclude that BN46/51 represents a unique nucleolar protein that can form specific complexes with fibrillarin and other nucleolar proteins. We suggest that the association of BN46/51 with the MTOC of basal bodies may reflect its role in connecting the nucleolus with the MTOC activity for the mitotic spindle. This would provide a mechanism for nucleolar segregation during the closed mitosis of Naegleria amebae.
Brodsky, J.L. (1999) Selectivity of the molecular chaperone-specific immunosuppressive agent 15-deoxyspergualin: modulation of Hsc70 ATPase activity without compromising DnaJ chaperone interactions. Biochem. Pharmacol. 57:877-880 The immunosuppressive and cytostatic agent 15-deoxyspergualin (DSG) binds to the Hsc70 class of molecular chaperones with a K(D) = 4 microM. Because Hsc70s represent a diverse group of cellular effectors and because Hsc70 function frequently requires a DnaJ molecular chaperone, the specificity of DSG for different Hsc70s and the ability of DSG to block the productive interaction between an Hsc70 and its DnaJ partner were examined. DSG stimulated the ATPase activity of a mammalian and yeast cytosolic Hsc70 from 20 to 40%, but was unable to elicit such a response in a homologous Hsc70, Binding Protein (BiP), that resides in the lumen of the endoplasmic reticulum. In addition, the DnaJ-stimulated Hsc70 ATPase activity and the DnaJ-mediated release of an unfolded polypeptide from an Hsc70 were unaffected by DSG. These results indicate that Hsc70s exhibit substrate selectivity for DSG and that DSG does not compromise Hsc70 functions that require DnaJs. Thus, the immunosuppressive and cytostatic effects of DSG may be specific for a subset of cellular Hsc70s and confined to DnaJ-independent Hsc70-mediated activities. Brodsky, J.L., E.D. Werner, M.E. Dubas, J.L. Goeckeler, K.B. Kruse, and A.A. McCracken (1999) The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct. J. Biol. Chem. 274:3453-3460 Polypeptide import into the yeast endoplasmic reticulum (ER) requires two hsp70s, Ssa1p in the cytosol and BiP (Kar2p) in the ER lumen. After import, aberrant polypeptides may be exported to the cytoplasm for degradation by the proteasome, and defects in the ER chaperone calnexin (Cne1p) compromise their degradation. Both import and export require BiP and the Sec61p translocation complex, suggesting that import and export may be mechanistically related. We now show that the cne1 and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro- factor is defective in kar2 mutant microsomes. Pulse-chase analysis indicates that A1PiZ, another substrate for degradation, is stabilized in the kar2 strains at the restrictive temperature. Because two of the kar2 mutants examined are proficient for polypeptide import, the roles of BiP during ER protein export and import differ, indicating that these processes must be mechanistically distinct. To examine whether Ssa1p drives polypeptides from the ER and is also required for degradation, we assembled reactions using strains either containing a mutation in SSA1 or in which the level of Ssa1p could be regulated. We found that pro-alpha factor and A1PiZ were degraded normally, indicating further that import and export are distinct and that other cytosolic factors may pull polypeptides from the ER.
Srinivasan, A., A. McClellan, J. Vartikar, I. Marks, P. Cantolupo, Y. Li, P. Whyte, K. Rundell, J.L. Brodsky, and J.M. Pipas (1998) The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain. Mol. Cell. Biol. 17:4761-4773 Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four- to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes. McClellan, A.J., J.B. Endres, J.P. Vogel, D. Palazzi, M.D. Rose, and J.L. Brodsky (1998) Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation into the yeast ER. Mol. Biol. Cell 9:3533-3545 The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST-63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST-63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by less than 2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST-63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.
Brodsky, J.L, J.G. Lawrence, and A.J. Caplan (1998) Mutations in the cytosolic DnaJ-homologue, YDJ1, delay and compromise the efficient translation of heterologous proteins in yeast. Biochemistry 37:18045-18055 The Saccharomyces cerevisiae YDJ1 gene encodes a yeast homologue of DnaJ, an E. coli molecular chaperone and regulator of Hsp70 function. We examined the function of Ydj1p in vivo by analyzing the activity and production of firefly luciferase (FFLux) and green fluorescent protein (GFP) after inducible expression in yeast strains containing a wild type or a mutant YDJ1 gene. Although FFLux and GFP mRNA levels were similar in the wild type and mutant strains, the FFLux protein was translated about half as efficiently in the ydj1-151 mutant compared to the wild type strain; the lower FFLux level was not the result of increased FFLux turnover in the mutant. In contrast, GFP translation was significantly delayed in the ydj1-151 mutant compared to the wild type strain. Surprisingly, we observed that FFLux and GFP mRNA bound efficiently to polysomes in the ydj1-151 mutant. Analysis of polysome profiles also revealed a modest increase in the amount of 60S ribosomal subunits in the ydj1-151 strain, consistent with a translation defect in the mutant, although Ydj1 protein was not found associated with polysomes. To determine whether the inducible expression of an endogenous yeast protein was also less efficient in the ydj1-151 strain, we examined the inducible synthesis of the yeast TATA-binding protein (TBP) but observed no translation defect. Statistical analysis of the FFLux, GFP, and TBP genes suggests that Ydj1p facilitates the expression of proteins that are poorly translated because both FFLux and GFP contain an abundance of codons that are rarely used in yeast.
Brodsky, J.L., M. Bauerle, M. Horst, and A.J. McClellan (1998) Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum. FEBS Lett. 435:183-186 To determine whether mitochondrial hsp70 (mHsp70) could substitute for the endoplasmic retuculum (ER) Hsp70 (BiP) during protein translocation, we assembled ER-derived reconstituted proteoliposomes supplemented with either protein. We found that only BiP restored translocation in kar2 mutant vesicles and stimulated translocation approximately 3-fold in wild type proteoliposomes. mHsp70 associated poorly with both a BiP binding (DnaJ) domain of Sec63p and an ER precursor, and its ATPase activity was poorly enhanced upon incubation with the DnaJ domain. In contrast, BiP bound to the Sec63p-DnaJ domain in an ATP-dependent manner and its ATPase activity was stimulated significantly by this polypeptide. We conclude that mHsp70 is unable to support protein translocation into the ER because it fails to associate productively with Sec63p and a precursor. Beswick, V., J.L. Brodsky, F. Kepes, J.M. Neumann, A. Sanson, and M. Garrigos (1998) Expression, purification, and characterization of Sss1p, an essential component of the yeast Sec61p protein translocation complex. Protein Expression and Purification 13:423-432 Sss1p, a 8.9-kDa membrane protein, is an essential component of the protein translocation complex involved in the transport of secretory proteins a |
Download the