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1.  Dependence of Endoplasmic Reticulum-associated Degradation on the Peptide Binding Domain and Concentration of BiP 
Molecular Biology of the Cell  2003;14(8):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.
doi:10.1091/mbc.E02-12-0847
PMCID: PMC181579  PMID: 12925775
2.  Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells 
Molecular Biology of the Cell  2012;23(5):955-964.
The endoplasmic reticulum (ER) unfolded protein response (UPR) is correlated with changes in unfolded secretory levels. A novel fluorescence biosensor now reports changes in the unfolded protein burden. This reporter reveals a form of ER stress—inositol withdrawal—that stimulates the UPR without changes in unfolded protein levels.
Accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) stress pathway. To enhance secretory protein folding and promote adaptation to stress, the UPR upregulates ER chaperone levels, including BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous reporter of the ER folding environment. Changes in Kar2p-sfGFP fluorescence levels directly correlate with UPR activity and represent a robust reporter for high-throughput analysis. A novel second feature of this reporter is that photobleaching microscopy (fluorescence recovery after photobleaching) of Kar2p-sfGFP mobility reports on the levels of unfolded secretory proteins in individual cells, independent of UPR status. Kar2p-sfGFP mobility decreases upon treatment with tunicamycin or dithiothreitol, consistent with increased levels of unfolded proteins and the incorporation of Kar2p-sfGFP into slower-diffusing complexes. During adaptation, we observe a significant lag between down-regulation of the UPR and resolution of the unfolded protein burden. Finally, we find that Kar2p-sfGFP mobility significantly increases upon inositol withdrawal, which also activates the UPR, apparently independent of unfolded protein levels. Thus Kar2p mobility represents a powerful new tool capable of distinguishing between the different mechanisms leading to UPR activation in living cells.
doi:10.1091/mbc.E11-12-0995
PMCID: PMC3290652  PMID: 22219379
3.  Essential Roles of the Kar2/BiP Molecular Chaperone Downstream of the UPR Pathway in Cryptococcus neoformans 
PLoS ONE  2013;8(3):e58956.
The endoplasmic reticulum (ER) is a central hub where secreted or membrane-bound proteins are maturated and folded properly in eukaryotes. Maintenance of ER homeostasis is particularly important for human fungal pathogens, such as Cryptococcus neoformans, which encounter a plethora of host-mediated stresses during infection. Our previous study demonstrated that the unfolded protein response (UPR) pathway, composed of the evolutionarily conserved Ire1 kinase and the unique Hxl1 transcription factor, has pleiotropic roles in ER stress response, thermotolerance, antifungal drug resistance, and virulence in C. neoformans. Here, we functionally characterized an ER-resident molecular chaperone, Kar2/BiP, in C. neoformans. Conditional expression of KAR2 by the copper-regulated promoter revealed that Kar2 is essential for the viability of C. neoformans. Constitutive expression of KAR2 by the strong histone H3 promoter partially restores resistance to ER stress, cell wall stress, thermotolerance, and genotoxic stress in ire1Δ and hxl1Δ mutants, suggesting that Kar2 mainly functions downstream of the UPR pathway. Furthermore, Kar2 appears to control azole resistance in C. neoformans downstream of the UPR pathway without regulation of ERG11 or ERG3. Interestingly, we discovered that azole treatment is sensed as ER-stress and subsequently activates the Ire1-dependent Hxl1 splicing event and induction of KAR2 by the UPR pathway. In contrast, the constitutive expression of Kar2 is not sufficient to restore the Ire1-mediated regulation of capsule production in C. neoformans UPR mutants. In conclusion, this study demonstrates that Kar2 is not only essential for vegetative growth but also required for response and adaptation to the environmental stresses and antifungal drugs downstream of the UPR pathway in C. neoformans.
doi:10.1371/journal.pone.0058956
PMCID: PMC3590199  PMID: 23484059
4.  Endoplasmic reticulum stress regulation of the Kar2p/BiP chaperone alleviates proteotoxicity via dual degradation pathways 
Molecular Biology of the Cell  2012;23(4):630-641.
ETOC: A procedure to uncouple the highly conserved target gene Kar2/BiP from UPR regulation is used to show that the primary function of its induction is to mediate the disposal of misfolded proteins that would otherwise be toxic.
The unfolded protein response (UPR) monitors and maintains protein homeostasis in the endoplasmic reticulum (ER). In budding yeast, the UPR is a transcriptional regulatory pathway that is quiescent under normal conditions. Under conditions of acute ER stress, activation of UPR targets is essential for cell viability. How individual target genes contribute to stress tolerance is unclear. Uncovering these roles is hampered because most targets also play important functions in the absence of stress. To differentiate stress-specific roles from everyday functions, a single target gene was uncoupled from UPR control by eliminating its UPR-specific regulatory element. Through this approach, the UPR remains intact, aside from its inability to induce the designated target. Applying the strategy to the major ER chaperone Kar2p/BiP revealed the physiological function of increasing its cellular concentration. Despite hundreds of target genes under UPR control, we show that activation of KAR2 is indispensable to alleviate some forms of ER stress. Specifically, activation is essential to dispose misfolded proteins that are otherwise toxic. Surprisingly, induced BiP/Kar2p molecules are dedicated to alleviating stress. The inability to induce KAR2 under stress had no effect on its known housekeeping functions.
doi:10.1091/mbc.E11-04-0297
PMCID: PMC3279391  PMID: 22190740
5.  BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the Unfolded Protein Response 
PLoS Biology  2010;8(7):e1000415.
Computational modeling and experimentation in the unfolded protein response reveals a role for the ER-resident chaperone protein BiP in fine-tuning the system's response dynamics.
The unfolded protein response (UPR) is an intracellular signaling pathway that counteracts variable stresses that impair protein folding in the endoplasmic reticulum (ER). As such, the UPR is thought to be a homeostat that finely tunes ER protein folding capacity and ER abundance according to need. The mechanism by which the ER stress sensor Ire1 is activated by unfolded proteins and the role that the ER chaperone protein BiP plays in Ire1 regulation have remained unclear. Here we show that the UPR matches its output to the magnitude of the stress by regulating the duration of Ire1 signaling. BiP binding to Ire1 serves to desensitize Ire1 to low levels of stress and promotes its deactivation when favorable folding conditions are restored to the ER. We propose that, mechanistically, BiP achieves these functions by sequestering inactive Ire1 molecules, thereby providing a barrier to oligomerization and activation, and a stabilizing interaction that facilitates de-oligomerization and deactivation. Thus BiP binding to or release from Ire1 is not instrumental for switching the UPR on and off as previously posed. By contrast, BiP provides a buffer for inactive Ire1 molecules that ensures an appropriate response to restore protein folding homeostasis to the ER by modulating the sensitivity and dynamics of Ire1 activity.
Author Summary
Secreted and membrane-spanning proteins constitute one of every three proteins produced by a eukaryotic cell. Many of these proteins initially fold and assemble in the endoplasmic reticulum (ER). A variety of physiological and environmental conditions can increase the demands on the ER, overwhelming the ER protein folding machinery. To restore homeostasis in response to ER stress, cells activate an intracellular signaling pathway called the unfolded protein response (UPR) that adjusts the folding capacity of the ER according to need. Its failure impairs cell viability and has been implicated in numerous disease states. In this study, we quantitatively interrogate the homeostatic capacity of the UPR. We arrive at a mechanistic model for how the ER stress sensor Ire1 cooperates with its binding partner BiP, a highly redundant ER chaperone, to fine-tune UPR activity. Moving between a predictive computational model and experiments, we show that BiP release from Ire1 is not the switch that activates Ire1; rather, BiP modulates Ire1 activation and deactivation dynamics. BiP binding to Ire1 and its dissociation in an ER stress-dependent manner buffers the system against mild stresses. Furthermore, BiP binding accelerates Ire1 deactivation when stress is removed. We conclude that BiP binding to Ire1 serves to fine-tune the dynamic behavior of the UPR by modulating its sensitivity and shutoff kinetics. This function of the interaction between Ire1 and BiP may be a general paradigm for other systems in which oligomer formation and disassembly must be finely regulated.
doi:10.1371/journal.pbio.1000415
PMCID: PMC2897766  PMID: 20625545
6.  Deletion of Yeast p24 Genes Activates the Unfolded Protein Response 
Molecular Biology of the Cell  2001;12(4):957-969.
Yeast cells lacking a functional p24 complex accumulate a subset of secretory proteins in the endoplasmic reticulum (ER) and increase the extracellular secretion of HDEL-containing ER residents such as Kar2p/BiP. We report that a loss of p24 function causes activation of the unfolded protein response (UPR) and leads to increased KAR2 expression. The HDEL receptor (Erd2p) is functional and traffics in p24 deletion strains as in wild-type strains, however the capacity of the retrieval pathway is exceeded. Other conditions that activate the UPR and elevate KAR2 expression also lead to extracellular secretion of Kar2p. Using an in vitro assay that reconstitutes budding from the ER, we detect elevated levels of Kar2p in ER-derived vesicles from p24 deletion strains and from wild-type strains with an activated UPR. Silencing the UPR by IRE1 deletion diminished Kar2p secretion under these conditions. We suggest that activation of the UPR plays a major role in extracellular secretion of Kar2p.
PMCID: PMC32279  PMID: 11294899
7.  The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum. 
Molecular and Cellular Biology  1993;13(2):877-890.
The endoplasmic reticulum (ER) of eukaryotic cells contains an abundant 78,000-Da protein (BiP) that is involved in the translocation, folding, and assembly of secretory and transmembrane proteins. In the yeast Saccharomyces cerevisiae, as in mammalian cells, BiP mRNA is synthesized at a high basal rate and is further induced by the presence of increased amounts of unfolded proteins in the ER. However, unlike mammalian BiP, yeast BiP is also induced severalfold by heat shock, albeit in a transient fashion. To identify the regulatory sequences that respond to these stimuli in the yeast KAR2 gene that encodes BiP, we have cloned a 1.3-kb segment of DNA from the region upstream of the sequences coding for BiP and fused it to a reporter gene, the Escherichia coli beta-galactosidase gene. Analysis of a series of progressive 5' truncations as well as internal deletions of the upstream sequence showed that the information required for accurate transcriptional regulation of the KAR2 gene in S. cerevisiae is contained within a approximately 230-bp XhoI-DraI fragment (nucleotides -245 to -9) and that this fragment contains at least two cis-acting elements, one (heat shock element [HSE]) responding to heat shock and the other (unfolded protein response element [UPR]) responding to the presence of unfolded proteins in the ER. The HSE and UPR elements are functionally independent of each other but work additively for maximum induction of the yeast KAR2 gene. Lying between these two elements is a GC-rich region that is similar in sequence to the consensus element for binding of the mammalian transcription factor Sp1 and that is involved in the basal expression of the KAR2 gene. Finally, we provide evidence suggesting that yeast cells monitor the concentration of free BiP in the ER and adjust the level of transcription of the KAR2 gene accordingly; this effect is mediated via the UPR element in the KAR2 promoter.
Images
PMCID: PMC358971  PMID: 8423809
8.  Separate domains of KAR1 mediate distinct functions in mitosis and nuclear fusion 
The Journal of Cell Biology  1992;117(6):1277-1287.
The yeast KAR1 gene is essential for mitotic growth and important for nuclear fusion. Mutations in KAR1 prevent duplication of the spindle pole body (SPB), and affect functions associated with both the nuclear and cytoplasmic microtubules. The localization of hybrid Kar1-lacZ proteins, described elsewhere (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press), suggest that the protein is associated with the SPB. In this paper, we report a deletion analysis demonstrating that the mitotic and karyogamy functions of KAR1 are separate and independent, residing in discrete functional domains. One region, here shown to be essential for mitosis, coincided with a part of the protein that is both necessary and sufficient to target Karl-lacZ hybrid proteins to the SPB (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Complementation testing demonstrated that deletions in this interval did not affect nuclear fusion. A second region, required only for karyogamy, was necessary for the localization of a Kar3-lacZ hybrid protein to the SPB. These data suggest a model for the roles of Kar1p and Kar3p, a kinesin-like protein, in nuclear fusion. Finally, a third region of KAR1 was found to be important for both mitosis and karyogamy. This domain included the hydrophobic carboxy terminus and is sufficient to target a lacZ-Kar1 hybrid protein to the nuclear envelope (Vallen E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Altogether, the essential mitotic regions of KAR1 comprised 20% of the coding sequence. We propose a model for Kar1p in which the protein is composed of several protein-binding domains tethered to the nuclear envelope via its hydrophobic tail.
PMCID: PMC2289497  PMID: 1607389
9.  Kar4p, a karyogamy-specific component of the yeast pheromone response pathway. 
Molecular and Cellular Biology  1996;16(8):3990-4002.
Karyogamy is the process whereby two haploid nuclei fuse to form a diploid nucleus during mating in Saccharomyces cerevisiae. Here, we describe the characterization of the KAR4 gene, previously identified in a screen for new nuclear fusion-defective mutants. During mating, kar4 mutants were defective for the microtubule-dependent movement of nuclei, a phenotype identical to that of mutations in KAR3 and CIK1. Consistent with its mutant phenotype, we found that the kar4 mutation resulted in failure to induce KAR3 and CIK1 mRNA during mating. Expression of KAR3 and CIK1 under independent regulatory control suppressed the kar4 defect, indicating that KAR4 is required primarily for the induction of KAR3 and CIK1. KAR4 was also required for meiosis, during which it may regulate KAR3; however, mitotic expression of KAR3 and CIK1 during S/G2 phase was independent of KAR4. A 30-bp region upstream of KAR3 conferred both KAR4- and STE12-dependent induction by mating pheromone. This region contained one moderate and two weak matches to the consensus pheromone response element to which the Ste12p transcriptional activator binds and five repeats of the sequence CAAA(A). Overproduction of Ste12p suppressed the kar4 defect in KAR3 induction and nuclear fusion. In contrast, Ste12p-independent expression of Kar4p did not alleviate the requirement for Ste12p during KAR3 induction. We propose that Kar4p assists Ste12p in the pheromone-dependent expression of KAR3 and CIK1. KAR4 defines a novel level of regulation for the pheromone response pathway, acting at a subset of Stel2p-inducible genes required for karyogamy.
PMCID: PMC231395  PMID: 8754797
10.  Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum 
Molecular and Cellular Biology  2000;20(18):6923-6934.
We previously characterized the SLS1 gene in the yeast Yarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903–30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between ΔScsls1 and translocation-deficient kar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding of ScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.
PMCID: PMC88768  PMID: 10958688
11.  Genetic Interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during Nuclear Fusion in Saccharomyces cerevisiae 
Molecular Biology of the Cell  1999;10(3):609-626.
During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Δ, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Δ and sec72Δ, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein–protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.
PMCID: PMC25191  PMID: 10069807
12.  Gcn4p and Novel Upstream Activating Sequences Regulate Targets of the Unfolded Protein Response 
PLoS Biology  2004;2(8):e246.
Eukaryotic cells respond to accumulation of unfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), a signal transduction pathway that communicates between the ER and the nucleus. In yeast, a large set of UPR target genes has been experimentally determined, but the previously characterized unfolded protein response element (UPRE), an upstream activating sequence (UAS) found in the promoter of the UPR target gene KAR2, cannot account for the transcriptional regulation of most genes in this set. To address this puzzle, we analyzed the promoters of UPR target genes computationally, identifying as candidate UASs short sequences that are statistically overrepresented. We tested the most promising of these candidate UASs for biological activity, and identified two novel UPREs, which are necessary and sufficient for UPR activation of promoters. A genetic screen for activators of the novel motifs revealed that the transcription factor Gcn4p plays an essential and previously unrecognized role in the UPR: Gcn4p and its activator Gcn2p are required for induction of a majority of UPR target genes during ER stress. Both Hac1p and Gcn4p bind target gene promoters to stimulate transcriptional induction. Regulation of Gcn4p levels in response to changing physiological conditions may function as an additional means to modulate the UPR. The discovery of a role for Gcn4p in the yeast UPR reveals an additional level of complexity and demonstrates a surprising conservation of the signaling circuit between yeast and metazoan cells.
The yeast unfolded protein response activates a large set of target genes, but a characterized element found in the promoter of one target, KAR2, cannot account for most targets. Using computational and experimental methods, the authors identify additional elements, as well a role for GCN4p in the response
doi:10.1371/journal.pbio.0020246
PMCID: PMC509306  PMID: 15314660
13.  SSI1 encodes a novel Hsp70 of the Saccharomyces cerevisiae endoplasmic reticulum. 
Molecular and Cellular Biology  1996;16(11):6444-6456.
The endoplasmic reticulum (ER) of the budding yeast Saccharomyces cerevisiae contains a well-characterized, essential member of the Hsp70 family of molecular chaperones, Kar2p. Kar2p has been shown to be involved in the translocation of proteins into the ER as well as the proper folding of proteins in that compartment. We report the characterization of a novel Hsp70 of the ER, Ssi1p. Ssi1p, which shares 24% of the amino acids of Kar2p, is not essential for growth under normal conditions. However, deletion of SSI1 results in cold sensitivity as well as enhanced resistance to manganese. The localization of Ssi1p to the ER, suggested by the presence of a conserved S. cerevisiae ER retention signal at its C terminus, was confirmed by subcellular fractionation, protease protection assays, and immunofluorescence. The SSI1 promoter contains an element with similarity to the unfolded protein response element of KAR2. Like KAR2, SSI1 is induced both in the presence of tunicamycin and in a kar2-159 mutant strain, conditions which lead to an accumulation of unfolded proteins in the ER. Unlike KAR2, however, SSI1 is not induced by heat shock. Deletion of SSI1 shows a complex pattern of genetic interactions with various conditional alleles of KAR2, ranging from synthetic lethality to synthetic rescue. Interestingly, SSI1 deletion strains show a partial block in translocation of multiple proteins into the ER, suggesting that Ssi1p plays a direct role in the translocation process.
PMCID: PMC231646  PMID: 8887673
14.  The Saccharomyces cerevisiae YFR041C/ERJ5 gene encoding a type I membrane protein with a J domain is required to preserve the folding capacity of the endoplasmic reticulum 
Biochimica et biophysica acta  2006;1773(2):232-242.
YFR041C/ERJ5 was identified in Saccharomyces cerevisiae as a gene regulated by the unfolded protein response pathway (UPR). The open reading frame of the gene has a J domain characteristic of the DnaJ chaperone family of proteins that regulate the activity of Hsp70 chaperones. We determined the expression and topology of Erj5p, a type I membrane protein with a J domain in the lumen of the endoplasmic reticulum (ER) that colocalizes with Kar2p, the major Hsp70 in the yeast ER. We identified synthetic interactions of Δerj5 with mutations in genes involved in protein folding in the ER (kar2-159, Δscj1Δjem1) and in the induction of the unfolded protein response (Δire1). Loss of Erj5p in yeast cells with impaired ER protein folding capacity increased sensitivity to agents that cause ER stress. We identified the ERJ5 mRNA and confirmed that agents that promote accumulation of misfolded proteins in the ER regulate its abundance. We found that loss of the non-essential ERJ5 gene leads to a constitutively induced UPR, indicating that ERJ5 is required for maintenance of an optimal folding environment in the yeast ER.
doi:10.1016/j.bbamcr.2006.10.011
PMCID: PMC1847348  PMID: 17157937
DnaJ; chaperones; protein folding; Saccharomyces cerevisiae; endoplasmic reticulum; unfolded protein response
15.  Regulation and Recovery of Functions of Saccharomyces cerevisiae Chaperone BiP/Kar2p after Thermal Insult 
Eukaryotic Cell  2005;4(12):2008-2016.
We described earlier a novel mode of regulation of Hsp104, a cytosolic chaperone directly involved in the refolding of heat-denatured proteins, and designated it delayed upregulation, or DUR. When Saccharomyces cerevisiae cells grown at the physiological temperature of 24°C, preconditioned at 37°C, and treated briefly at 50°C were shifted back to 24°C, Hsp104 expression was strongly induced after 2.5 h of recovery and returned back to normal after 5 h. Here we show that the endoplasmic reticulum (ER) chaperones BiP/Kar2p and Lhs1p and the mitochondrial chaperone Hsp78 were also upregulated at the physiological temperature during recovery from thermal insult. The heat shock element (HSE) in the KAR2 promoter was found to be sufficient to drive DUR. The unfolded protein element could also evoke DUR, albeit weakly, in the absence of a functional HSE. BiP/Kar2p functions in ER translocation and assists protein folding. Here we found that the synthesis of new BiP/Kar2p molecules was negligible for more than an hour after the shift of the cells from 50°C to 24°C. Concomitantly, ER translocation was blocked, suggesting that preexisting BiP/Kar2p molecules or other necessary proteins were not functioning. Translocation resumed concomitantly with enhanced synthesis of BiP/Kar2p after 3 h of recovery, after which ER exit and protein secretion also resumed. For a unicellular organism like S. cerevisiae, conformational repair of denatured proteins is the sole survival strategy. Chaperones that refold proteins in the cytosol, ER, and mitochondria of S. cerevisiae appear to be subject to DUR to ensure survival after thermal insults.
doi:10.1128/EC.4.12.2008-2016.2005
PMCID: PMC1317487  PMID: 16339719
16.  BiP Clustering Facilitates Protein Folding in the Endoplasmic Reticulum 
PLoS Computational Biology  2014;10(7):e1003675.
The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER): translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as ‘clusters’). Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.
Author Summary
The misfolding of proteins carries important implications for diseases such as Alzheimer's, Parkinson's, cancer, and diabetes. Once misfolded, proteins tend to associate into aggregates that pose a toxic threat to the cell. Chaperones are proteins that rescue the cell from an accumulation of these maladjusted proteins through dissociation of toxic oligomers and proper (re)folding. The endoplasmic reticulum (ER) is an organelle that serves as the staging ground for the chaperone activities of protein transport, folding, and maturation in the early secretory pathway. We have developed a computational model to investigate potential mechanisms that enable multiple ER-resident molecules working in concert to effectively fold peptides and transport nascent proteins across the ER membrane. Although previous models focused on chaperone interactions with peptides, we have explored the influence of cooperativity among chaperone molecules to assist in protein folding and maturation. We found that chaperone cooperation led to a higher yield of folded molecules compared to when chaperones bound to peptides in a 1∶1 stoichiometry. We have concluded that the clustering or multiple binding of chaperones may facilitate protein folding in vivo.
doi:10.1371/journal.pcbi.1003675
PMCID: PMC4081015  PMID: 24991821
17.  Interaction between BiP and Sec63p is required for the completion of protein translocation into the ER of Saccharomyces cerevisiae 
The Journal of Cell Biology  1995;131(5):1163-1171.
To clarify the roles of Kar2p (BiP) and Sec63p in translocation across the ER membrane in Saccharomyces cerevisiae, we have utilized mutant alleles of the essential genes that encode these proteins: kar2-203 and sec63-1. Sanders et al. (Sanders, S. L., K. M. Whitfield, J. P. Vogel, M. D. Rose, and R. W. Schekman. 1992. Cell. 69:353-365) showed that the translocation defect of the kar2-203 mutant lies in the inability of the precursor protein to complete its transit across the membrane, suggesting that the lumenal hsp70 homologue Kar2p (BiP) binds the transiting polypeptide in order to facilitate its passage through the pore. We now show that mutation of a conserved residue (A181-->T) (Nelson, M. K., T. Kurihara, and P. Silver. 1993. Genetics. 134:159- 173) in the lumenal DnaJ box of Sec63p (sec63-1) results in an in vitro phenotype that mimics the precursor stalling defect of kar2-203. We demonstrate by several criteria that this phenotype results specifically from a defect in the lumenal interaction between Sec63p and BiP: Neither a sec62-1 mutant nor a mutation in the cytosolically exposed domain of Sec63p causes precursor stalling, and interaction of the sec63-1 mutant with the membranebound components of the translocation apparatus is unimpaired. Additionally, dominant KAR2 suppressors of sec63-1 partially relieve the stalling defect. Thus, proper interaction between BiP and Sec63p is necessary to allow the precursor polypeptide to complete its transit across the membrane.
PMCID: PMC2120636  PMID: 8522580
18.  Nucleotide Binding by Lhs1p Is Essential for Its Nucleotide Exchange Activity and for Function in Vivo* 
The Journal of Biological Chemistry  2009;284(46):31564-31571.
Protein translocation and folding in the endoplasmic reticulum of Saccharomyces cerevisiae involves two distinct Hsp70 chaperones, Lhs1p and Kar2p. Both proteins have the characteristic domain structure of the Hsp70 family consisting of a conserved N-terminal nucleotide binding domain and a C-terminal substrate binding domain. Kar2p is a canonical Hsp70 whose substrate binding activity is regulated by cochaperones that promote either ATP hydrolysis or nucleotide exchange. Lhs1p is a member of the Grp170/Lhs1p subfamily of Hsp70s and was previously shown to function as a nucleotide exchange factor (NEF) for Kar2p. Here we show that in addition to this NEF activity, Lhs1p can function as a holdase that prevents protein aggregation in vitro. Analysis of the nucleotide requirement of these functions demonstrates that nucleotide binding to Lhs1p stimulates the interaction with Kar2p and is essential for NEF activity. In contrast, Lhs1p holdase activity is nucleotide-independent and unaffected by mutations that interfere with ATP binding and NEF activity. In vivo, these mutants show severe protein translocation defects and are unable to support growth despite the presence of a second Kar2p-specific NEF, Sil1p. Thus, Lhs1p-dependent nucleotide exchange activity is vital for ER protein biogenesis in vivo.
doi:10.1074/jbc.M109.055160
PMCID: PMC2797226  PMID: 19759005
19.  Stable Binding of ATF6 to BiP in the Endoplasmic Reticulum Stress Response 
Molecular and Cellular Biology  2005;25(3):921-932.
Endoplasmic reticulum (ER) stress-induced activation of ATF6, an ER membrane-bound transcription factor, requires a dissociation step from its inhibitory regulator, BiP. It has been generally postulated that dissociation of the BiP-ATF6 complex is a result of the competitive binding of misfolded proteins generated during ER stress. Here we present evidence against this model and for an active regulatory mechanism for dissociation of the complex. Contradictory to the competition model that is based on dynamic binding of BiP to ATF6, our data reveal relatively stable binding. First, the complex was easily isolated, in contrast to many chaperone complexes that require chemical cross-linking. Second, ATF6 bound at similar levels to wild-type BiP and a BiP mutant form that binds substrates stably because of a defect in its ATPase activity. Third, ER stress specifically induced the dissociation of BiP from ER stress transducers while the competition model would predict dissociation from any specific substrate. Fourth, the ATF6-BiP complex was resistant to ATP-induced dissociation in vitro when isolated without detergents, suggesting that cofactors stabilize the complex. In favor of an active dissociation model, one specific region within the ATF6 lumenal domain was identified as a specific ER stress-responsive sequence required for ER stress-triggered BiP release. Together, our results do not support a model in which competitive binding of misfolded proteins causes dissociation of the BiP-ATF6 complex in stressed cells. We propose that stable BiP binding is essential for ATF6 regulation and that ER stress dissociates BiP from ATF6 by actively restarting the BiP ATPase cycle.
doi:10.1128/MCB.25.3.921-932.2005
PMCID: PMC543992  PMID: 15657421
20.  A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome 
The Journal of Cell Biology  1993;123(6):1355-1363.
Reconstituted proteoliposomes derived from solubilized yeast microsomes are able to translocate a secreted yeast mating pheromone precursor (Brodsky, J. L., S. Hamamoto, D. Feldheim, and R. Schekman. 1993. J. Cell Biol. 120:95-107). Reconstituted proteoliposomes prepared from strains with mutations in the SEC63 or KAR2 genes are defective for translocation; the kar2 defect can be overcome by the addition of purified BiP (encoded by the KAR2 gene). We now show that addition of BiP to wild-type reconstituted vesicles increases their translocation efficiency three-fold. To identify other ER components that are required for translocation, we purified a microsomal membrane protein complex that contains Sec63p. We found that the complex also includes BiP, Sec66p (gp31.5), and Sec67p (p23). The Sec63p complex restores translocation activity to reconstituted vesicles that are prepared from a sec63-1 strain, or from cells in which the SEC66 or SEC67 genes are disrupted. BiP dissociates from the complex when the purification is performed in the presence of ATP gamma S or when the starting membranes are from yeast containing the sec63-1 mutation. We conclude that the purified Sec63p complex is active and required for protein translocation, and that the association of BiP with the complex may be regulated in vivo.
PMCID: PMC2290880  PMID: 8253836
21.  Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction 
Background
The analysis of transcriptional levels of the genes involved in protein synthesis and secretion is a key factor to understand the host organism's responses to recombinant protein production, as well as their interaction with the cultivation conditions. Novel techniques such as the sandwich hybridization allow monitoring quantitatively the dynamic changes of specific RNAs. In this study, the transcriptional levels of some genes related to the unfolded protein response (UPR) and central metabolism of Pichia pastoris were analysed during batch and fed-batch cultivations using an X-33-derived strain expressing a Rhizopus oryzae lipase under control of the formaldehyde dehydrogenase promoter (FLD1), namely the alcohol oxidase gene AOX1, the formaldehyde dehydrogenase FLD1, the protein disulfide isomerase PDI, the KAR2 gene coding for the BiP chaperone, the 26S rRNA and the R. oryzae lipase gene ROL.
Results
The transcriptional levels of the selected set of genes were first analysed in P. pastoris cells growing in shake flask cultures containing different carbon and nitrogen sources combinations, glycerol + ammonium, methanol + methylamine and sorbitol + methylamine. The transcriptional levels of the AOX1 and FLD1 genes were coherent with the known regulatory mechanism of C1 substrates in P. pastoris, whereas ROL induction lead to the up-regulation of KAR2 and PDI transcriptional levels, thus suggesting that ROL overexpression triggers the UPR. This was further confirmed in fed-batch cultivations performed at different growth rates. Transcriptional levels of the analysed set of genes were generally higher at higher growth rates. Nevertheless, when ROL was overexpressed in a strain having the UPR constitutively activated, significantly lower relative induction levels of these marker genes were detected.
Conclusion
The bead-based sandwich hybridization assay has shown its potential as a reliable instrument for quantification of specific mRNA species in P. pastoris cells grown in fed-batch cultures. As a proof-of-principle, the influence of the carbon and nitrogen sources, the specific growth rate, as well as the ROL overexpression on the transcriptional levels of a reduced set of bioprocess-relevant genes has been quantitatively studied, revealing that ROL overexpression and secretion seems to trigger the UPR in P. pastoris, resulting in a physiological bottleneck for the production process.
doi:10.1186/1475-2859-6-21
PMCID: PMC1950523  PMID: 17634115
22.  BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast 
The Journal of Cell Biology  1995;130(1):41-49.
Although transiently associated with numerous newly synthesized proteins, BiP has not been shown to be an essential component directly linked to the folding and oligomerization of newly synthesized proteins in the endoplasmic reticulum. To determine whether it is needed as a molecular chaperone, we analyzed the maturation of an endogenous yeast glycoprotein, carboxypeptidase Y (CPY) in several yeast strains with temperature-sensitive mutations in BiP. These kar2 mutant strains have previously been found to be defective in translocation at the nonpermissive temperature (Vogel, J. P., L. M. Misra, and M. D. Rose, 1990. J. Cell Biol, 110:1885-1895). To circumvent the translocation block, we used DTT at permissive temperature to delay folding and intracellular transport. We then followed the maturation of the ER- retained CPY after shifting to the nonpermissive temperature and dilution of the DTT. Without the functional chaperone, CPY aggregated, failed to be oxidized, and remained in the ER. In contrast to wild-type cells, in which BiP binding was transient with no more than 10-15% of labeled CPY associated at any time, 30-100% of the CPY remained associated with BiP in the mutant strains. In a heterozygous diploid strain, CPY matured and exited the ER normally. Taken together, the results provide clear evidence that BiP plays a critical role as a molecular chaperone in CPY folding.
PMCID: PMC2120506  PMID: 7790376
23.  BTB-Kelch Proteins and Ubiquitination of Kainate Receptors 
Kainate receptors (KAR) form a class of glutamate receptors that have been implicated in epilepsy, stroke, Alzheimer’s and neuropathic pain.1 KAR subtypes are known to be segregated to specific locations within neurons and play significant roles in synaptic transmission and plasticity.2 Increasing evidence suggests a the role for ubiqutination in regulating the number of synaptic neurotransmitter receptors.3–5 The ubiquitin pathway consists of activation (E1), conjugation (E2) and ligation (E3). Cullins form the largest family of E3 ligase complexes. We have recently shown that the BTB/Kelch domain proteins, actinfilin and mayven, bind both Cul3 and specific KAR subtypes (GluR6 and GluR5-2b) to target these KARs for ubiquitination and degradation.5 In this chapter we will review how these interactions occur, what they mean for the stability of KARs and their associated proteins and how, in turn, they may affect synaptic functions in the central nervous system.
doi:10.1007/978-1-4419-9557-5_10
PMCID: PMC3929045  PMID: 21713671
24.  Kar9p Is a Novel Cortical Protein Required for Cytoplasmic Microtubule Orientation in Yeast  
The Journal of Cell Biology  1998;140(2):377-390.
kar9 was originally identified as a bilateral karyogamy mutant, in which the two zygotic nuclei remained widely separated and the cytoplasmic microtubules were misoriented (Kurihara, L.J., C.T. Beh, M. Latterich, R. Schekman, and M.D. Rose. 1994. J. Cell Biol. 126:911–923.). We now report a general defect in nuclear migration and microtubule orientation in kar9 mutants. KAR9 encodes a novel 74-kD protein that is not essential for life. The kar9 mitotic defect was similar to mutations in dhc1/dyn1 (dynein heavy chain gene), jnm1, and act5. kar9Δ dhc1Δ, kar9Δ jnm1Δ, and kar9Δ act5Δ double mutants were synthetically lethal, suggesting that these genes function in partially redundant pathways to carry out nuclear migration. A functional GFP-Kar9p fusion protein localized to a single dot at the tip of the shmoo projection. In mitotic cells, GFP-Kar9p localized to a cortical dot with both mother–daughter asymmetry and cell cycle dependence. In small-budded cells through anaphase, GFP-Kar9p was found at the tip of the growing bud. In telophase and G1 unbudded cells, no localization was observed. By indirect immunofluorescence, cytoplasmic microtubules intersected the GFP-Kar9p dot. Nocodazole experiments demonstrated that Kar9p's cortical localization was microtubule independent. We propose that Kar9p is a component of a cortical adaptor complex that orients cytoplasmic microtubules.
PMCID: PMC2132572  PMID: 9442113
25.  The unfolded protein response (UPR) pathway in Cryptococcus 
Virulence  2013;5(2):341-350.
Unique and evolutionarily conserved signaling pathways allow an organism to sense, respond to, and adapt to internal and external environmental cues at its biological niche. In eukaryotic cells, the unfolded protein response (UPR) pathway regulates endoplasmic reticulum (ER) homeostasis upon exposure to environmental changes causing ER stress. The UPR pathway of Cryptococcus neoformans, an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals, consists of the evolutionarily conserved Ire1 kinase, a unique bZIP transcription factor, Hxl1, and the ER-resident molecular chaperone Kar2/BiP. Although the Cryptococcus UPR pathway regulates ER stress, antifungal drug resistance, and virulence in an Ire1/Hxl1-dependent manner, Ire1 has Hxl1-independent roles in capsule biosynthesis and thermotolerance. In this review, we highlight the conserved and unique features of the Cryptococcus UPR pathway compared with other fungal UPR systems and its importance in the pathogenesis of cryptococcosis and discuss future challenges in this field.
doi:10.4161/viru.26774
PMCID: PMC3956512  PMID: 24504058
Cryptococcus neoformans; ER stress; unfolded protein response; Ire1; Hxl1

Results 1-25 (1313042)