Deletion of yeast p24 genes activates the unfolded protein response

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.     

Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells

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.     

Unfolded protein response-induced BiP/Kar2p production protects cell growth against accumulation of misfolded protein aggregates in the yeast endoplasmic reticulum

Overproduction of delta(pro), a mutated secretory proteinase derived from a filamentous fungus Rhizopus niveus, results in formation of gross aggregates (delta(pro) aggregates) in the yeast endoplasmic reticulum (ER) lumen, activation of the unfolded protein response (UPR) and ER membrane proliferation. To investigate the roles of the UPR against the delta(pro) aggregates, we constructed an IRE1-deleted ((delta)ire1) strain. In contrast to wild-type cells, (delta)ire1 cells ceased to grow several hours after the overproduction of (delta)pro. Two lines of evidence argued against the possibility that the growth defect was due to the inability to make extra ER membrane which accommodates the (delta)pro aggregates. First, by electron microscopy, ER membrane proliferation was observed in (delta)ire1 cells overproducing (delta)pro. Second, disruption of the OPI1 gene in the (delta)ire1 mutant, which is considered to derepress the activities of phospholipid-synthesizing enzymes, did not restore the growth upon the overproduction of (delta)pro. Instead, the growth was restored when an extra copy of the KAR2 gene, which encodes yeast BiP, was introduced, indicating that an increase in the amount of BiP is essential for cell growth when the (delta)pro aggregates accumulate in the ER. Since BiP is included in the insoluble (delta)pro aggregates, it is likely that the amount of free BiP in the ER lumen is insufficient without the UPR to fully exert its functions. Consistently, overproduction of (delta)pro impaired protein translocation and folding in (delta)ire1 cells but not in wild-type cells. The tunicamycin sensitivity of (delta)ire1 cells was also suppressed by extra expression of KAR2, suggesting that BiP plays a principal role in protecting cell growth against misfolded proteins accumulated in the ER.     

Saccharomyces cerevisiae Rot1p is an ER-localized membrane protein that may function with BiP/Kar2p in protein folding

The 70-kDa heat shock protein (Hsp70) family of molecular chaperones cooperates with cofactors to promote protein folding, assembly of protein complexes and translocation of proteins across membranes. Although many cofactors of cytosolic Hsp70s have been identified, knowledge about cofactors of BiP/Kar2p, an endoplasmic reticulum (ER)-resident Hsp70, is still poor. Here we propose the Saccharomyces cerevisiae protein Rot1p as a possible cofactor of BiP/Kar2p involved in protein folding. Rot1p was found to be an essential, ER-localized membrane protein facing the lumen. ROT1 genetically interacted with several ER chaperone genes including KAR2, and the rot1-2 mutation triggered the unfolded protein response. Rot1p associated with Kar2p, especially under conditions of ER stress, and maturation of a model protein, a reduced form of carboxypeptidaseY, was impaired in a kar2-1 rot1-2 double mutant. These findings suggest that Rot1p participates in protein folding with Kar2p. Morphological analysis of rot1-2 cells revealed cell wall defects and accumulation of autophagic bodies in the vacuole. This implies that the protein folding machinery in which Rot1p is involved chaperones proteins acting in various physiological processes including cell wall synthesis and lysis of autophagic bodies.     

Interaction between BiP and Sec63p is required for the completion of protein translocation into the ER of Saccharomyces cerevisiae

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.     

Herp is dually regulated by both the endoplasmic reticulum stress-specific branch of the unfolded protein response and a branch that is shared with other cellular stress pathways

The mammalian unfolded protein response (UPR) includes two major branches: one(s) specific to ER stress (Ire1/XBP-1 and ATF6-dependent), and one(s) shared by other cellular stresses (PERK/eIF-2alpha phosphorylation-dependent). Here, we demonstrate that the ER-localized protein Herp represents a second target, in addition to CHOP, that is dually regulated by both the shared and the ER stress-specific branches during UPR activation. For the first time, we are able to assess the contribution of each branch of the UPR in the induction of these targets. We demonstrate that activation of the shared branch of the UPR alone was sufficient to induce Herp and CHOP. ATF4 was not required during ER stress when both branches were used but did contribute significantly to their induction. Conversely, stresses that activated only the shared branch of the UPR were completely dependent on ATF4 for CHOP and Herp induction. Thus, the shared and the ER stress-specific branches of the UPR diverge to regulate two groups of targets, one that is ATF6 and Ire1/XBP-1-dependent, which includes BiP and XBP-1, and another that is eIF-2alpha kinase-dependent, which includes ATF4 and GADD34. The two branches also converge to maximally up-regulate targets like Herp and CHOP. Finally, our studies reveal that a PERK-dependent target other than ATF4 is contributing to the cross-talk between the two branches of the UPR that has previously been demonstrated.     

Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish

The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease.KEY WORDS: Unfolded protein response, Steatosis, Zebrafish, Tunicamycin, Thapsigargin, ER stress, Fatty liver disease     

BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the Unfolded Protein Response

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.     

Genetic interactions between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum.

KAR2 encodes the yeast homologue of mammalian BiP, the endoplasmic reticulum (ER) resident member of the HSP70 family. Kar2p has been shown to be required for the translocation of proteins across the ER membrane as well as nuclear fusion. Sec63, an ER integral membrane protein that shares homology with the Escherichia coli DnaJ protein, is also required for translocation. In this paper we describe several specific genetic interactions between these two proteins, Kar2p and Sec63p. First, temperature-sensitive mutations in KAR2 and SEC63 form synthetic lethal combinations. Second, dominant mutations in KAR2 are allele-specific suppressors for the temperature-sensitive growth and translocation defect of sec63-1. Third, the sec63-1, unlike other translocation defective mutations, results in the induction of KAR2 mRNA levels. Taken together, these genetic interactions suggest that Kar2p and Sec63p interact in vivo in a manner similar to that of the E. coli HSP70, DnaK, and DnaJ. We propose that the interaction between these two proteins is critical to their function in protein translocation.     

Regulation and recovery of functions of Saccharomyces cerevisiae chaperone BiP/Kar2p after thermal insult

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 degrees C, preconditioned at 37 degrees C, and treated briefly at 50 degrees C were shifted back to 24 degrees 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 degrees C to 24 degrees 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.     

未折叠蛋白响应的激活机制

未折叠蛋白在内质网（endoplasmic reticulum,ER）腔中累积造成ER应激，此时细胞启动未折叠蛋白响应（unfolded protein response,UPR）以恢复蛋白质稳态。目前已知有三种UPR感受器，即IRE1、PERK和ATF6，它们均为ER跨膜蛋白，在ER应激时被激活并启动下游UPR信号通路。虽然UPR感受器最早是在研究细胞如何应对ER应激时发现的，但它们如何感知ER应激至今未得到完满的回答。随着研究的深入，人们发现UPR的功能不仅限于维持蛋白质稳态，而UPR感受器也不是只对未折叠蛋白累积作出响应。本文对UPR的发现及其经典通路作一介绍，着重阐述目前已知的UPR感受器的激活机制，并就UPR和ER应激关系以及该领域存在的问题进行讨论。     

Purification and characterization of BiP/Kar2 protein from Saccharomyces cerevisiae.

Using specific anti-BiP/Kar2 antibody as the probe, we have developed an efficient purification method of BiP/Kar2 protein from the total cell extract of Saccharomyces cerevisiae. Overproduction of BiP/Kar2 protein was achieved by the cloning of the KAR2 gene on multicopy plasmids and the treatment of cells harboring the cloned KAR2 gene with tunicamycin. Freeze-thaw treatment, hydroxyapatite high pressure liquid chromatography, and ATP-agarose column chromatography of crude extract yielded homogeneous BiP/Kar2 protein (including less than 0.2% of degradative derivative) with a 430-fold purification and 28% recovery. Edman degradation of purified BiP/Kar2 suggests that the mature protein corresponds to a processed product with the removal of a 42-amino acid presequence. It is active as a homodimer and exhibits ATPase activity with a specific activity of 2 pmol/min/micrograms of protein. Protease susceptibility indicated that the ADP form of BiP/Kar2 is more resistant than the ATP form to the chymotrypsin digestion and that BiP/Kar2 required the presence of ATP to avoid the irreversible denaturation. Synthesis of BiP/Kar2 was induced by the inducible expression of an aberrant heterologous protein, yeast killer prepro-signal mouse alpha-amylase fusion protein.     

SSI1 encodes a novel Hsp70 of the Saccharomyces cerevisiae endoplasmic reticulum.

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.