The transmembrane domain of the molecular chaperone Cosmc directs its localization to the endoplasmic reticulum

The molecular basis for retention of integral membrane proteins in the endoplasmic reticulum (ER) is not well understood. We recently discovered a novel ER molecular chaperone termed Cosmc, which is essential for folding and normal activity of the Golgi enzyme T-synthase. Cosmc, a type II single-pass transmembrane protein, lacks any known ER retrieval/retention motifs. To explore specific ER localization determinants in Cosmc we generated a series of Cosmc mutants along with chimeras of Cosmc with a non-ER resident type II protein, the human transferrin receptor. Here we show that the 18 amino acid transmembrane domain (TMD) of Cosmc is essential for ER localization and confers ER retention to select chimeras. Moreover, mutations of a single Cys residue within the TMD of Cosmc prevent formation of disulfide-bonded dimers of Cosmc and eliminate ER retention. These studies reveal that Cosmc has a unique ER-retention motif within its TMD and provide new insights into the molecular mechanisms by which TMDs of resident ER proteins contribute to ER localization.     

Oxidative protein folding by an endoplasmic reticulum-localized peroxiredoxin

Endoplasmic reticulum (ER) oxidation 1 (ERO1) transfers disulfides to protein disulfide isomerase (PDI) and is essential for oxidative protein folding in simple eukaryotes such as yeast and worms. Surprisingly, ERO1-deficient mammalian cells exhibit only a modest delay in disulfide bond formation. To identify ERO1-independent pathways to disulfide bond formation, we purified PDI oxidants with a trapping mutant of PDI. Peroxiredoxin IV (PRDX4) stood out in this list, as the related cytosolic peroxiredoxins are known to form disulfides in the presence of hydroperoxides. Mouse embryo fibroblasts lacking ERO1 were intolerant of PRDX4 knockdown. Introduction of wild-type mammalian PRDX4 into the ER rescued the temperature-sensitive phenotype of an ero1 yeast mutation. In the presence of an H(2)O(2)-generating system, purified PRDX4 oxidized PDI and reconstituted oxidative folding of RNase A. These observations implicate ER-localized PRDX4 in a previously unanticipated, parallel, ERO1-independent pathway that couples hydroperoxide production to oxidative protein folding in mammalian cells.     

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here, we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase (OST) complex, the key enzyme of N-glycosylation. The deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity toward compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas the deletion of OST6, a functional homolog of OST3, has no effect. OST activity in vitro determined in membranes from rot1-1ost3Δ cells was found to be decreased to 45% compared with wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase Y, Gas1 or dipeptidyl aminopeptidase B, displayed an underglycosylation pattern. By affinity chromatography, a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the O-mannosylation process, as the glycosylation of distinct glycoproteins of this type were affected as well. Altogether, the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation.     

Co-translational function of Cosmc, core 1 synthase specific molecular chaperone, revealed by a cell-free translation system

The core 1 structure of the mucin type O-glycan is synthesized by core 1 β1,3-galactosyltransferase (C1GalT). Core 1 synthase specific molecular chaperone (Cosmc), a molecular chaperone specific for C1GalT, is essential for the expression of functional C1GalT in mammalian cells. In this study, we have established a procedure for detecting the chaperone activity of Cosmc by using a wheat germ cell-free translation system. Active C1GalT was expressed following simultaneous translation with Cosmc or translation in the presence of recombinant Cosmc protein. Moreover, we show that recombinant Cosmc must be present during the translation of C1GalT, as it is ineffective when added after translation. These results indicate that Cosmc mediates the co-translational activation of C1GalT and that it may prevent the unfavorable aggregation of C1GalT.     

Immunocytochemical analysis for intracellular dynamics of C1GalT associated with molecular chaperone, Cosmc

The core 1 structure Galβ1-3GalNAcα1-Ser/Thr (T antigen), the major constituent of O-glycan core structure, is synthesized by cooperation of core 1 synthase (C1GalT) and its specific molecular chaperone, Cosmc. The chaperone function of Cosmc has been well investigated biochemically. In this study, we established monoclonal antibodies specifically recognizing either C1GalT or Cosmc, respectively, and investigated the sub-cellular localization of each protein to elucidate how they cooperate to synthesize the core 1 structure.A sequential immunocytochemical analysis of the human colon cancer cell line, LSB, demonstrated different localization of two proteins. C1GalT was localized in Golgi apparatus, while Cosmc was localized in endoplasmic reticulum. In contrast, the LSC cells, which do not have core 1 synthase activity due to a missense mutation in the Cosmc gene, did not express the C1GalT protein. Although the treatment with a proteasome inhibitor, lactacystin, of LSC cells resulted in the increased expression of C1GalT protein, the distribution of C1GalT was not in Golgi apparatus as seen in LSB cells. On the contrary, overexpression of Cosmc but not C1GalT lead to precise localization of C1GalT protein, which distributed in Golgi apparatus and recovered the core 1 synthase activity in LSC cells. These results suggest that the intracellular dynamics of C1GalT is controlled by its specific molecular chaperon, Cosmc, in association with core 1 synthase activity.     

The p88 molecular chaperone is identical to the endoplasmic reticulum membrane protein, calnexin.

We previously described a novel molecular chaperone (designated p88) that participates in the assembly of murine class I histocompatibility molecules (Degen, E., and Williams, D. B. (1991) J. Cell Biol. 112, 1099-1115). Our findings suggest that p88 may either promote proper assembly of class I molecules or retain them, probably within the endoplasmic reticulum (ER), until assembly of the ternary complex of heavy chain, beta 2-microglobulin, and peptide ligand is complete. In this report, we compare p88 to calnexin, a calcium-binding 90-kDa phosphoprotein of the ER membrane (Wada, I., Rindress, D., Cameron, P. H., Ou, W.-J., Doherty, J.-J., II, Louvard, D., Bell, A.W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). We show that p88 and calnexin share antigenic epitopes defined by a polyclonal anti-calnexin antiserum. Furthermore, both proteins were immunoprecipitated in association with an intracellularly retained variant of the class I H-2Kb molecule. Since p88 and calnexin were also indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, were resistant to digestion with endoglycosidase H, and exhibited virtually identical patterns of peptide fragments following digestion with either V8 protease or trypsin, we conclude that p88 and calnexin represent the same protein. The identification of the p88 chaperone as a phosphorylated, calcium-binding protein of the ER membrane suggests possible means whereby its interaction with class I molecules may be regulated.     

Ubiquitin-independent proteasomal degradation of endoplasmic reticulum-localized connexin43 mediated by CIP75

Connexin43 (Cx43) is a transmembrane protein that forms gap junction channels. Regulation of Cx43 turnover is one mechanism to control the level of intercellular communication that occurs through gap junction channels. Proteasomal degradation of Cx43 is regulated in part through CIP75, a ubiquitin-like and ubiquitin-associated domain containing protein. CIP75 interacts with endoplasmic reticulum-localized Cx43, as demonstrated through co-immunoprecipitation and immunofluorescence microscopy experiments. CIP75 also binds to free monoubiquitin and lysine 48-linked tetraubiquitin chains in vitro and binds to ubiquitinated proteins in cellular lysates. However, analysis of Cx43 that immunoprecipitated with CIP75 demonstrated that the Cx43 associated with CIP75 was not ubiquitinated, and a mutant form of Cx43 that lacked lysines capable of ubiquitination retained the capacity to interact with CIP75. These results suggest that although CIP75 can interact with ubiquitinated cellular proteins, its interaction with Cx43 and stimulation of Cx43 proteasomal degradation does not require the ubiquitination of Cx43.     

Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90

In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.     

The heat shock protein 70 molecular chaperone network in the pancreatic endoplasmic reticulum - a quantitative approach

Traditionally, the canine pancreatic endoplasmic reticulum (ER) has been the workhorse for cell-free studies on protein transport into the mammalian ER. These studies have revealed multiple roles for the major ER-luminal heat shock protein (Hsp) 70, IgG heavy chain-binding protein (BiP), at least one of which also involves the second ER-luminal Hsp70, glucose-regulated protein (Grp) 170. In addition, at least one of these BiP activities depends on Hsp40. Up to now, five Hsp40s and two nucleotide exchange factors, Sil1 and Grp170, have been identified in the ER of different mammalian cell types. Here we quantified the various proteins of this chaperone network in canine pancreatic rough microsomes. We also characterized the various purified proteins with respect to their affinities for BiP and their effect on the ATPase activity of BiP. The results identify Grp170 as the major nucleotide exchange factor for BiP, and the resident ER-membrane proteins ER-resident J-domain protein 1 plus ER-resident J-domain protein 2/Sec63 as prime candidates for cochaperones of BiP in protein transport in the pancreatic ER. Thus, these data represent a comprehensive analysis of the BiP chaperone network that was recently linked to two human inherited diseases, polycystic liver disease and Marinesco-Sj?gren syndrome.     

Regulation of mRNA translation by protein folding in the endoplasmic reticulum

The process of protein secretion is intimately linked to the rate and potential of proper folding and assembly of secretory proteins. The efficiency of protein folding is communicated to the cytoplasm via several signal transduction pathways. This regulates the rate of polypeptide chain synthesis and induction of genes encoding functions that reduce protein-folding load on the endoplasmic reticulum (ER). This review summarizes recent insights into the mechanisms that couple protein translation with protein folding in the ER.     

Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast

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.     

Cell surface expression of calnexin, a molecular chaperone in the endoplasmic reticulum

The folding and assembly of nascent proteins in the endoplasmic reticulum are assisted by the molecular chaperone calnexin, which is itself retained within the endoplasmic reticulum. It was up to now assumed that calnexin was selectively expressed on the surface of immature thymocytes because of a particular characteristic of the protein sorting machinery in these cells. We now report that a small fraction of calnexin is normally expressed on the surface of various cells such as mastocytoma cells, murine splenocytes, fibroblast cells, and human HeLa cells. Surface biotinylation followed by chase culture of living cells revealed that calnexin is continuously delivered to the cell surface and then internalized for lysosomal degradation. These results suggest that there is continuous exocytosis and endocytosis of calnexin, and the amount of calnexin on the plasma membrane results from the balance of the rates of these two events. To study the structural requirement of calnexin for surface expression, we created deletion mutants of calnexin and found that the luminal domain, particularly the glycoprotein binding domain, is necessary. These findings suggest that the surface expression of calnexin depends on the association with glycoproteins and that calnexin may play a certain role as a chaperone on the plasma membrane as well.     

Saccharomyces cerevisiae Rot1 is an essential molecular chaperone in the endoplasmic reticulum

Molecular chaperones prevent aggregation of denatured proteins in vitro and are thought to support folding of diverse proteins in vivo. Chaperones may have some selectivity for their substrate proteins, but knowledge of particular in vivo substrates is still poor. We here show that yeast Rot1, an essential, type-I ER membrane protein functions as a chaperone. Recombinant Rot1 exhibited antiaggregation activity in vitro, which was partly impaired by a temperature-sensitive rot1-2 mutation. In vivo, the rot1-2 mutation caused accelerated degradation of five proteins in the secretory pathway via ER-associated degradation, resulting in a decrease in their cellular levels. Furthermore, we demonstrate a physical and probably transient interaction of Rot1 with four of these proteins. Collectively, these results indicate that Rot1 functions as a chaperone in vivo supporting the folding of those proteins. Their folding also requires BiP, and one of these proteins was simultaneously associated with both Rot1 and BiP, suggesting that they can cooperate to facilitate protein folding. The Rot1-dependent proteins include a soluble, type I and II, and polytopic membrane proteins, and they do not share structural similarities. In addition, their dependency on Rot1 appeared different. We therefore propose that Rot1 is a general chaperone with some substrate specificity.     

Regulation of nucleocytoplasmic localization of the atDjC6 chaperone protein

Summary. The sequence of the atDjC6 chaperone protein includes three potential nuclear localization signal (NLS) sequences (A–C) and three potential nuclear export signal (NES) sequences (X–Z). The subcellular localization of atDjC6 was studied by scanning laser confocal microscopy of chimera with the green-fluorescent protein (GFP) transiently expressed in tobacco BY-2 cells. The localization of the atDjC6::GFP chimera was coincident with that of the nuclear stain propidium iodide. Site-directed mutagenesis was used to verify the predicted NLS sequences. Each was individually fused to GFP and tested for protein localization. The individual NLS sequences were sufficient to direct partial nuclear localization of GFP, although the targeting information within NLS-B is apparently conformation sensitive. Site-directed mutagenesis of the NES sequences increased the amount of each chimera that was nuclearly localized, indicating a decrease in nuclear export. When any pair of NLS sequences were appended to GFP, the chimera were entirely nuclearly localized. Quantitative two-hybrid analysis was used to verify that the decoding of NLS sequence information involves interaction with the NLS-receptor protein importin-. Each of the NLS sequences is flanked by a site of potential Ser phosphorylation, and recombinant atDjC6 could be phosphorylated in vitro. Mutagenesis of Ser residues to the P-Ser mimic Asp interfered with nuclear targeting, apparently by preventing recognition or binding by importin-. Our results are consistent with a regulated nucleocytoplasmic localization of the atDjC6 chaperone protein.Correspondence and reprints: Plant Genetics Research Unit, USDA Agricultural Research Service, 108 Curtis Hall, University of Missouri, Columbia, MO 65211, U.S.A.     

Brefeldin A induces endoplasmic reticulum-associated O-glycosylation of galactosyltransferase

Recent data from several laboratories show that Brefeldin A (BFA) induces a microtubule-dependent back-flow of Golgi components to the endoplasmic reticulum (ER) thereby causing disassembly of the Golgi apparatus and its fusion with ER membranes. In order to delineate the effect of BFA on resident Golgi proteins, we investigated its effect on biosynthesis, maturation and intracellular transport of galactosyltransferase (gal-T), an established trans-Golgi enzyme. Using a protocol of metabolic labeling/immunoprecipitation followed by electrophoretic/fluorographic analysis, we show that in the presence of BFA, gal-T matures to a molecular form of 48.5 kD, a size intermediate between the 2 precursor forms of 44 and 47 and the mature form of 54 kD (Strous and Berger: J. Biol. Chem., 257:7623-28, 1982). Little mature form was detectable in the presence of BFA even after prolonged chase times of up to 28 hr. The intermediate form was sensitive to O-glycanase and endoglycosidase H, indicating early O-glycosylation without sialylation and lack of complex N-glycosylation, respectively. In order to define the compartment responsible for O-glycosylation in the presence of BFA, a temperature block of 25 degrees C was applied which inhibited recovery of Golgi elements from BFA-induced fusion with ER. At this temperature and in absence of BFA, biosynthesis of gal-T was not appreciably affected, while maturation was completely inhibited as indicated by the presence of unmodified precursor forms of gal-T. After 60 min preincubation with BFA, a time period sufficient to demonstrate complete fusion of Golgi with ER, subsequent biosynthesis of gal-T at 25 degrees C in absence of BFA led to the intermediate form, while precursor forms were not detectable. These data provide direct evidence for BFA-induced redistribution to the EF of Golgi enzymes involved in O-glycosylation and their early functional involvement in biosynthesis of newly synthesized gal-T.     

The rough endoplasmic reticulum-resident FK506-binding protein FKBP65 is a molecular chaperone that interacts with collagens

The rough endoplasmic reticulum-resident FK-506-binding protein FKBP65 can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. The peptidylprolyl cis-trans-isomerase activity of FKBP65 was previously shown to have only marginal effects on the rate of triple helix formation (Zeng, B., MacDonald, J. R., Bann, J. G., Beck, K., Gambee, J. E., Boswell, B. A., and B?chinger, H. P. (1998) Biochem. J. 330, 109-114). Here we show that FKBP65 is a monomer in solution and acts as a chaperone molecule when tested with two classic chaperone assays: FKBP65 inhibits the thermal aggregation of citrate synthase and is active in the denatured rhodanese refolding and aggregation assay. The chaperone activity is comparable to that of protein-disulfide isomerase, a well characterized chaperone. FKBP65 delays the in vitro fibril formation of type I collagen, indicating that FKBP65 is also able to interact with triple helical collagen, and acts as a collagen chaperone.     

Regulation of protein biogenesis at the endoplasmic reticulum membrane

The biogenesis of most secretory and membrane proteins involves targeting the nascent protein to the endoplasmic reticulum (ER), translocation across or integration into the ER membrane and maturation into a functional product. The essential machinery that directs these events for model secretory and membrane proteins has been identified, shifting the focus of studies towards the molecular mechanisms by which these core components function. By contrast, regulatory mechanisms that allow certain proteins to serve multiple functions within a cell remain entirely unexplored. This article examines each stage of protein biogenesis as a potential site of regulation that could be exploited by the cell to effectively increase the diversity of functional gene expression.     

Regulation of immature protein dynamics in the endoplasmic reticulum

The quality of nascent protein folding in vivo is influenced by the microdynamics of the proteins. Excessive collisions between proteins may lead to terminal misfolding, and the frequency of protein interactions with molecular chaperones determines their folding rates. However, it is unclear how immature protein dynamics are regulated. In this study, we analyzed the diffusion of immature tyrosinase in the endoplasmic reticulum (ER) of non-pigmented cells by taking advantage of the thermal sensitivity of the tyrosinase. The diffusion of tyrosinase tagged with yellow fluorescence protein (YFP) in living cells was directly measured using fluorescent correlation spectroscopy. The diffusion of folded tyrosinase in the ER of cells treated with brefeldin A, as measured by fluorescent correlation spectroscopy, was critically affected by the expression level of tyrosinase-YFP. Under defined conditions in which random diffusional motion of folded protein was allowed, we found that the millisecond-order diffusion rate observed for folded tyrosinase almost disappeared for the misfolded molecules synthesized at a nonpermissive high temperature. This was not because of enhanced aggregation at the high temperature, as terminally misfolded tyrosinase synthesized in the absence of calnexin interactions showed comparable, albeit slightly slower, diffusion. Yet, the thermally misfolded tyrosinase was not immobilized when measured by fluorescence recovery after photobleaching. In contrast, terminally misfolded tyrosinase synthesized in cells in which alpha-glucosidases were inhibited showed extensive immobilization. Hence, we suggest that the ER represses random fluctuations of immature tyrosinase molecules while preventing their immobilization.