Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP-mannose and cis-prenyltransferase activity

Protein O-mannosylation has been postulated to be critical for production and secretion of glycoproteins in fungi. Therefore, understanding the regulation of this process and the influence of heterologous expression of glycoproteins on the activity of enzymes engaged in O-glycosylation are of considerable interest. In this study we expressed cellobiohydrolase II (CBHII) of T. reesei, which is normally highly O-mannosylated, in Saccharomyces cerevisiae pmt mutants partially blocked in O-mannosylation. We found that the lack of Pmt1 or Pmt2 protein O-mannosyltransferase activity limited the glycosylation of CBHII, but it did not affect its secretion. The S. cerevisiae pmt1Delta and pmt2Delta mutants expressing T. reesei cbh2 gene showed a decrease of GDP-mannose level and a very high activity of cis-prenyltransferase compared to untransformed strains. On the other hand, elevation of cis-prenyltransferase activity by overexpression of the S. cerevisiae RER2 gene in these mutants led to an increase of dolichyl phosphate mannose synthase activity, but it did not influence the activity of O-mannosyltransferases. Overexpression of the MPG1 gene increased the level of GDP-mannose and stimulated the activity of mannosyltransferases elongating O-linked sugar chains, leading to partial restoration of CBHII glycosylation.     

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.     

Role of protein O-mannosyltransferase Pmt4 in the morphogenesis and virulence of Cryptococcus neoformans

Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen Cryptococcus neoformans. Disruption of the C. neoformans PMT4 gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the pmt4 mutant compared to wild-type cells. The pmt4 mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the pmt4 mutant, as well as a global decrease in protein mannosylation. Heterologous expression of C. neoformans PMT4 in a Saccharomyces cerevisiae pmt1pmt4 mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in C. neoformans was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of C. neoformans.     

Roles of O-mannosylation of aberrant proteins in reduction of the load for endoplasmic reticulum chaperones in yeast

The protein quality control system in the endoplasmic reticulum (ER) ensures that only properly folded proteins are deployed throughout the cells. When nonnative proteins accumulate in the ER, the unfolded protein response is triggered to limit further accumulation of nonnative proteins and the ER is cleared of accumulated nonnative proteins by the ER-associated degradation (ERAD). In the yeast ER, aberrant nonnative proteins are mainly directed for the ERAD, but a distinct fraction of them instead receive O-mannosylation. In order to test whether O-mannosylation might also be a mechanism to process aberrant proteins in the ER, here we analyzed the effect of O-mannosylation on two kinds of model aberrant proteins, a series of N-glycosylation site mutants of prepro-alpha-factor and a pro-region-deleted derivative of Rhizopus niveus aspartic proteinase-I (Deltapro) both in vitro and in vivo. O-Mannosylation increases solubilities of the aberrant proteins and renders them less dependent on the ER chaperone, BiP, for being soluble. The release from ER chaperones allows the aberrant proteins to exit out of the ER for the normal secretory pathway transport. When the gene for Pmt2p, responsible for the O-mannosylation of these aberrant proteins, and that for the ERAD were simultaneously deleted, the cell exhibited enhanced unfolded protein response. O-Mannosylation may therefore function as a fail-safe mechanism for the ERAD by solubilizing the aberrant proteins that overflowed from the ERAD pathway and reducing the load for ER chaperones.     

Pmt-mediated O mannosylation stabilizes an essential component of the secretory apparatus, Sec20p, in Candida albicans

Sec20p is an essential endoplasmic reticulum (ER) membrane protein in yeasts, functioning as a tSNARE component in retrograde vesicle traffic. We show that Sec20p in the human fungal pathogen Candida albicans is extensively O mannosylated by protein mannosyltransferases (Pmt proteins). Surprisingly, Sec20p occurs at wild-type levels in a pmt6 mutant but at very low levels in pmt1 and pmt4 mutants and also after replacement of specific Ser/Thr residues in the lumenal domain of Sec20p. Pulse-chase experiments revealed rapid degradation of unmodified Sec20p (38.6 kDa) following its biosynthesis, while the stable O-glycosylated form (50 kDa) was not formed in a pmt1 mutant. These results suggest a novel function of O mannosylation in eukaryotes, in that modification by specific Pmt proteins will prevent degradation of ER-resident membrane proteins via ER-associated degradation or a proteasome-independent pathway.     

Sel1p/Ubx2p participates in a distinct Cdc48p-dependent endoplasmic reticulum-associated degradation pathway

The endoplasmic reticulum (ER) serves a critical role in the biogenesis of secretory proteins. Folding of nascent polypeptides occurs in the ER before anterograde transport through the secretory pathway, whereas terminally misfolded secretory proteins are recognized and eliminated by ER-associated degradation (ERAD). Here, we investigated the role of the ubiquitin regulatory X (UBX) domain-containing protein Sel1p in ER quality control and transport. Mutant sel1Delta yeast displayed a constitutively active unfolded protein response and a mildly reduced rate of secretory protein transport from the ER. Immunoisolation of Sel1p from detergent-solubilized ER microsomes revealed a protein complex containing both Cdc48p and Npl4p and suggested a direct role for Sel1p in ERAD. In cells that lack Sel1p, we observed a reduction in the level of Cdc48p bound to ER membranes and a decrease in the turnover rate of two model ERAD substrates, carboxypeptidase Y* and Ste6*. In addition, we found that Sel1p and a second UBX domain-containing protein, Shp1p, associated with Cdc48p in a mutually exclusive manner. Interestingly, the association of Sel1p with Cdc48p was regulated by ATP, while the interaction of Shp1p with Cdc48p was not influenced by ATP. Based on these findings, we conclude that Sel1p operates in the ERAD pathway by coupling Cdc48p to ER membranes and that Shp1p acts in a distinct Cdc48p-dependent protein degradation pathway.     

Pkc1p modifies CPY* degradation in the ERAD pathway

The process of endoplasmic reticulum-associated degradation (ERAD) involved in the degradation of misfolded N-linked glycoproteins utilizes Cdc48p which extracts misfolded glycoproteins from the lumen to the cytosol to present them for deglycosylation and degradation. Pkc1p has been identified as a component of the ERAD pathway, because deletion of the pkc1 gene impairs ERAD and causes accumulation of CPY* in the lumen of the ER, most probably because of the mislocalization of Cdc48p. In addition, we show that Cdc48p interacts in the cytosol with the deglycosylation enzyme, PNGase, only when Cdc48p is associated with a misfolded glycoprotein.     

Sec61p is part of the endoplasmic reticulum‐associated degradation machinery

Endoplasmic reticulum‐associated degradation (ERAD) is a cellular pathway for the disposal of misfolded secretory proteins. This process comprises recognition of the misfolded proteins followed by their retro‐translocation across the ER membrane into the cytosol in which polyubiquitination and proteasomal degradation occur. A variety of data imply that the protein import channel Sec61p has a function in the ERAD process. Until now, no physical interactions between Sec61p and other essential components of the ERAD pathway could be found. Here, we establish this link by showing that Hrd3p, which is part of the Hrd‐Der ubiquitin ligase complex, and other core components of the ERAD machinery physically interact with Sec61p. In addition, we study binding of misfolded CPY^* proteins to Sec61p during the process of degradation. We show that interaction with Sec61p is maintained until the misfolded proteins are ubiquitinated on the cytosolic side of the ER. Our observations suggest that Sec61p contacts an ERAD ligase complex for further elimination of ER lumenal misfolded proteins.     

Characterization of a fission yeast SUMO-1 homologue, pmt3p, required for multiple nuclear events, including the control of telomere length and chromosome segregation

Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. Here we describe the identification of the SUMO-1 homologue of fission yeast, which we show to be required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of the pmt3(+) gene, the Schizosaccharomyces pombe homologue of SMT3, was not lethal, but mutant cells carrying the disrupted gene grew more slowly. The pmt3Delta cells showed various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, we found that pmt3(+) is required for telomere length maintenance. Loss of Pmt3p function caused a striking increase in telomere length. When Pmt3p synthesis was restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase- and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation.     

Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins

Many misfolded endoplasmic reticulum (ER) proteins are eliminated by ERAD, a process in which substrates are polyubiquitylated and moved into the cytosol for proteasomal degradation. We have identified in S. cerevisiae distinct ubiquitin-ligase complexes that define different ERAD pathways. Proteins with misfolded ER-luminal domains use the ERAD-L pathway, in which the Hrd1p/Hrd3p ligase forms a near stoichiometric membrane core complex by binding to Der1p via the linker protein Usa1p. This core complex associates through Hrd3p with Yos9p, a substrate recognition protein in the ER lumen. Substrates with misfolded intramembrane domains define a pathway (ERAD-M) that differs from ERAD-L by being independent of Usa1p and Der1p. Membrane proteins with misfolded cytosolic domains use the ERAD-C pathway and are directly targeted to the Doa10p ubiquitin ligase. All three pathways converge at the Cdc48p ATPase complex. These results lead to a unifying concept for ERAD that may also apply to mammalian cells.     

Identification of O-mannosylated Virulence Factors in Ustilago maydis

The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens Candida albicans or Cryptococcus neoformans as well as in the phytopathogenic fungus Ustilago maydis. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in pmt4 mutants. Taking advantage of the characteristics described for Pmt4 substrates in Saccharomyces cerevisiae, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus U. maydis and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of pmt4 and msb2 showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in pmt4 mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of pmt4 mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis.     

A conserved acidic motif is crucial for enzymatic activity of protein O-mannosyltransferases

Protein O-mannosylation is an essential modification in fungi and mammals. It is initiated at the endoplasmic reticulum by a conserved family of dolichyl phosphate mannose-dependent protein O-mannosyltransferases (PMTs). PMTs are integral membrane proteins with two hydrophilic loops (loops 1 and 5) facing the endoplasmic reticulum lumen. Formation of dimeric PMT complexes is crucial for mannosyltransferase activity, but the direct cause is not known to date. In bakers' yeast, O-mannosylation is catalyzed largely by heterodimeric Pmt1p-Pmt2p and homodimeric Pmt4p complexes. To further characterize Pmt1p-Pmt2p complexes, we developed a photoaffinity probe based on the artificial mannosyl acceptor substrate Tyr-Ala-Thr-Ala-Val. The photoreactive probe was preferentially cross-linked to Pmt1p, and deletion of the loop 1 (but not loop 5) region abolished this interaction. Analysis of Pmt1p loop 1 mutants revealed that especially Glu-78 is crucial for binding of the photoreactive probe. Glu-78 belongs to an Asp-Glu motif that is highly conserved among PMTs. We further demonstrate that single amino acid substitutions in this motif completely abolish activity of Pmt4p complexes. In contrast, both acidic residues need to be exchanged to eliminate activity of Pmt1p-Pmt2p complexes. On the basis of our data, we propose that the loop 1 regions of dimeric complexes form part of the catalytic site.     

Inositol deacylation by Bst1p is required for the quality control of glycosylphosphatidylinositol-anchored proteins

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytosol, and degraded by the proteasome. A number of proteins are processed and modified by a glycosylphosphatidylinositol (GPI) anchor in the ER, but the quality control mechanisms of GPI-anchored proteins remain unclear. Here, we report on the quality control mechanism of misfolded GPI-anchored proteins. We have constructed a mutant form of the beta-1,3-glucanosyltransferase Gas1p (Gas1*p) as a model misfolded GPI-anchored protein. Gas1*p was modified with a GPI anchor but retained in the ER and was degraded rapidly via the proteasome. Disruption of BST1, which encodes GPI inositol deacylase, caused a delay in the degradation of Gas1*p. This delay was because of an effect on the deacylation activity of Bst1p. Disruption of genes involved in GPI-anchored protein concentration and N-glycan processing caused different effects on the degradation of Gas1*p and a soluble misfolded version of carboxypeptidase Y. Furthermore, Gas1*p associated with both Bst1p and BiP/Kar2p, a molecular chaperone, in vivo. Our data suggest that GPI inositol deacylation plays important roles in the quality control and ER-associated degradation of GPI-anchored proteins.     

Transmembrane topology of pmt1p, a member of an evolutionarily conserved family of protein O-mannosyltransferases

The identification of the evolutionarily conserved family of dolichyl-phosphate-D-mannose:protein O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential role in a number of physiologically important processes. Strikingly, all members of the Pmt protein family share almost identical hydropathy profiles; a central hydrophilic domain is flanked by amino- and carboxyl-terminal sequences containing several putative transmembrane helices. This pattern is of particular interest because it diverges from structural models of all glycosyltransferases characterized so far. Here, we examine the transmembrane topology of Pmt1p, an integral membrane protein of the endoplasmic reticulum, from Saccharomyces cerevisiae. Structural predictions were directly tested by site-directed mutagenesis of endogenous N-glycosylation sites, by fusing a topology-sensitive monitor protein domain to carboxyl-terminal truncated versions of the Pmt1 protein and, in addition, by N-glycosylation scanning. Based on our results we propose a seven-transmembrane helical model for the yeast Pmt1p mannosyltransferase. The Pmt1p amino terminus faces the cytoplasm, whereas the carboxyl terminus faces the lumen of the endoplasmic reticulum. A large hydrophilic segment that is oriented toward the lumen of the endoplasmic reticulum is flanked by five amino-terminal and two carboxyl-terminal membrane spanning domains. We could demonstrate that this central loop is essential for the function of Pmt1p.     

Substrate recognition in ER-associated degradation mediated by Eps1, a member of the protein disulfide isomerase family

Pma1-D378N is a misfolded plasma membrane protein in yeast that is prevented from delivery to the cell surface and targeted instead for ER-associated degradation (ERAD). Degradation of Pma1-D378N is dependent on the ubiquitin ligase Doa10 and the ubiquitin chaperone Cdc48. Recognition of Pma1-D378N by the ERAD pathway is dependent on Eps1, a transmembrane member of the protein disulfide isomerase (PDI) oxidoreductase family. Eps1 has two thioredoxin-like domains containing a CPHC and a CDKC active site. Although Eps1 interaction with wild-type Pma1 was not detected, Eps1 co-immunoprecipitates with Pma1-D378N. Eps1 interaction with Pma1-D378N requires the CPHC motif, although both thioredoxin-like domains appear to cooperate in substrate recognition. In the absence of the native transmembrane domain and cytoplasmic tail of Eps1, degradation of Pma1-D378N is slowed, suggesting that Eps1 facilitates presentation of substrate to membrane-bound components of the degradation machinery. Genetic interactions with other mutants of the ERAD machinery and induction of the unfolded protein response in eps1Delta cells support a general role for Eps1 as a recognition component of the ERAD pathway.     

Single, context-specific glycans can target misfolded glycoproteins for ER-associated degradation

The endoplasmic reticulum (ER) maintains an environment essential for secretory protein folding. Consequently, the premature transport of polypeptides would be harmful to the cell. To avert this scenario, mechanisms collectively termed "ER quality control" prevent the transport of nascent polypeptides until they properly fold. Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway. To better understand the relationship between quality control and ERAD, we studied a new misfolded variant of carboxypeptidase Y (CPY). The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway. Systematic analysis revealed that a single, specific N-linked glycan of CPY was required for sorting into the pathway. The determinant is dependent on the putative lectin-like receptor Htm1/Mnl1p. The discovery of a similar signal in misfolded proteinase A supported the generality of the mechanism. These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold.