Protein O-mannosylation is crucial for cell wall integrity, septation and viability in fission yeast

Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, which are of fundamental importance in eukaryotes. The PMT family, which is classified into PMT1, PMT2 and PMT4 subfamilies, is evolutionarily conserved. Despite the fact that PMTs are crucial for viability of baker's yeast as well as of mouse, recent studies suggested that there are significant differences in the organization and properties of the O-mannosylation machinery between yeasts and mammals. In this study we identified and characterized the PMT family of the archaeascomycete Schizosaccharomyces pombe. Unlike Saccharomyces cerevisiae where the PMT family is highly redundant, in S. pombe only one member of each PMT subfamily is present, namely, oma1+ (protein O-mannosyltransferase), oma2+ and oma4+. They all act as protein O-mannosyltransferases in vivo. oma1+ and oma2+ form heteromeric protein complexes and recognize different protein substrates compared to oma4+, suggesting that similar principles underlie mannosyltransfer reaction in S. pombe and budding yeast. Deletion of oma2+, as well as simultaneous deletion of oma1+ and oma4+ is lethal. Characterization of the viable S. pombe oma1Delta and oma4Delta single mutants showed that a lack of O-mannosylation results in abnormal cell wall and septum formation, thereby severely affecting cell morphology and cell-cell separation.     

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.     

Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP

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.     

O-mannosylation is required for degradation of the endoplasmic reticulum-associated degradation substrate Gas1*p via the ubiquitin/proteasome pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, protein O-mannosylation, which is executed by protein O-mannosyltransferases, is essential for a variety of biological processes as well as for conferring solubility to misfolded proteins. To determine if O-mannosylation plays an essential role in endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins, we used a model misfolded protein, Gas1*p. The O-mannose content of Gas1*p, which is transferred by protein O-mannosyltransferases, was higher than that of Gas1p. Both Pmt1p and Pmt2p, which do not transfer O-mannose to correctly folded Gas1p, participated in the O-mannosylation of Gas1*p. Furthermore, in a pmt1 Delta pmt2 Delta double-mutant background, degradation of Gas1*p is altered from a primarily proteasome dependent to a vacuolar protease-dependent pathway. This process is in a manner dependent on a Golgi-to-endosome sorting function of the VPS30 complex II. Collectively, our data suggest that O-mannosylation plays an important role for proteasome-dependent degradation of Gas1*p via the ERAD pathway and when O-mannosylation is insufficient, Gas1*p is degraded in the vacuole. Thus, we propose that O-mannosylation by Pmt1p and Pmt2p might be a key step in the targeting of some misfolded proteins for degradation via the proteasome-dependent ERAD pathway.     

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.     

ER stress induces alternative nonproteasomal degradation of ER proteins but not of cytosolic ones

Inhibition of protein folding in the endoplasmic reticulum (ER) causes ER stress, which triggers the unfolded protein response (UPR). To decrease the biosynthetic burden on the ER, the UPR inhibits in its initial stages protein synthesis. At later stages it upregulates components of ER-associated degradation (ERAD) and of the ubiquitin/proteasome system, which targets ER as well as cytosolic proteins for disposal. Here we report that, at later stages, the UPR also activates an alternative nonproteasomal pathway of degradation, which is resistant to proteasome inhibitors and is specific for ER substrates (assessed with uncleaved precursor of asialoglycoprotein receptor H2a and unassembled CD3delta) and not for cytosolic ones (p53). To mimic the initial inhibition of translation during UPR, we incubated cells with cycloheximide. After this treatment, degradation of ERAD substrates was no longer effected by proteasomal inhibition, similarly to the observed outcome of UPR. The degradation also became insensitive to abrogation of ubiquitination in a cell line carrying a thermosensitive E1 ubiquitin activating enzyme mutant. Of all protease inhibitors tested, only the metal chelator o-phenanthroline could block this nonproteasomal degradation. Preincubation of o-phenanthroline with Mn2+ or Co2+, but not with other cations, reversed the inhibition. Our results suggest that, upon inhibition of translation, an alternative nonproteasomal pathway is activated for degradation of proteins from the ER. This involves a Mn2+/Co2+-dependent metalloprotease or other metalloprotein. The alternative pathway selectively targets ERAD substrates to reduce the ER burden, but does not affect p53, the levels of which remain dependent on proteasomal control.     

The yeast G protein alpha subunit Gpa1 transmits a signal through an RNA binding effector protein Scp160

In yeast Saccharomyces cerevisiae the G protein betagamma subunits (Ste4/Ste18) have long been known to transmit the signal required for mating. Here we demonstrate that GTPase-deficient mutants of Galpha (Gpa1) directly activate the mating response pathway. We also show that signaling by activated Gpa1 requires direct coupling to an RNA binding protein Scp160. These findings suggest an additional role for Gpa1 and reveal Scp160 as a component of the mating response pathway in yeast.     

Members of the evolutionarily conserved PMT family of protein O-mannosyltransferases form distinct protein complexes among themselves

Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, an essential protein modification. Since PMTs are evolutionarily conserved in fungi but are absent in green plants, the PMT family is a putative target for new antifungal drugs, particularly in fighting the threat of phytopathogenic fungi. The PMT family is phylogenetically classified into PMT1, PMT2, and PMT4 subfamilies, which differ in protein substrate specificity. In the model organism Saccharomyces cerevisiae as well as in many other fungi the PMT family is highly redundant, and only the simultaneous deletion of PMT1/PMT2 and PMT4 subfamily members is lethal. In this study we analyzed the molecular organization of PMT family members in S. cerevisiae. We show that members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p) and that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms. Under certain physiological conditions, however, Pmt1p interacts also with Pmt3p, and Pmt5p with Pmt2p, suggesting a compensatory cooperation that guarantees the maintenance of O-mannosylation. Unlike the PMT1/PMT2 subfamily members, the single member of the PMT4 subfamily (Pmt4p) acts as a homomeric complex. Using mutational analyses we demonstrate that the same conserved protein domains underlie both heteromeric and homomeric interactions, and we identify an invariant arginine residue of transmembrane domain two as essential for the formation and/or stability of PMT complexes in general. Our data suggest that protein-protein interactions between the PMT family members offer a point of attack to shut down overall protein O-mannosylation in fungi.     

The endoplasmic reticulum-associated degradation is necessary for plant salt tolerance

Eukaryotic organisms have quality-control mechanisms that allow misfolded or unassembled proteins to be retained in the endoplasmic reticulum (ER) and subsequently degraded by ER-associated degradation (ERAD). The ERAD pathway is well studied in yeast and mammals; however, the biological functions of plant ERAD have not been reported. Through molecular and cellular biological approaches, we found that ERAD is necessary for plants to overcome salt stress. Upon salt treatment ubiquitinated proteins increased in plant cells, especially unfolded proteins that quickly accumulated in the ER and subsequently induced ER stress responses. Defect in HRD3A of the HRD1/HRD3 complex of the ERAD pathway resulted in alteration of the unfolded protein response (UPR), increased plant sensitivity to salt, and retention of ERAD substrates in plant cells. Furthermore, we demonstrated that Ca(2+) release from the ER is involved in the elevation of UPR and reactive oxygen species (ROS) participates the ERAD-related plant salt response pathway.     

Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation

Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be refolded or degraded to maintain the homeostasis of the ER. Components of both productive folding and ER-associated degradation (ERAD) mechanisms are known to be up-regulated by the unfolded protein response (UPR). We describe two novel components of mammalian ERAD, Derlin-2 and -3, which show weak homology to Der1p, a transmembrane protein involved in yeast ERAD. Both Derlin-2 and -3 are up-regulated by the UPR, and at least Derlin-2 is a target of the IRE1 branch of the response, which is known to up-regulate ER degradation enhancing alpha-mannosidase-like protein (EDEM) and EDEM2, receptor-like molecules for misfolded glycoprotein. Overexpression of Derlin-2 or -3 accelerated degradation of misfolded glycoprotein, whereas their knockdown blocked degradation. Derlin-2 and -3 are associated with EDEM and p97, a cytosolic ATPase responsible for extraction of ERAD substrates. These findings indicate that Derlin-2 and -3 provide the missing link between EDEM and p97 in the process of degrading misfolded glycoproteins.     

A regulatory link between ER-associated protein degradation and the unfolded-protein response

Ubiquitin conjugation during endoplasmic-reticulum-associated degradation (ERAD) depends on the activity of Ubc7. Here we show that Ubc1 acts as a further ubiquitin-conjugating enzyme in this pathway. Absence of both enzymes results in marked stabilization of an ERAD substrate and induction of the unfolded-protein response (UPR). Furthermore, basic ERAD activity is sufficient to eliminate unfolded proteins under normal conditions. However, when stress is applied, the UPR is required to increase ERAD activity. We thus demonstrate, for the first time, a regulatory loop between ERAD and the UPR, which is essential for normal growth of yeast cells.     

Cellular tolerance of prion protein PrP in yeast involves proteolysis and the unfolded protein response

Secretory proteins undergo a stringent quality control process in the endoplasmic reticulum (ER). Misfolded ER proteins are returned to the cytosol and destroyed by the proteasome. Prion protein PrP is degraded by the proteasome in mammalian cells. However, the significance of proteolysis on PrP-induced cell death is controversial. Moreover, the specific pathway involved in PrP degradation remains unknown. Here, we demonstrate that the unglycosylated form of human PrP is subjected to the ER-associated protein degradation (ERAD) process in the yeast Saccharomyces cerevisiae. We also show that unglycosylated PrP is degraded by the Hrd1-Hrd3 pathway. Accumulation of misfolded proteins triggers the unfolded protein response (UPR), which promotes substrate refolding. Interestingly, we find that the expression of PrP leads to growth impairment in cells deficient in UPR and ERAD. These findings raise the possibility that decreased UPR activity and proteolysis may contribute to the pathogenesis of some prion-related diseases.     

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.     

The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants.

We are investigating the transport and turnover of the multispanning membrane protein Ste6. The Ste6 protein is a member of the ABC-transporter family and is required for the secretion of the yeast mating pheromone a-factor. In contrast to the prevailing view that Ste6 is a plasma membrane protein, we found that Ste6 is mainly associated with internal membranes and not with the cell surface. Fractionation and immunofluorescence data are compatible with a Golgi localization of Ste6. Despite its mostly intracellular localization, the Ste6 protein is in contact with the cell surface, as demonstrated by the finding that Ste6 accumulates in the plasma membrane in endocytosis mutants. The Ste6 protein which accumulates in the plasma membrane in endocytosis mutants is ubiquitinated. Ste6 is thus the second protein in yeast besides MAT alpha 2 for which ubiquitination has been demonstrated. Ste6 is a very unstable protein (half-life 13 min) which is stabilized approximately 3-fold in a ubc4 ubc5 mutant, implicating the ubiquitin system in the degradation of Ste6. The strongest stabilizing effect on Ste6 is, however, observed in the vacuolar pep4 mutant (half-life > 2 h), suggesting that most of Ste6 is degraded in the vacuole. Secretory functions are required for efficient degradation of Ste6, indicating that Ste6 enters the secretory pathway and is transported to the vacuole by vesicular carriers.     

Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast.

STE20 encodes a protein kinase related to mammalian p65Pak which functions in several signal transduction pathways in yeast, including those involved in pseudohyphal and invasive growth, as well as mating. In addition, Ste20 plays an essential role in cells lacking Cla4, a kinase with significant homology to Ste20. It is not clear how the activity of Ste20 is regulated in response to these different signals in vivo, but it has been demonstrated recently that binding of the small GTP binding protein Cdc42 is able to activate Ste20 in vitro. Here we show that Ste20 functionally interacts with Cdc42 in a GTP-dependent manner in vivo: Ste20 mutants that can no longer bind Cdc42 were unable to restore growth of ste20 cla4 mutant cells. They were also defective for pseudohyphal growth and agar invasion, and displayed reduced mating efficiency when mated with themselves. Surprisingly, however, the kinase activity of such Ste20 mutants was normal when assayed in vitro. Furthermore, these alleles were able to fully activate the MAP kinase pathway triggered by mating pheromones in vivo, suggesting that binding of Cdc42 and Ste20 was not required to activate Ste20. Wild-type Ste20 protein was visualized as a crescent at emerging buds during vegetative growth and at shmoo tips in cells arrested with alpha-factor. In contrast, a Ste20 mutant protein unable to bind Cdc42 was found diffusely throughout the cytoplasm, suggesting that Cdc42 is required to localize Ste20 properly in vivo.     

Endoplasmic reticulum-associated degradation mediated by MoHrd1 and MoDer1 is pivotal for appressorium development and pathogenicity of Magnaporthe oryzae

Most secretory proteins are folded and modified in the endoplasmic reticulum (ER); however, protein folding is error-prone, resulting in toxic protein aggregation and cause ER stress. Irreversibly misfolded proteins are subjected to ER-associated degradation (ERAD), modified by ubiquitination, and degraded by the 26S proteasome. The yeast ERAD ubiquitin ligase Hrd1p and multispanning membrane protein Der1p are involved in ubiquitination and transportation of the folding-defective proteins. Here, we performed functional characterization of MoHrd1 and MoDer1 and revealed that both of them are localized to the ER and are pivotal for ERAD substrate degradation and the ER stress response. MoHrd1 and MoDer1 are involved in hyphal growth, asexual reproduction, infection-related morphogenesis, protein secretion and pathogenicity of M. oryzae. Importantly, MoHrd1 and MoDer1 mediated conidial autophagic cell death and subsequent septin ring assembly at the appressorium pore, leading to abnormal appressorium development and loss of pathogenicity. In addition, deletion of MoHrd1 and MoDer1 activated the basal unfolded protein response (UPR) and autophagy, suggesting that crosstalk between ERAD and two other closely related mechanisms in ER quality control system (UPR and autophagy) governs the ER stress response. Our study indicates the importance of ERAD function in fungal development and pathogenesis of M. oryzae.     

AAA-ATPase p97/Cdc48p, a Cytosolic Chaperone Required for Endoplasmic Reticulum-Associated Protein Degradation

Endoplasmic reticulum-associated degradation (ERAD) disposes of aberrant proteins in the secretory pathway. Protein substrates of ERAD are dislocated via the Sec61p translocon from the endoplasmic reticulum to the cytosol, where they are ubiquitinated and degraded by the proteasome. Since the Sec61p channel is also responsible for import of nascent proteins, this bidirectional passage should be coordinated, probably by molecular chaperones. Here we implicate the cytosolic chaperone AAA-ATPase p97/Cdc48p in ERAD. We show the association of mammalian p97 and its yeast homologue Cdc48p in complexes with two respective ERAD substrates, secretory immunoglobulin M in B lymphocytes and 6myc-Hmg2p in yeast. The membrane 6myc-Hmg2p as well as soluble lumenal CPY*, two short-lived ERAD substrates, are markedly stabilized in conditional cdc48 yeast mutants. The involvement of Cdc48p in dislocation is underscored by the accumulation of ERAD substrates in the endoplasmic reticulum when Cdc48p fails to function, as monitored by activation of the unfolded protein response. We propose that the role of p97/Cdc48p in ERAD, provided by its potential unfoldase activity and multiubiquitin binding capacity, is to act at the cytosolic face of the endoplasmic reticulum and to chaperone dislocation of ERAD substrates and present them to the proteasome.     

The Leu-132 of the Ste4(Gbeta) subunit is essential for proper coupling of the G protein with the Ste2 alpha factor receptor during the mating pheromone response in yeast

In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered alpha pheromone response in yeast cells. Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the beta propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and alpha cells. The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor. These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromone-stimulated receptor and that this domain could be an important point for physical interaction between the Gbeta and the Galpha subunits.