Evidence for interaction of yeast protein kinase C with several subunits of oligosaccharyl transferase

Oligosaccharyltransferase (OT) in Saccharomyces cerevisiae is an enzyme complex consisting of 8 transmembrane proteins located in the endoplasmic reticulum (ER). Studies on potential protein-protein interactions in OT using a two-hybrid library screen revealed that protein kinase C (Pkc1p) interacted with the lumenal domains of several OT subunits. Additional genetic experiments revealed that overexpression of two OT subunits rescued the growth defect caused by overexpression of a Pkc1 active site mutant, implying that there are specific genetic interactions between PKC1 and OT. These in vivo findings were complemented by in vitro studies that showed that several of the OT subunits bound to a fusion protein consisting of glutathione S-transferase linked via its C-terminus to Pkc1p. Assays of OT activity, in which glycosylation of a simple acceptor peptide was assayed in microsomes from wild-type and a pkc1 null revealed a 50% reduction in activity in the microsomes from the null strain. In contrast, strains containing null mutations of two other genes known to be downstream of Pkc1p in the PKC1-MAP kinase pathway had a level of OT activity comparable to that of wild-type cells. These in vivo and in vitro experiments suggest that in yeast cells Pkc1p may be involved in regulation of the N-glycosylation of proteins.     

New findings on interactions among the yeast oligosaccharyl transferase subunits using a chemical cross-linker

At present, there is very limited knowledge about the structural organization of the yeast oligosaccharyl transferase (OT) complex and the function of each of its nine subunits. Because of the failure of the yeast two-hybrid system to reveal interactions between luminal domains of these subunits, we utilized a membrane permeable, thiocleavable cross-linking reagent dithiobis-succinimidyl propionate to biochemically study the interactions of various OT subunits. Four essential gene products, Ost1p, Wbp1p, Swp1p, and Stt3p were shown to be cross-linked to each other in a pairwise fashion. In addition, Ost1p was found to be cross-linked to all other eight OT subunits individually. This led us to propose that Ost1p may reside in the core of the OT complex and could play an important role in its assembly. Ost4p and Ost5p were found to only interact with specific components of the OT complex and may function as an additional anchor for optimal stability of Stt3p and Ost1p in the membrane, respectively. Interestingly, Ost3p and Ost6p subunits exhibited a surprisingly identical pattern of cross-linking to other subunits, which is consistent with their proposed redundant function. Based on these findings, we analyzed the distribution of the lysine residues that are likely to be involved in cross-linking of OT subunits and propose that the OT subunits interact with each other through either their transmembrane domains and/or a region proximal to it, rather than through their luminal or cytoplasmic domains.     

Subunits of the translocon interact with components of the oligosaccharyl transferase complex

Following initiation of translocation across the membrane of the endoplasmic reticulum via the translocon, polypeptide chains are N-glycosylated by the oligosaccharyl transferase (OT) enzyme complex. Translocation and N-glycosylation are concurrent events and would be expected to require juxtaposition of the translocon and the OT complex. To determine whether any of the subunits of the OT complex and translocon mediate interactions between the two complexes, we initiated a systematic study in budding yeast using the split-ubiquitin approach. Interestingly, the OT subunit Stt3p was found to interact only with Sec61p, whereas another OT subunit, Ost4p, was found to interact with all three components of the translocon, Sec61p, Sbh1p, and Sss1p. The OT subunit Wbp1p was found to interact very strongly with Sec61p and Sbh1p and weakly with Sss1p. Other OT subunits, Ost1p, Ost2p, and Swp1p were found to interact with Sec61p and either Sbh1p or Sss1p. Ost3p exhibited a weak interaction with Sec61p and Sbh1p, whereas Ost5p and Ost6p interacted very weakly with Sec61p and failed to interact with Sbh1p or Sss1p. We were able to confirm these split-ubiquitin findings by a chemical cross-linking technique. Based on our findings using these two techniques, we conclude that the association of these two complexes is stabilized via multiple protein-protein contacts. Based on extrapolation of the structural parameters of the crystal structure of the prokaryotic Sec complex to the eukaryotic complex, we propose a working model to understand the organization of the translocon-OT supercomplex.     

Oligosaccharyltransferase: a complex multisubunit enzyme of the endoplasmic reticulum

The attachment of N-linked oligosaccharide chains to proteins is an important cotranslational process. These chains can, in some cases, serve to stabilize the protein, while in other cases they function as recognition elements. A key enzyme in the N-glycosylation process is oligosaccharyltransferase (OT). In yeast this enzyme, which is found in the endoplasmic reticulum, consists of nine different transmembrane protein subunits. Our general aim is to learn more about the functions of the multiple subunits of yeast OT and their mode of interaction with each other. Using a combination of biochemical and genetic techniques the subunit Ost1p has been shown to recognize Asn-X-Ser/Thr glycosylation sites. The principle tool used in the identification process was a benzophenone-based glycosylation site peptide that was shown to be crosslinked to Ost1p. Our current objective is to identify the domain in the primary structure that is involved in recognition of the glycosylation site sequence. By use of bifunctional crosslinkers, the possible interaction of Ost1p with other subunits of OT will be studied. This work and other studies on the OT subunits are concisely summarized.     

Studies on the N-glycosylation of the subunits of oligosaccharyl transferase in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, oligosaccharyl transferase (OT) consists of nine different subunits. Three of the essential gene products, Ost1p, Wbp1p, and Stt3p, are N-linked glycoproteins. To study the function of the N-glycosylation of these proteins, we prepared single or multiple N-glycosylation site mutations in each of them. We established that the four potential N-glycosylation sites in Ost1p and the two potential N-glycosylation sites in Wbp1p were occupied in the mature proteins. Interestingly, none of the N-glycosylation sites in these two proteins was conserved, and no defect in growth or OT activity was observed when the N-glycosylation sites were mutated to block N-glycosylation in either subunit. However, in the third glycosylated subunit, Stt3p, there are two adjacent potential N-glycosylation sites (N(535)NTWN(539)NT) that, in contrast to the other subunits, are highly conserved in eukaryotic organisms. Mass spectrometric analysis of a tryptic digest of Stt3p showed that the peptide containing the two adjacent N-glycosylation sites was N-glycosylated at one site. Furthermore, the glycan chain identified as Man(8)GlcNAc(2) is found linked only to Asn(539). Mutation experiments were carried out at these two sites. Four single amino acid mutations blocking either N-glycosylation site (N535Q, T537A, N539Q, and T541A) resulted in strains that were either lethal or extremely temperature sensitive. However, other mutations in the two N-glycosylation sites N(535)NTWN(539)NT (N536Q, T537S, N540Q, and T541S), did not exhibit growth defects. Based on these studies, we conclude that N-glycosylation of Stt3p at Asn(539) is essential for its function in the OT complex.     

Two oligosaccharyl transferase complexes exist in yeast and associate with two different translocons

Oligosaccharyl transferase (OT) scans and selectively glycosylates -Asn-X-Thr/Ser-motifs in nascent polypeptide chains in the endoplasmic reticulum (ER). Several groups have reported different results for the composition of this enzyme complex. In this study, using a membrane protein two-hybrid approach, the split-ubiquitin system, we show that except for Ost3p and Ost6p, all of the other subunits of OT exist as dimers or oligomers in the yeast, Saccharomyces cerevisiae. Ost3p and Ost6p behave strikingly similar in a series of genetic and biochemical assays, but clearly do not exist in the same OT complex. This observation, as well as the results in an accompanying study to analyze the composition of OT complex by blue native gel electrophoresis using a series of wild-type and mutant yeast strains strongly suggests that two isoforms of the OT complex exist in the ER, differing only in the presence of Ost3p or Ost6p. Each of these two isoforms of the OT complex specifically interacts with two structurally similar, but functionally different translocon complexes: the Sec61 and the Ssh1 translocon complexes.     

Structure of the oligosaccharyl transferase complex at 12 A resolution

Oligosaccharyl transferase (OT) catalyzes the transfer of a lipid-linked oligosaccharide to the nascent polypeptide emerging from the translocon. Currently, there is no structural information on the membrane-embedded OT complex, which consists of eight different polypeptide chains. We report a 12 A resolution cryo-electron microscopy structure of OT from yeast. We mapped the locations of four essential OT subunits through a maltose-binding protein fusion strategy. OT was found to have a large domain in the lumenal side of endoplasmic reticulum where the catalysis occurs. The lumenal domain mainly comprises the catalytic Stt3p, the donor substrate-recognizing Wbp1p, and the acceptor substrate-recognizing Ost1p. A prominent groove was observed between these subunits, and we propose that the nascent polypeptide from the translocon threads through this groove while being scanned by the Ost1p subunit for the presence of the glycosylation sequon.     

Studies on oligosaccharyl transferase in yeast

In yeast, OT consists of nine different subunits, all of which contain one or more predicted transmembrane segments. In yeast, five of these proteins are encoded by essential genes, Swp1p, Wbp1p, Ost2p, Ost1p and Stt3p. Four others are not essential Ost3p, Ost4p, Ost5p, Ost6p. All yeast OT subunits have been cloned and sequenced (Kelleher et al., 1992; 2003; Kelleher & Gilmore, 1997; Kumar et al., 1994; 1995; Breuer & Bause, 1995) and the structure of one of them, Ost4p, has been solved by NMR (Zubkov et al., 2004). Very recently, the preliminary crystal structure of the lumenal domain of an archaeal Stt3p homolog has been reported (Mayumi et al., 2007). Homologs of all OT subunits have been identified in higher eukaryotic organisms (Kelleher et al., 1992; 2003; Kumar et al., 1994; Kelleher & Gilmore, 1997).     

A novel and simple method of production and biophysical characterization of a mini-membrane protein, Ost4p: a subunit of yeast oligosaccharyl transferase

Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction. In eukaryotes, oligosaccharyl transferase (OT), a multi-subunit membrane-associated enzyme complex, catalyzes this reaction in newly synthesized proteins. In Saccharomyces cerevisiae, OT consists of nine nonidentical membrane proteins. Ost4p, the smallest subunit, bridges the catalytic subunit Stt3p with Ost3p. Mutation of transmembrane residues 18-24 in Ost4p has negative effect on OT activity, disrupts the Stt3p-Ost4p-Ost3p complex, results in temperature-sensitive phenotype, and hypoglycosylation. Heterologous expression and purification of integral membrane proteins are the bottleneck in membrane protein research. The authors report the cloning, successful overexpression and purification of recombinant Ost4p with a novel but simple method producing milligram quantities of pure protein. GB1 protein was found to be the most suitable tag for the large scale production of Ost4p. The cleavage of Ost4p conveniently leaves GB1 protein in solution eliminating further purification. The precipitated pure Ost4p is reconstituted in appropriate membrane mimetic. The recombinant protein is highly helical as indicated by the far-UV CD spectrum. The well-dispersed heteronuclear single quantum coherence spectrum indicates that this minimembrane protein is well-folded. The successful production of pure recombinant Ost4p with a novel yet simple method may have important ramification for the production of other membrane proteins.     

A specific segment of the transmembrane domain of Wbp1p is essential for its incorporation into the oligosaccharyl transferase complex

Wbp1p, a type I transmembrane protein, is an essential component of oligosaccharyl transferase (OT), which consists of nine different subunits in yeast. It has been proposed that three subunits, Wbp1p, Ost2p, and Swp1p, physically interact with each other, but the mechanism of these interactions is unknown. To explore the mode of interaction, we have focused on the single-transmembrane protein, Wbp1p, and made several deletions and mutations within the short cytosolic domain and the transmembrane domain. Our results show that the deletion of the cytosolic domain has no effect on cell growth, but mutation of all 17 amino acids in the transmembrane domain to 17 Leu residues or replacement of the transmembrane and cytosolic domains with the counterparts of Ost1p results in lethality. Immunoprecipitation experiments show that Wbp1p mutated in these two ways is not incorporated into the OT complex. This finding suggests that the transmembrane domain of Wbplp may mediate its association with the other subunits. A series of mutations of the transmembrane domain have revealed that block alterations in the half of the transmembrane domain facing the lumen of the endoplasmic reticulum (ER) impaired cell viability. Seven single-Lys mutants in the same domain were temperature sensitive for growth at 37 degrees C. In contrast, block mutations in the other half of the transmembrane domain facing the cytosol did not result in lethality and indicated that this portion of the transmembrane domain was not involved in stable incorporation of Wbp1p into the OT complex.     

Studies on the function of oligosaccharyl transferase subunits. Stt3p is directly involved in the glycosylation process

In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT) is composed of nine different transmembrane proteins. Using a glycosylatable peptide containing a photoprobe, we previously found that only one essential subunit, Ost1p, was specifically labeled by the photoprobe and recently have shown that it does not contain the recognition domain for the glycosylatable sequence Asn-Xaa-Thr/Ser. In this study we utilized additional glycosylatable peptides containing two photoreactive groups and found that these were linked to Stt3p and Ost3p. Stt3p is the most conserved subunit in the OT complex, and therefore 21 block mutants in the lumenal region were prepared. Of the 14 lethal mutant proteins only two, as well as one temperature-sensitive mutant protein, were incorporated into the OT complex. However, using microsomes prepared from these three strains, the labeling of Ost1p was markedly decreased upon photoactivation with the Asn-Bpa-Thr photoprobe. Based on the block mutants single amino acid mutations were prepared and analyzed. From all of these results, we conclude that the sequence from residues 516 to 520, WWDYG in Stt3p, plays a central role in glycosylatable peptide recognition and/or the catalytic glycosylation process.     

Protein-protein interactions in the subunits of ribonuclease P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of RNA and five protein subunits (Ph1481p, Ph1496p, Ph1601p, Ph1771p, and Ph1877p). In vivo interactions among five protein subunits of RNase P in P. horikoshii OT3 were examined using a yeast two-hybrid system. The analysis indicates that proteins Ph1481p and Ph1601p interact strongly with Ph1877p and Ph1771p respectively, whereas Ph1481p interacts moderately with Ph1601p. In contrast, no interaction was detected between Ph1496p and the other four proteins. Co-immunoprecipitation analysis confirmed the interactions obtained by yeast two-hybrid assay.     

Assembly of the Yeast Vacuolar Proton-Translocating ATPase

The yeast vacuolar proton-translocating ATPase (V-ATPase) is the bestcharacterized member of the V-ATPase family. Biochemical and genetic screensled to the identification of a large number of genes in yeast, designatedVMA, encoding proteins required to assemble a functional V-ATPase. Atotal of thirteen genes encode subunits of the final enzyme complex. Inaddition to subunit-encoding genes, we have identified three genes that codefor proteins that are not part of the final V-ATPase complex yet required forits assembly. We refer to these nonsubunit Vma proteins as assembly factors,since their function is dedicated to assembling the V-ATPase. The assemblyfactors, Vma12p, Vma21p, and Vma22p are localized to the endoplasmicreticulum (ER) and aid the assembly of newly synthesized V-ATPase subunitsthat are translocated into the ER membrane. At least two of these proteins,Vma12p and Vma22p, function together in an assembly complex and interactdirectly with nascent V-ATPase subunits.     

Identification of Sec36p,Sec37p,and Sec38p: components of yeast complex that contains Sec34p and Sec35p

The Saccharomyces cerevisiae proteins Sec34p and Sec35p are components of a large cytosolic complex involved in protein transport through the secretory pathway. Characterization of a new secretion mutant led us to identify SEC36, which encodes a new component of this complex. Sec36p binds to Sec34p and Sec35p, and mutation of SEC36 disrupts the complex, as determined by gel filtration. Missense mutations of SEC36 are lethal with mutations in COPI subunits, indicating a functional connection between the Sec34p/sec35p complex and the COPI vesicle coat. Affinity purification of proteins that bind to Sec35p-myc allowed identification of two additional proteins in the complex. We call these two conserved proteins Sec37p and Sec38p. Disruption of either SEC37 or SEC38 affects the size of the complex that contains Sec34p and Sec35p. We also examined COD4, COD5, and DOR1, three genes recently reported to encode proteins that bind to Sec35p. Each of the eight genes that encode components of the Sec34p/sec35p complex was tested for its contribution to cell growth, protein transport, and the integrity of the complex. These tests indicate two general types of subunits: Sec34p, Sec35p, Sec36p, and Sec38p seem to form the essential core of a complex to which Sec37p, Cod4p, Cod5p, and Dor1p seem to be peripherally attached.     

Functional mapping of the fission yeast DNA polymerase δ B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis

Background  

DNA polymerase δ plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol δ is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol δ comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively.     

Insight into functional aspects of Stt3p, a subunit of the oligosaccharyl transferase. Evidence for interaction of the N-terminal domain of Stt3p with the protein kinase C cascade

Over a decade ago, the gene STT3 was identified in a staurosporine and temperature sensitivity screen of yeast. Subsequently the product of this gene was shown to be a subunit of the endoplasmic reticulum-localized oligosaccharyl transferase (OT) complex. Although stt3 mutants are known to be staurosporine-sensitive, we found that mutants of other OT subunits (except ost4 Delta) are staurosporine-resistant, which indicates that this phenotype of stt3 mutants is not simply a consequence of their defect in glycosylation, as previously speculated. Staurosporine sensitivity was found to be an allele-specific phenotype restricted to cells harboring mutations in highly conserved residues in the N-terminal domain of the STT3 protein. Cells bearing mutations in one of the cytosolic-oriented loops (amino acids 158-168) in the N terminus of Stt3p were found to be specifically susceptible to staurosporine. Staurosporine is a specific inhibitor of Pkc1p, and a genetic link had previously been suggested between PKC1 and STT3. It is known that overexpression of PKC1 suppresses the staurosporine sensitivity of the stt3 mutants in an allele-specific manner, which is typical of mutants of Pkc1p cascade. It has been shown that the pkc1 null mutant exhibits lowered OT activity. Our results combined with these previous observations indicate that the N-terminal domain of Stt3p may interact with members of the Pkc1p cascade and consequently mutations in this domain result in staurosporine sensitivity. We further speculate that the Pkc1p regulates OT activity through the N-terminal domain of Stt3p, the C-terminal domain of which possesses the recognition and/or catalytic site of the OT complex.     

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification,apoptosis, and localized lipid peroxidation

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O₂^?-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91^phox and p22^phox) forming the catalytic core, three cytosolic proteins (p67^phox, p47^phox and p40^phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called ‘Trimera’, composed of the essential domains of the cytosolic proteins p47^phox (aa 1–286), p67^phox (aa 1–212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.     

Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping

Decapping is a key step in mRNA turnover. However, the composition and regulation of the human decapping complex is poorly understood. Here, we identify three proteins that exist in complex with the decapping enzyme subunits hDcp2 and hDcp1: hEdc3, Rck/p54, and a protein in decapping we name Hedls. Hedls is important in decapping because it enhances the activity of the catalytic hDcp2 subunit and promotes complex formation between hDcp2 and hDcp1. Specific decapping factors interact with the mRNA decay activators hUpf1 and TTP, and TTP enhances decapping of a target AU-rich element (ARE) RNA in vitro. Each decapping protein localizes in cytoplasmic processing bodies (PBs), and overexpression of Hedls produces aberrant PBs and concomitant accumulation of a deadenylated ARE-mediated mRNA decay intermediate. These observations suggest that multiple proteins involved in human decapping are important subunits of PBs and are activated on ARE-mRNAs by the protein TTP.     

The yeast Ccr4-Not complex controls ubiquitination of the nascent-associated polypeptide (NAC-EGD) complex

In this work, we determine that the Saccharomyces cerevisiae Ccr4-Not complex controls ubiquitination of the conserved ribosome-associated heterodimeric EGD (enhancer of Gal4p DNA binding) complex, which consists of the Egd1p and Egd2p subunits in yeast and is named NAC (nascent polypeptide-associated complex) in mammals. We show that the EGD complex subunits are ubiquitinated proteins, whose ubiquitination status is regulated during cell growth. Egd2p has a UBA domain that is not essential for interaction with Egd1p but is required for stability of Egd2p and Egd1p. Ubiquitination of Egd1p requires Not4p. Ubiquitination of Egd2p also requires Not4p, an intact Not4p RING finger domain, and all other subunits of the Ccr4-Not complex tested. In the absence of Not4p, Egd2p mislocalizes to punctuate structures. Finally, the EGD complex can be ubiquitinated in vitro by Not4p and Ubc4p, one of the E2 enzymes with which Not4p can interact. Taken together our results reveal that the EGD ribosome-associated complex is ubiquitinated in a regulated manner, and they show a new role for the Ccr4-Not complex in this ubiquitination.