Department of Biochemistry and Cell Biology, and Institute for Cell and Developmental Biology, SUNY at Stony Brook, Stony Brook, New York 11794, USA
Received on December 10, 1999; revised on February 3, 2000; accepted on February 3, 2000.
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Abstract |
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Key words: asparagine-linked glycosylation/endoplasmic reticulum/Oligosaccharyltransferase/protein kinase C
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Introduction |
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Our overall interest is to determine the mode of interaction of the subunits of OT and their function. Recently we have studied the function of Ost1p in glycosylation site recognition (Yan et al., 1999). With respect to the former issue, we took the yeast two hybrid approach to determine if we could detect subunit interaction of the OT subunits with each other, or with other proteins. To do this we initially used the lumenal domain of Wbp1p as a "target" and asked if protein(s) in several yeast DNA libraries could be found to interact with the target and thereby activate reporter genes (ß-galactosidase, HIS3). For a review of the two-hybrid system, see Fields and Sternglanz (1994)
. Using this approach we were very surprised to find that in two different genomic DNA libraries the only protein found to interact with Wbp1p in library screening was Pkc1p, the yeast homolog of mammalian protein kinase C (Antonsson et al., 1994
; Watanabe et al., 1994
). Three different but overlapping segments of Pkc1p were found to interact specifically with several OT subunits, namely Wbp1p, Swp1p, Stt3p, Ost1p, and Ost3p. In yeast, Pkc1p is involved in signal transduction pathways and in its absence cell wall defects due to impairment of glycan synthesis result (for review, see Mellor and Parker, 1998
). In addition to the two-hybrid and other in vivo genetic experiments, we carried out several types of in vitro experiments. In the first of these we found evidence for interaction of Pkc1p and several of the subunits in vitro. Furthermore, we found that Pkc1p, previously known to be involved in the MAP kinase signal transduction pathway that modulates yeast cell wall synthesis, affected the activity of OT since microsomes from a pkc1 null strain had only about 50% of the OT activity of the wild-type, as measured in vitro using the peptide glycosylation assay. In contrast null mutations in two kinases located downstream of Pkc1p in this pathway, bck1 and mpk1, had no effect on OT activity. Taken together, these findings suggest that Pkc1p may regulate the activity of the N-glycosylation of proteins.
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Results |
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Discussion |
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Since Pkc1p, the yeast homolog of mammalian protein kinase C, has been shown to be involved in the MAP kinase signaling pathway (Lee et al., 1993; Levin et al., 1994
), it has been assumed that Pkc1p is exclusively a cytoplasmic enzyme. If this were the case it would be very difficult to understand how it could interact with one or more of the lumenal domains of OT. In this context it is of interest that in the case of mammalian systems, although protein kinase C isoforms have not been shown directly to be present in the lumen of the ER, there are several reports that certain isoforms of PKC become associated with the ER upon activation (for example, by adding phorbol ester) (Chida et al., 1994
; Goodnight et al., 1995
).
Interestingly, STT3, the gene that encodes Stt3p, is a member of a family of ten STT genes. One of the members of the family, STT1, was subsequently shown to be PKC1 (Yoshida et al., 1992). STT3 was first identified as one of the ten STT genes; an stt3-2 mutation exhibited osmotic fragility, as well as staurosporine (a specific protein kinase C inhibitor) sensitivity and temperature sensitivity (stt). The osmotic phenotype could be overcome by 1 M sorbitol and the stt phenotype could be overcome by overexpression of PKC1 (Yoshida et al., 1995
). Independently, the STT3 gene was shown to encode a subunit of OT (Zufferey et al., 1995
). Thus, these earlier studies already provided genetic evidence for interaction between the genes encoding Pkc1p and Stt3p.
To further test for the functional interaction between Pkc1p and OT, we took a genetic approach using PKC1 and two of the OT subunits, WBP1 and SWP1. Wbp1p has been implicated to play an important role in the catalytic activity of OT and to interact with several other OT subunits (te Heesen et al., 1993). Overexpression of both Pkc1p and either Wbp1p or Swp1p in a wild-type yeast strain had no effect on the growth rate. On the other hand, overexpression of a Pkc1 kinase mutant (pkc1[K893R]p) in a wild-type Pkc1p background impaired growth, and this defect was reversed when either Wbp1p or Swp1p was co-overexpressed. This result implies that an excess of the mutant that is inactive in kinase activity disrupts the interaction between the wild-type Pkc1p and OT components, resulting in decreased OT activity and impaired growth. On the other hand, co-overexpression of either Wbp1p or Swp1p may sequester the overexpressed mutant protein and thereby restored normal OT activity and growth. Interestingly, we found that pkc1[K853R]p had a much reduced level when Wbp1p or Swp1p were co-overexpressed. Thus, it appears that overexpression of these OT subunits does not merely sequester the mutant kinase, but promotes its degradation. No such down-regulation (or impairment of growth) was observed when wild-type Pkc1p was co-overexpressed (data not shown). How regulation of the level of pkc1[K853R]p is controlled by OT subunits will be an interesting question to answer.
Another important question that remains unanswered is why interaction between the various subunits of OT could not be detected by the two-hybrid approach using the lumenal domain of the target protein. A simple explanation may be that the lumenal domains of the proteins tested are not involved in the interactions between the subunits of OT that have been detected by immunological methods (e.g., immunoprecipitation of OT complex). This would imply that these interactions are mainly mediated by the transmembrane and/or the cytoplasmic domains of these subunits. In fact, in the case of glycophorin, which exists as a dimer in the red blood cell membrane, the transmembrane domain has been shown to mediate the dimerization (Lemmon et al., 1992). Also, we have found that mutations in the transmembrane domain of Ost4p disrupt its interactions with two other OT subunits, Stt3p and Ost3p (Kim et al., 2000
). As noted earlier, Fu et al. (1997)
did detect two hybrid interactions among mammalian OT subunits using lumenal domain constructs. Why we could not detect interactions between the yeast homologs using the library screen approach is not clear. One explanation is that the limited lumenal domains of the rat proteins used as probes by Fu et al. (1997)
have low sequence similarity to their counterparts in yeast, and that in this simple eukaryote the subunit interactions may not occur via the lumenal domains, where Pkc1p binds, but in the transmembrane domains.
Having obtained genetic evidence for interactions between Pkc1p and OT, we turned to in vitro binding experiments using a GST-Pkc1 fusion protein and various OT subunits. Specific binding was observed with the same three subunits that had been detected in the two-hybrid system, namely, Wbp1p, Swp1p, and Stt3p. In addition, using this assay it was found that full length Ost1p was able to bind to Pkc1p. This finding is in contrast to the negative result obtained in the two-hybrid experiments, in which we used the lumenal domain of Ost1p instead of the full length protein. Perhaps the truncated protein (lexA-OST1) has an altered conformation that lacked binding activity. Since Ost1p is predicted to have a very short C-terminal cytoplasmic domain, it seems likely that it is the lumenal domain of Ost1p that interacts with Pkc1p.
In S.cerevisiae there is only a single Pkc1p that is a homolog of mammalian protein kinase C, which consists of many different isoforms in higher organisms. In mammals the various protein kinase C isoforms participate in different cellular functions depending on specific cell types. Perhaps in the case of yeast the single Pkc1p carries out diverse functions. For example, Pkc1p is known to be involved in the MAP kinase pathway in yeast, and mutations in the genes involved in this pathway that are downstream of PKC1 exhibit cell lysis defects like that observed in the pkc1 mutant. However, because these defects of the downstream proteins are less severe than those of pkc1 mutants, it has been suggested that there are yet still other unknown functions of Pkc1p (Mellor and Parker, 1998). Indeed earlier it had been proposed that PKC1 genetically interacts with STT3 and thereby regulates its function (Yoshida et al., 1995
). Since OT, a key enzyme for N-glycosylation, is involved in assembly of cell wall glycans, Pkc1p might function to regulate OT activity. In fact, it has been reported that PKC1 is involved in the regulation of several cell wall biosynthetic genes in yeast (Igual et al., 1996
). By studying peptide glycosylation in vitro using microsomes prepared from a pkc1 null mutant we found that functional Pkc1p is required for maximal OT activity. Microsomes prepared from a pkc1 null mutant exhibited only 50% of the activity of wild-type microsomes. This same level of inhibition was observed with microsomes from the wild-type strain when two different kinase inhibitors were added to in vitro OT assays. This clearly was the result of down-regulation of OT to a basal level of activity since (1) no further inhibition was observed with wild microsomes when the inhibitor concentration was increased 2-fold and (2) the basal value of 50% OT activity observed in the kinase null strain was not lowered further by addition of the inhibitors. In contrast to the pkc1 null mutant, the microsomes of two strains containing null mutations in two kinases downstream of Pkc1p, Bck1p and Mpk1p, had wild-type OT activity, indicating that the interaction with OT is specific for Pkc1p. This suggests that Pkc1p may be at a branch point, not only being a component of the MAP kinase pathway, but also participating in modulation of OT activity (Figure 6). However, it is clear that Pkc1p is not absolutely essential for OT activity, because even without Pkc1p, OT activity is at a level 50% of wild type.
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Materials and methods |
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Plasmid and library DNAs
pGAD1, 2, and 3 yeast genomic DNA libraries were used for yeast two-hybrid screening (James et al., 1996). Library DNAs were kindly provided by Dr. Philip James of University of Wisconsin. Target plasmids for two-hybrid screenings were as follows: plexA-WBP146321, plexA-STT3466718, and plexA-SWP120195. For in vitro binding assays, pGST-PKC1114880 was constructed from a library DNA isolated from two-hybrid screening using plexA-WBP146321 as a target. For overexpression of OT subunits, OST1, WBP1, and STT3 were HA-tagged and then subcloned into a pET vector. For Swp1p overexpression, SWP125195-HA was subcloned into the pET vector. Two PKC1 plasmids were gifts from Dr. David E.Levin of the Johns Hopkins University. They were pl293 (pGAL-PKC1::HA) and pl295 (pGAL-pkc1[K853R]::HA) (Watanabe et al., 1994
). pHP166 is a pGFP-C-FUS (Niedenthal et al., 1996
) derivative in which URA3 marker is replaced with TRP1 marker. pHP167 (pMET25-WBP1::GFP) was constructed from pHP166 for studies in the cellular localization of Wbp1p as well as genetic experiments. pHP170 (pMET25-SWP1, constructed from pHP166) was used for genetic interaction study.
Yeast two-hybrid library screening
The two-hybrid experiments were performed as previously described (Park and Sternglanz, 1998) with some modifications. Strain L4O was transformed with plexA-WBP1 (target plasmid) and a two-hybrid genomic DNA library (GAD-library), and His+ ß-galactosidase+ transformants identified. Target plasmids and library GAD plasmids were isolated from the transformants and used for retransformation. When the His+ ß-galactosidase+ phenotype was reproduced from the retransformants of the original target plasmid and the GAD-library DNA, the library DNAs were pursued further. Library DNAs were then transformed into strain L4O along with each of several plasmids encoding various lexA-hybrids. Only those candidates that showed the His+ phenotype with the original target plasmid, and not with other unrelated lexA-hybrids, were sequenced and identified as specific candidates. In case of library screening using plexA-SWP1 as target, 25mM 3-aminotriazole was added to the screening plates to suppress HIS3 transcription because lexA-SWP1 itself showed weak transcriptional activation of the reporter gene. The other OT subunits tested showed no transcriptional activation.
Yeast cell lysis
Yeast cells were collected either from liquid media or from the surface of plate. Collected cells were resuspended in yeast lysis buffer (20 mM Tris-Cl, pH 7.5, 100 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF). Acid-washed glass beads equivalent to 1/3 volume of the cell suspension were added and the cells were disrupted by sonication (three times for 15 s, with 20 sec intervals on ice). After centrifugation supernatants were collected and subjected for Western blot analysis.
In vitro binding assay
In vitro binding assays were performed as previously described (Park and Sternglanz, 1999) with slight modifications. Purified GST-Pkc1p was mixed with glutathione agarose beads and unbound GST-Pkc1p was removed by washing the beads. Then E.coli lysates containing various different overexpressed subunits of OT (or lumenal domains of these subunits) having 3 HA epitopes at their C-termini were added to the beads. Following overnight incubation at 4°C, the beads were washed 34 times with PBS buffer with or without 1% Triton X-100, depending upon the different OST-HAs. To detect bound OT subunits, SDS sample buffer was added to the beads. After heating at 100°C for 5 min, the supernatant fractions recovered from the beads were subjected to SDSPAGE and then transferred to nitrocellulose filter paper. OST-HA proteins were detected on the nitrocellulose using anti-HA antibody (12CA5).
In vitro peptide glycosylation assay (OT assay)
Microsomes were prepared as previously described (Brodsky and Schekman, 1993) and then resuspended in glycosylation buffer (50 mM TrisHCl, pH 7.4, 10 mM MnCl2, 1 mM 2-mercaptoethanol). [3H]Ac-Asn-BPA-Thr-amide acceptor peptide (Yan et al., 1998
) was then added and the glycosylation reaction was allowed to proceed for 20 min at room temperature. To assess formation of [3H]-labeled glycopeptide the reaction was stopped by addition of concanavalin A agarose beads (Sigma) diluted 1:1 with ConA buffer (50 mM TrisHCl, pH 7.5, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, 1% NP-40). The beads were washed three times with ConA buffer and glycopeptide formation was quantitated by determining the amount of [3H]-labeled glycopeptide bound to the beads by counting them in a liquid scintillation counter (LKB).
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Acknowledgments |
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Footnotes |
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References |
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