Glycobiology, 2000, Vol. 10, No. 7 737-744
© 2000 Oxford University Press
Evidence for interaction of yeast protein kinase C with several subunits of oligosaccharyl transferase
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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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.
Key words: asparagine-linked glycosylation/endoplasmic reticulum/Oligosaccharyltransferase/protein kinase C
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Introduction |
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Although the in vitro N-glycosylation of unfolded proteins and nascent polypeptides in yeast as well as in higher eukaryotes has been studied over the past several decades (Pless and Lennarz, 1977
; Bause, 1984; Gavel and von Heijne, 1990; Herscovics and Orlean, 1993; Allen et al., 1995; Holst et al., 1996), it has only been in the last decade that the enzyme oligosaccharyltransferase (OT) has actually been isolated and characterized at the molecular level. Remarkably, this enzyme, unlike other membrane-associated glycosyl transferases, was found in both yeast and mammals to consist of a complex of unique polypeptides, ranging from 4 in mammals to 8 (or 9) in yeast (Kelleher et al., 1992; Silberstein and Gilmore, 1996; Spirig et al., 1997; Sanjay et al., 1998; Knauer and Ludwig, 1999). This conclusion was based on experiments involving detergent solubilization followed by immunoprecipitation of a complex containing all of the subunits. The idea of a complex was initially suggested by a number of genetic experiments in yeast. In the first of these it was established that a temperature-sensitive defect in a mutant of one of the subunits, WBP1, was suppressed by overexpression of another subunit, SWP1. Subsequent experiments showed that Wbp1p and Swp1p can be biochemically cross-linked, indicating that these two proteins physically interact (te Heesen et al., 1993). Recently, it has been established that a complex of S. cerevisiae OT isolated by immunoprecipitation contains all 8 known subunits and that there are three subcomplexes within the OT complex; Ost1p-Ost5p, Ost2p-Wbp1p-Swp1p, and Stt3p-Ost4p-Ost3p (Karaoglu et al., 1997; Spirig et al., 1997). In addition to these biochemical studies, a variety of genetic studies are consistent with interactions between the subunits (Silberstein et al., 1995; Zufferey et al., 1995; Reiss et al., 1997; Spirig et al., 1997). These findings of physical interactions correlate well with the results of genetic interaction studies.
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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Specific interaction of Pkc1p with several OT subunits
After extensive two-hybrid library screening (>1 x 107 transformants/library) using plexA-WBP1 as the initial target plasmid, we recovered four GAD-library plasmids that showed a His+ ß-galactosidase+ phenotype; two of them showed the same phenotype when re-transformed into L4O, the two-hybrid host strain containing the original target plasmid. Further, these two plasmids showed interaction with two different lexA-WBP1 plasmids (Figure 1). These candidate library plasmids also showed interaction with a lexA-STT3 plasmid, but not with a lexA-OST1 plasmid.
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Sequencing of the inserts from two candidate library DNAs showed that they consisted of two different Pkc1p fragments fused in-frame to the GAD sequence (Figure 2A,B). From the two-hybrid library screening using plexA-SWP1 as the target, one transformant showed the same His+ ß-galactosidase+ phenotype (data not shown), and was found to consist of another Pkc1p fragment fused in frame to the GAD (Figure 2C). Thus, several Pkc1p fragments spanning similar regions of the ORF, and lacking the kinase active site, showed interaction with three different subunits of OT: Wbp1p, Swp1p, and Stt3p. They did not show interaction with three other presumably irrelevant S.cerevisiae proteins fused to lexA, namely, topo I, Sir4p, and lamin. Both lexA-lamin and lexA-SIR4 (which contains the C-terminal domain of Sir4p) have been used to test for potential false positives in two-hybrid library screening (Park and Sternglanz, 1998
). These negative controls were especially important since two of these proteins (lamin and Sir4p) contain coiled-coil motifs that mediate protein–protein interactions (Fields and Sternglanz, 1994).
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Growth dependence between PKC1 and OT
To further investigate functional relationships between PKC1 and OT, genetic interactions between PKC1 and two of OT subunits, WBP1 and SWP1, were explored in co-overexpression experiments (Figure 3). Conditions were established so that PKC1 expression was under the control of the GAL1 promoter (pl293 and pl295) and WBP1 expression was dependent on the MET25 promoter (pHP167). Two different forms of Pkc1p were tested: (1) wild-type Pkc1p, (2) a kinase mutant, pkc1-K853R, that has a point mutation at a potential ATP-binding site in the kinase domain and lacks kinase activity (Watanabe et al., 1994
). This mutant protein cannot complement the cell lysis defect of pkc1 null mutant. No impairment of growth was observed when either Pkc1p, Wbp1p, or Swp1p alone was overexpressed in a wild-type strain (Figure 3A, rows 4, 2, and 3, respectively). Also, co-overexpression of Pkc1p and Wbp1p or Swp1p showed no change in growth rate (rows 5 and 6). In contrast, overexpression of the catalytically inactive point mutant, pkc1-K853Rp, in a wild-type (PKC1) background resulted in a growth defect on -ura-trp-met media supplemented with galactose (the condition for overexpression of Pkc1p) (row 7). However, co-overexpression of Wbp1p or Swp1p in cells overexpressing the kinase defective pkc1-K853Rp suppressed the growth defect in these cells (rows 8 and 9). This may be explained by the finding by Western blot analysis that overexpression of Wbp1p or Swp1p in transformants overexpressing pkc1-K853Rp resulted in a reduced level of the mutant kinase (Figure 3B).
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Several of the OT subunits bind to Pkc1p in vitro
To determine if these in vivo findings could be validated using in vitro methods, we fused GST to the N-terminus of one of the Pkc1p fragments isolated from two-hybrid library screening using lexA-WBP1 as the target (candidate A in Figure 2). After binding this fusion protein to glutathione beads we carried out in vitro binding experiments to ask if HA-tagged Stt3p, Wbp1p, Swp1p, and Ost1p could be shown to bind to the immobilized fusion protein. In the case of Stt3p-HA the glutathione agarose beads were washed three times with PBS buffer after addition of Stt3HAp, whereas for Ost1HAp, Wbp1HAp, and Swp1HAp, the beads were washed three to four times with PBS buffer containing 1% Triton X-100. As shown in Figure 4, in all cases a significant level of in vitro binding of these four OT subunits to immobilized GST-Pkc1p was detected (lane 2 in Figure 4A and 1anes 2, 5, and 8 in Figure 4B) when compared to control beads containing either no GST-Pkc1p (lane 1 in Figure 4A and lanes 1, 4, and 7 in Figure 4B) or GST alone (lane 3 in Figure 3A, and lanes 3, 6, and 9 in Figure 4B).
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Microsomes from a pkc1 null mutant strain exhibit reduced OT activity
Having obtained evidence by both in vivo and in vitro assays that Pkc1p interacts with several of the OT subunits, we asked if these interactions affect in vitro protein glycosylation activity. To do this, microsomes were prepared from a pkc1 null mutant strain (DL394) and a wild-type strain (DL102). Microsomes were prepared from each strain and compared with respect to their ability to catalyze peptide glycosylation. Because growth of the null strains requires an osmotic stabilizer, all of the strains were grown in YPAD liquid media supplemented with 1 M sorbitol to normalize the culture conditions. As shown in Figure 5, microsomes of the pkc1 null mutant contained only about 50% of the activity OT found in an equal quantity of microsomes from wild-type strain. Further, when two specific protein kinase C inhibitors, staurosporine or bis-indolylmaleimide (GF 109203X), were added to wild-type microsomes, a 50% reduction in OT activity was observed. Treatment with either drug had no effect on the OT activity of microsomes from the pkc1 null mutant. Thus, the inhibition observed with PKC inhibitors lowered the OT activity to the same basal level seen with the pkc1 null mutant. To test if known kinases downstream of the PKC1-MAP kinase pathway had any effect on OT activity, microsomes from a bck1 null mutant (DL251) and a mpk1 mutant (DL456) were analyzed for their ability to catalyze in vitro peptide glycosylation. As shown in Figure 5, microsomes from both mutants were found to have OT activity comparable to that of microsomes from the wild-type strain. The OT activity of both these mutants was inhibited 50% by the PKC inhibitors (staurosporine, bis-indolylmaleimide), as expected because both contain wild-type Pkc1p.
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Discussion |
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In the current study we initially carried out a yeast two-hybrid library screen in an effort to identify potential proteins that interact with the various OT subunits. We used the lumenal domains of several OT subunits as "targets." Because several of the OT subunits have long lumenal domains and very short cytoplasmic tails (e.g., Wbp1p, Stt3p, Ost1p, and Swp1p), it seems reasonable that these lumenal domains might be involved in protein–protein interactions. In the case of mammalian OT, Fu et al. (1997)
using lumenal domains for direct two-hybrid assays, showed that OST48 (a homolog of yeast Wbp1p) interacts with ribophorin I (a homolog of yeast Ost1p) and ribophorin II (Swp1p homolog), and that DAD1 (Ost2p homolog) interacts with OST48. Using extensive two-hybrid library screening or direct two-hybrid assays, we failed to detect lumenal interactions among the yeast OT subunits. However, to our surprise, we identified two Pkc1p fragments that interacted with the lumenal domain of Wbp1p; in another series of experiments these same fragments were found to interact with Stt3p. Yet another Pkc1p fragment was isolated when a library screening was performed using the lumenal domain of another OT subunit, Swp1p. All three Pkc1p fragments were found to overlap and have in common the absence of the C-terminal domain that contains the kinase active site. We later found that the lumenal domain of Ost3p also showed two-hybrid interaction with the same Pkc1p fragments (data not shown).
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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The results of the two-hybrid experiments, the in vitro binding experiments, and the effect of Pkc1p on OT activity measured in vitro suggest that Pkc1p interacts with the lumenal domains of several OT subunits and these interactions modulate OT activity. Clearly, for direct physical interaction, Pkc1p would have to be present in the ER lumen. There have been several studies suggesting a kinase activity in the lumen of the ER (Cala and Jones, 1994
; Chen et al., 1996) and some ER lumenal proteins are known to be phosphorylated (Quemeneur et al., 1994; Csermely et al., 1995). Although studies on mammalian PKCs have suggested that some isoforms can be localized in the ER (Chida et al., 1994; Goodnight et al., 1995), and one of the sequence analysis programs (TopPred2: http://www.biokemi.su.se/~server/toppred2/toppredServer.cgi) predicts a transmembrane segment in yeast Pkc1p, there is no direct evidence for the presence of this kinase in the lumen of the yeast ER. In fact, in yeast there has been no systematic study of the precise subcellular localization of Pkc1p. Subcellular fractionation to resolve this issue appears to be difficult, since it has been reported that Pkc1p is present in the particulate fraction and cannot be liberated by mild detergent treatment (Antonsson et al., 1994; Watanabe et al., 1994). These findings have led to the proposal that it is a cytoplasmic protein bound to the cytoskeleton. Clearly, the next phase in our studies must be to determine if any Pkc1p is present in the lumen of endoplasmic reticulum and, if so, to determine exactly how Pkc1p is involved in an interaction with the multiple subunits of OT that results in modulation of its activity in peptide glycosylation. |
Materials and methods |
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Yeast strains, media and handling
Strain L40 (MATa ade2 leu2 ura3 his3 trp1 URA3::lexAop-LacZ LYS2::lexAop-HIS3) (Hollenberg et al., 1995
) was used for the two-hybrid experiments. Strain DL102 (MAT a/
leu2 trp1 ura3 his4 can1r) was the diploid parental strain for a pkc1 null (pkc1::LEU2, DL394) (Watanabe et al., 1994), a bck1 null (bck1::URA3, DL251) (Lee and Levin , 1992), and a mpk1 null (mpk1::TRP1, DL456) (Lee et al., 1993). DL251, DL394, DL456, and DL102 strains were generous gifts from Dr. David E.Levin of Johns Hopkins University. W303–1a (MATa ade2 leu2 trp1 his4 ura3 can1r) and W303–1b (W303–1a MAT) strains were used as wild-type strains. For yeast growth and other experiments, we followed previously described methods (Guthrie and Fink, 1991).
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-WBP146–321, plexA-STT3466–718, and plexA-SWP120–195. For in vitro binding assays, pGST-PKC1114–880 was constructed from a library DNA isolated from two-hybrid screening using plexA-WBP146–321 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, SWP125–195-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 3–4 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 SDS–PAGE 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 Tris–HCl, 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 Tris–HCl, 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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We thank Drs. Rolf Sternglanz and Aaron Neiman for suggestions and critical reading of the manuscript. We thank Dr. David E.Levin for various PKC1 plasmids and strains, and Ms. Qi Yan for the assay of OT activity. This work was supported by National Institutes of Health Grant GM33185 to W.J.L.
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Footnotes |
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1 To whom correspondence should be addressed
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References |
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