Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M403234200 on September 17, 2004

J. Biol. Chem., Vol. 279, Issue 48, 49762-49772, November 26, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/48/49762    most recent
M403234200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakatsukasa, K.
Right arrow Articles by Endo, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakatsukasa, K.
Right arrow Articles by Endo, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Roles of O-Mannosylation of Aberrant Proteins in Reduction of the Load for Endoplasmic Reticulum Chaperones in Yeast*

Kunio Nakatsukasa{ddagger}§, Shigeo Okada{ddagger}, Kyohei Umebayashi¶, Ryoichi Fukuda||, Shuh-ichi Nishikawa{ddagger}, and Toshiya Endo{ddagger}**{ddagger}{ddagger}§§

From the {ddagger}Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan, the Department of Cell Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan, the ||Department of Biotechnology, the University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, **Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan, and the {ddagger}{ddagger}Institute for Advanced Research, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan

Received for publication, March 23, 2004 , and in revised form, August 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ({Delta}pro) 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The endoplasmic reticulum (ER)1 is the entrance for the protein secretory pathway in eukaryotic cells. Nascent secretory and membrane proteins translocating into the ER undergo folding, oligomerization, and maturation, including glycosylation and disulfide bridge formation, with the aid of molecular chaperones and folding enzymes. Quality control mechanisms in the ER monitor these processes to ensure that defective proteins that failed to acquire correct functional structures are not deployed throughout the cells (1). Aberrant proteins are continuously exposed to chaperones and folding enzymes to achieve their native conformations. If this protein repair is not successful, aberrant proteins are cleared from the ER by a mechanism called ER-associated degradation (ERAD) (2, 3).

Nonnative proteins have such features as the exposure of hydrophobic regions, exposed free cysteines, and the tendency to aggregate. These features are readily recognized by the ER chaperones and folding enzymes including molecular chaperones of the Hsp70 family, calnexin/calreticulin, and thiol oxidoreducatases of the protein-disulfide isomerase (PDI) family (4). In the ER of yeast Saccharomyces cerevisiae, BiP, an ER member of the Hsp70 family, facilitates folding and assembly of nonnative proteins by preventing their aggregate formation, in cooperation with its partner proteins Jem1p and Scj1p (5, 6). Interactions of nonnative proteins with these ER chaperones and folding enzymes during the "chaperone-mediated folding process" may well contribute to the block of their deployment out of the ER (1).

Both correctly folded proteins and nonnative proteins with N-linked glycans receive trimming of glucose by glucosidases in the ER lumen. If glycoproteins cannot acquire native conformations within an appropriate time window, they are targeted to the ERAD pathway for disposal (7, 8). Entry for the ERAD pathway requires trimming of a single mannose by the ER {alpha}1,2-mannosidase I and subsequent recognition of the Man8 moiety by a putative lectin Mnlp1/Htm1p, a yeast counterpart of mammalian EDEM (9–11). Trimming of terminal mannose by mannosidase I is a slow process, so that it regulates the protein flux for the ERAD pathway. PDI was shown to be responsible for directing nonnative proteins to the ERAD pathway in yeast (12). Nonnative proteins that enter the ERAD pathway are not degraded in the ER lumen but instead translocated back to the cytosol for degradation (13). BiP, a yeast ER member of the Hsp70 family, in cooperation with its partner proteins Jem1p and Scj1p facilitates the ERAD process by preventing aggregation of misfolded proteins prior to the retrotranslocation (5). The retrotranslocation of proteins across the ER membrane to the cytosol is mediated by the Sec61 translocon channel, the same translocon used for import (13), and/or Derlin-1 (Der1p in yeast) (14, 15) with the aid of such cytosolic components as the AAA ATPase, Cdc48, and its partner proteins (16). Most of the ERAD substrates are ubiquitinated in the cytosol and are finally degraded by the 26 S proteasome (13). In parallel with the ERAD process, accumulation of nonnative proteins in the ER leads to activation of the unfolded protein response (UPR), which induces transcription of genes encoding a set of proteins that can process aberrant proteins, and translational attenuation to reduce the amount of proteins imported newly into the ER (17).

In addition to N-linked glycochains, O-linked carbohydrate chains are attached to proteins in the ER (18). In yeast, O-linked glycosylation starts by the addition of the initial mannose from dolichol-phosphate mannose to Ser/Thr by one of the seven O-mannosyl transferases (Pmt1–7p), although no consensus amino acid sequence was identified as an O-mannosylation site. Roles of O-mannosylation in the protein secretion (18, 19), cell wall integrity (20), budding process (21), and stabilization of cell surface proteins (22) have been suggested. Recently, several aberrant proteins were found to receive O-mannosylation in the yeast ER; when N-glycosylation site mutants of prepro-{alpha}-factor (pp{alpha}F) were translocated into isolated yeast microsomes, subsequent incubation with cytosol led to O-mannosylation of imported and cleaved pro-{alpha}-factor (p{alpha}F) (23). Mutated bovine pancreatic trypsin inhibitor received O-mannosylation if artificially retained in the ER (24). KHN (Kar2-hemagglutinin neuraminidase) also received O-mannosylation during its degradation by the ERAD pathway (25). However, the biological significance of O-mannosylation of aberrant proteins remains unclear.

Although bulky hydrophilic N-linked glycans could increase solubilities of glycoproteins and suppress their irreversible aggregation (26–28), few studies have been carried out to test whether O-linked glycans have similar properties. In the present study, we analyzed the effect of O-mannosylation on distinct 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 ({Delta}pro). We found that O-mannosylated p{alpha}F mutants and {Delta}pro became soluble and partly secreted out of the cell through the normal secretory pathway. O-Mannosylation rendered p{alpha}F mutants less dependent on the ER chaperone, BiP, for being soluble. The impairment of O-mannosylation, especially with that of the ERAD, led to the enhanced UPR. Thus, we propose a new model in which O-mannosylation reduces the load for ER chaperones to contribute to the maintenance of ER homeostasis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains, Plasmids, and Culturing Conditions—Yeast strains used in this study are listed in Table I. For construction of the pmt2::LEU2 allele, the PMT2 gene was cloned by PCR using primers 5'-GCG GAA TTC GTT CTT GAT GTT CAC AGC CAT-3' and 5'-GCG AAG CTT TCG TGT AAG TCT TGA GGC GTT-3', and the amplified DNA fragment was introduced into the EcoRI and HindIII sites of pUC118. pJJ283 (29) was cut with XbaI and BamHI, and a resulting 2-kb fragment containing the LEU2 gene was introduced into the XbaI and BglII sites of the PMT2 gene. The pmt2::kanMX4 allele was generated by PCR using the kanMX4 gene (30) as a template and using primers 5'-ATG TCC TCG TCT TCG TCT ACC GGG TAC AGC AAA AAC AAT GCC GCC CAC ATT AAG CAA GAG TTA AGG CGC GCC AGA TCT GT-3' and 5'-TCA TGC TTC TTG CTT GTC GGC AAT GTC CCA AGT GGA AAA CCA GTT TAA GTA GCG GAA GTT ATC ATC GAT GAA TTC GAG CTC-3'. The der1::LEU2 allele and cue1::LEU2 allele were constructed as described previously (31, 32). The erg6::LEU2 and doa4::LEU2 alleles were constructed as described (5). Disruption of the genes was confirmed by PCR. Multiple mutants were generated by the second round of transformation.


View this table:
[in this window]
[in a new window]
  		TABLE I
Yeast strains used in this study

 
Plasmids encoding p(NNQ)p{alpha}F, p(NQN)p{alpha}F, and p(QNN)p{alpha}F (pGem2{alpha}FNNQ, pGem2{alpha}FNQN, and pGem2{alpha}FQNN) were generated by PCR using pDJ100 (33) as a template and using primers 5'-ATA CAA ACT ACT ATT GCC AGC AT-3' and 5'-AAA CAA TAA CCC GTT ATT TGT-3'; 5'-TCC CAA AGC ACA AAT AAC GGG TTA-3' and 5'-AAA TGG CAA AAC AGC AAC ATC GA-3'; and 5'-GTC CAG ACT ACA ACA GAA GAT GAA A-3' and 5'-TGG AGC AGC TAA TGC GGA GGA T-3', respectively. Plasmids encoding p(NQQ)p{alpha}F, p(QNQ)p{alpha}F, and p(QQN)p{alpha}F (pGem2{alpha}FNQQ, pGem2{alpha}FQNQ, and pGem2{alpha}FQQN) were generated by PCR using pGem2{alpha}F3Q (34) as a template and using primers 5'-GTC AAC ACT ACA ACA GAA GAT-3' and 5'-TGG AGC AGC TAA TGC GGA GGA T-3';5'-TCC AAC AGC ACA AAT AAC GGG TT-3' and 5'-AAA TGG CAA AAC AGC AAC ATC GA-3'; and 5'-ATA AAT ACT ACT ATT GCC AGC-3' and 5'-AAA CAA TAA CCC GTT ATT TGT-3', respectively. Plasmids encoding p(NNN)p{alpha}F, p(NQQ)p{alpha}F, and p(QQQ)p{alpha}F for a halo assay were constructed as follows. A genomic clone containing the MF{alpha}1-coding sequence was cut with EcoRI, and the resultant 1.8-kb EcoRI fragment was inserted into the EcoRI site of pUC18 to give pUCMF{alpha}1NNN. For construction of pUCMF{alpha}1QQQ, a 0.47-kb fragment was amplified by PCR using pGem2{alpha}F3Q as a template and using primers 5'-CAG ACT ACA ACA GAA GAT GAA A-3' and 5'-GCG GTC GAC TTT GTT ACA TCT ACA CTG-3' and cut with SalI. The resultant 0.47-kb fragment was introduced into HincII-SalI sites of pUCMF{alpha}1NNN. For constructing pUCMF{alpha}1NQQ, pGem2{alpha}FNQQ was cut with SalI and subsequently with HincII, and the resultant 0.47-kb HincII-SalI fragment was introduced into the HincII-SalI site of pUCMF{alpha}1NNN. Inserts containing the promoter region and the open reading frame of p(NNN)p{alpha}F, p(NQQ)p{alpha}F, or p(QQQ)p{alpha}F was introduced into pRS316 (35).

Cells were grown in YPD (1% yeast extract, 2% polypeptone, and 2% glucose), YPGal (1% yeast extract, 2% polypeptone, and 2% galactose), SCGal (0.67% yeast nitrogen base without amino acids, 2% galactose, 0.5% casamino acids), SCD (0.67% yeast nitrogen base without amino acids, 2% glucose, 0.5% casamino acids), and SD (0.67% yeast nitrogen base without amino acids and 2% glucose)

In Vitro Translocation of p{alpha}F Mutants and {Delta}pro into Yeast Microsomes and ERAD Assay—In vitro translocation into isolated yeast microsomes and ERAD assays of p{alpha}F mutants were performed as described (5, 34). The ATP mix contains 40 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, 1 mM ATP, 50 µM GDP-mannose (GDPMan). In order to detect a [14C]mannose-labeled form of {Delta}G(QQQ)p{alpha}F, nonlabeled {Delta}G(QQQ)p{alpha}F-loaded membranes were incubated with ATP mix and the yeast cytosol in the presence of 50 µM [14C]GDPMan instead of cold GDPMan. After incubation, proteins were subjected to immunoprecipitation with anti-pp{alpha}F antibodies.

In vitro translation and translocation into isolated yeast microsomes of {Delta}pro were performed as above, except that translation and translocation were coupled. Briefly, microsomes were added to the translation reaction mixture 10 min after initiation of the translation at 20 °C and further incubated for 60 min at 20 °C. {Delta}pro-loaded microsomes were collected by centrifugation and subjected to ERAD assays. Proteins bound to ConA-Sepharose were eluted with 1 M {alpha}-methylmannoside in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA for 1 h at 30 °C. Proteins were analyzed by SDS-PAGE and radioimaging with a Storm 860 image analyzer (Amersham Biosciences).

Sucrose Density Gradient Centrifugation—Sucrose density gradient centrifugation for proteins in microsomes and yeast cell lysate was performed as in Ref. 5.

Halo Assay—A halo assay was carried out according to Nishikawa and Nakano (36). Briefly, ~5 x 105 tester cells (BC180), which are supersensitive to {alpha}-factor, were spread onto SCD plates that contained appropriate supplements and were buffered at pH 3.5. Cells to be tested for {alpha}-factor production were spotted on the plates with sterile toothpicks. The secreted {alpha}-factor was quantified according to the following equation: halo size (mm) = 1.8 ln(amount of {alpha}-factor (ng)) + 5.5.2

Pulse-Chase Experiments—Metabolic labeling of yeast cells with Tran35S-label (ICN) and preparation of cell extracts were performed essentially as described (37). {Delta}pro was expressed from the chromosome at the URA3 locus under the GAL1 promoter. Before labeling, {Delta}pro was induced in the minimal medium containing 2% galactose. Immunoprecipitation of {Delta}pro with anti-RNAP-I antibodies and the second round of immunoprecipitation with anti-{alpha}1,3-mannose antibodies were performed as described previously (9, 38). The immunoprecipitated proteins were analyzed by SDS-PAGE with or without 6 M urea and radioimaging.

Assay of Cellular {beta}-Galactosidase Activity—UPR induction was measured by using a single copy (CEN) plasmid that harbored the CYC1 promoter containing the UPR element fused to the lacZ gene (pSCZ-Y, a gift from K. Mori). {beta}-Galactosidase activity of cells was determined as described (39).

Immunofluorescent Microscopy—Immunofluorescent staining of yeast cells was performed as described previously (40) with minor modifications. Incubation with primary antibodies was performed as follows: 1:250 dilution of the rabbit anti-BiP polyclonal antibody (41), 1:50 dilution of anti-RNAP-I antibody (42), and 1:250 dilution of anti-PDI antibody (a gift from H. Tachikawa). Incubation with secondary antibodies was performed as follows: 1:10 dilution of fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG antibody F(ab')2 fragment.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
N-Glycosylation Mutants of Pro-{alpha}-factor Receive O-Mannosylation—Prepro-{alpha}-factor is the precursor form of a yeast mating pheromone and consists of an ER-targeting signal sequence that is cleaved off in the ER, a pro-region, and four tandem repeats of {alpha}-factor peptides, all of which are cleaved in the Golgi complex. Since the pro-region has three potential N-glycosylation sites (Asn-23, Asn-57, and Asn-67) (Fig. 1A), we designate it as p(NNN)p{alpha}F. p(QQQ)p{alpha}F, a mutant form of p(NNN)p{alpha}F that has three glutamine residues substituted for the N-glycosyl acceptor sites (Fig. 1A) and therefore receives no N-glycosylation in the ER, has been used as a model aberrant protein for in vitro ERAD assays with yeast microsomes (34, 43, 44). Besides, it was reported that a fraction of (QQQ)p{alpha}F imported into yeast microsomes in vitro receives O-mannosylation in the ER lumen instead of being retrotraslocated to the cytosol for degradation under the ERAD conditions, although its biological relevance remained unclear (23).



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 1.
N-glycosylation mutants of p{alpha}F receive O-mannosylation. A, pp{alpha}F is schematically outlined. The triangles indicate asparagine residues that receive N-glycosylation. N-Glycosylation mutants of p{alpha}F used in this study are listed. Wild-type pp{alpha}F was designated as p(NNN)p{alpha}F. For designation of the N-glycosylation mutants, see "N-Glycosylation Mutants of Pro-{alpha}-factor Receive O-Mannosylation" (in "Results"). pre, cleavable signal sequence; pro, pro-sequence. B, radiolabeled pp{alpha}F and its N-glycosylation mutants were translocated into wild-type (SEY6210) microsomes at 30 °C for 60 min. After reisolation, the membranes were subjected to incubation with ATP mix and the yeast cytosol fraction at 30 °C for the indicated times (chase). Proteins were analyzed by SDS-PAGE. (NNN)p{alpha}F, a signal cleaved form of p(NNN)p{alpha}F, receives three N-linked glycans to form 3G(NNN)p{alpha}F (0 min of NNN). The bands for 1G(QNQ)p{alpha}F and 1G(QQN)p{alpha}F migrate faster than that for 1G(NQQ)p{alpha}F (45) and are overlapped by those for p(QNQ)p{alpha}F and p(QQN)p{alpha}F (0 min of QNQ and QQN). The asterisks indicate O-mannosylated forms. {Delta}Gp, 1Gp, 2Gp, and 3Gp, p{alpha}F mutants with 0, 1, 2, or 3 N-linked glycan(s), respectively. m{Delta}Gp and m1Gp indicate O-mannosylated p{alpha}F mutants with 0 or 1 N-linked glycan, respectively. pp, pp{alpha}F; p, p{alpha}F. C, radiolabeled p(NQQ)p{alpha}F and p(QQQ)p{alpha}F were translocated into wild-type (SEY6210) microsomes at 30 °C for 60 min. After reisolation, the membranes were incubated with ATP mix and the yeast cytosol fraction at 30 °C for 60 min and subsequently solubilized with 1% SDS. The proteins were precipitated with ConA-Sepharose (lanes 1 and 2) or first treated with endoglycosidase H and then precipitated with ConA-Sepharose (lanes 3 and 4). 1G(NQQ)p{alpha}F(1Gp), the O-mannosylated form of 1G(NQQ)p{alpha}F(m1Gp), and the O-mannosylated form of {Delta}G(QQQ)p{alpha}F (m{Delta}Gp) were precipitated with ConA-Sepharose (lanes 1 and 2). After treatment with endoglycosidase H, O-mannosylated (NQQ)p{alpha}F, in which N-linked glycan had been removed by endoglycosidase H, and O-mannosylated {Delta}G(QQQ)p{alpha}F were precipitated with ConA-Sepharose (lanes 3 and 4). D, nonlabeled p(QQQ)p{alpha}F were translocated into wild-type (SEY6210) microsomes at 30 °C for 60 min. After reisolation, the {Delta}G(QQQ)p{alpha}F-loaded membranes were subjected to incubation with the yeast cytosol fraction and ATP mix with 50 µM [14C]GDPMan instead of cold GDP-Man at 30 °C for 50 min. Proteins were subjected to immunoprecipitation (IP) with anti-pp{alpha}F antibodies and analyzed by SDS-PAGE and radioimaging (lane 2; 14C-Man). Lane 1, control ERAD assay with [35S]methionine-labeled p(QQQ)p{alpha}F similar to B. E, translocation and ERAD assays similar to B except that pmt2{Delta} (KNY2) microsomes were used instead of wild-type (SEY6210) microsomes. The weak band between those labeled with p and pp for p(NNN)p{alpha}F (NNN) probably arose from partially N-glycosylated forms.

 
When radiolabeled wild-type p(NNN)p{alpha}F and mutant p(QQQ)p{alpha}F, synthesized in yeast lysates (pp in Fig. 1B, NNN and QQQ) were incubated with isolated yeast microsomes, they were efficiently translocated into microsomes. p(NNN)p{alpha}F received signal sequence cleavage and N-glycosylation to form 3G(NNN)p{alpha}F(3Gp in Fig. 1B, NNN) in the ER lumen, whereas p(QQQ)p{alpha}F received signal sequence cleavage, but no N-glycosylation and formed {Delta}G(QQQ)p{alpha}F({Delta}Gp in Fig. 1B, QQQ), since it lacked all of the N-glycosyl acceptor sites. The microsomes were then washed and incubated with the yeast cytosol fraction and "ATP mix" (the ERAD conditions; see "Experimental Procedures"). The amount of 3G(NNN)p{alpha}F remained the same even after 60 min incubation with the cytosol. On the other hand, the amount of {Delta}G(QQQ)p{alpha}F decreased, but a smeared band (asterisk in Fig. 1B, QQQ) concomitantly appeared above the band for {Delta}G(QQQ)p{alpha}F and shifted upward with increasing time of incubation, which appeared to reflect O-mannosylation (23). Indeed, the {Delta}G(QQQ)p{alpha}F species responsible for the smeared band was precipitated with ConA-Sepharose beads (m{Delta}Gp in Fig. 1C, lane 2) and even after treatment with endoglycosidase H, which removes N-linked glycans (m{Delta}Gp in Fig. 1C, lane 4). When nonlabeled p(QQQ)p{alpha}F, instead of radiolabeled p(QQQ)p{alpha}F, was translocated into the microsomes and subsequently incubated with the cytosol and ATP mix in the presence of [14C]GDPMan, a [14C]mannose-labeled form of {Delta}G(QQQ)p{alpha}F was detected by radioimaging after immunoprecipitation with anti-pp{alpha}F antibodies (m{Delta}Gp in Fig. 1D). This confirms that mannose was transferred to {Delta}G(QQQ)p{alpha}F from GDPMan as a donor.

Since {Delta}G(QQQ)p{alpha}F received O-mannosylation whereas 3G(NNN)p{alpha}F did not, we tried to assess the relationship between the number of N-glycosylated moieties and the extent of O-mannosylation on p{alpha}F derivatives. We thus made a series of p(NNN)p{alpha}F mutants in which one or two asparagine residue(s) in the N-glycosyl acceptor sites was converted to glutamine residue(s) (Fig. 1A). p(NNN)p{alpha}F mutants with an Asn -> Gln substitution at position 67, 57, or 23 are designated as p(NNQ)p{alpha}F, p(NQN)p{alpha}F, and p(QNN)p{alpha}F, respectively, and those with double Asn -> Gln substitutions at positions 57 and 67, 23 and 67, or 23 and 57 are designated as p(NQQ)p{alpha}F, p(QNQ)p{alpha}F, and p(QQN)p{alpha}F, respectively. The relative mobilities of signal cleaved and N-glycosylated forms of single glycosylation site mutants (2G(NNQ)p{alpha}F, 2G(NQN)p{alpha}F, and 2G(QNN)p{alpha}F) and double glycosylation site mutants (1G(NQQ)p{alpha}F, 1G(QNQ)p{alpha}F, and 1G(QQN)p{alpha}F) to those of 3G(NNN)p{alpha}F and {Delta}G(QQQ)p{alpha}F are consistent with one another (pp in Fig. 1B). The bands for 1G(QNQ)p{alpha}F and 1G(QQN)p{alpha}F migrated faster than that for 1G(NQQ)p{alpha}F (45) and were overlapped by those for p(QNQ)p{alpha}F and p(QQN)p{alpha}F (1Gp and pp in Fig. 1B, 0 min of chase of QNQ and QQN). During incubation with the cytosol and ATP mix, 1G(NQQ)p{alpha}F, 1G(QNQ)p{alpha}F, and 1G(QQN)p{alpha}F were partly converted to higher molecular weight forms (asterisk in Fig. 1B, NQQ, QNQ, and QQN), whereas 2G(NNQ)p{alpha}F, 2G(NQN)p{alpha}F, and 2G(QNN)p{alpha}F were not (Fig. 1B). These higher molecular weight forms arose from O-mannosylation, since they were not observed with microsomes prepared from the pmt2{Delta} strain, which lacks the PMT2 gene, one of the seven PMT genes for mannosyl transferases in yeast (Fig. 1E). O-Mannosylation of double glycosylation site mutants was also confirmed by precipitation with ConA-Sepharose beads after treatment with endoglycosidase H (Fig. 1C, lanes 1 and 3). Therefore, although 3G(NNN)p{alpha}F and the single glycosylation site mutants (2G(NNQ)p{alpha}F, 2G(NQN)p{alpha}F, and 2G(QNN)p{alpha}F) did not receive O-mannosylation, double glycosylation site mutants (1G(NQQ)p{alpha}F, 1G(QNQ)p{alpha}F, and 1G(QQN)p{alpha}F) and {Delta}G(QQQ)p{alpha}F were significantly O-mannosylated. The O-mannosylated forms were resistant to externally added protease but were degraded by protease upon disruption of the membranes with detergent (data not shown), suggesting that they remained in the ER lumen.

O-Mannosylation Renders Aggregation-prone p{alpha}F Mutants Soluble—In contrast to 3G(NNN)p{alpha}F, which is soluble in the ER lumen, {Delta}G(QQQ)p{alpha}F is prone to aggregation and remains soluble only in the presence of the ER luminal Hsp70, BiP (5). We therefore reasoned that removal of single or double N-glycosylation sites may affect solubilities of the p{alpha}F mutants differently, which would in turn lead to different levels of O-mannosylation of the p{alpha}F mutants.

To test this hypothesis, we first asked if O-mannosylation of {Delta}G(QQQ)p{alpha}F required functional BiP, since aggregation-prone proteins tend to interact with BiP to remain soluble (5) (Fig. 2). Radiolabeled p(QQQ)p{alpha}F was translocated into microsomes prepared from wild-type cells or kar2-159 yeast cells at 23 °C ("translocation"). The {Delta}G(QQQ)p{alpha}F-loaded microsomes were incubated at 23 or 37 °C for 30 min in the presence of ATP mix ("first incubation") but in the absence of the cytosol. During this incubation, BiP in kar2-159 microsomes is fully functional at permissive temperature (23 °C) but becomes defective at restrictive temperature (37 °C). The microsomes were then incubated with both the cytosol and ATP mix at the same temperature as the first incubation for 50 min ("O-mannosylation"). Whereas {Delta}G(QQQ)p{alpha}F was efficiently O-mannosylated (m{Delta}Gp) in wild-type microsomes in the presence of the cytosol and ATP mix both at the permissive temperature of 23 and 37 °C (asterisk in Fig. 2, lanes 2 and 4) and in the kar2-159 microsomes at 23 °C (asterisk in Fig. 2, lane 6), it was hardly O-mannosylated in the kar2-159 microsomes at the restrictive temperature of 37 °C (Fig. 2, lane 8). This result indicates that functional BiP is required for O-mannosylation of {Delta}G(QQQ)p{alpha}F, probably because substrates for O-mannosylation should be soluble in the ER lumen.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 2.
Function of BiP is required for O-mannosylation of {Delta}G(QQQ)p{alpha}F. Radiolabeled p(QQQ)p{alpha}F was translocated into wild-type (RSY607) or kar2-159 (RSY579) microsomes at 23 °C for 60 min. The reaction mixtures were subsequently split in half, and the {Delta}G(QQQ)p{alpha}F-loaded membranes were isolated. Samples were subjected to incubation with ATP mix at 23 or 37 °C for 30 min. After reisolation, the membranes were subjected to the second incubation (O-mannosylation) with ATP mix and the yeast cytosol fraction at 23 °C or 37 °C for 0 min (odd-numbered lanes) or 50 min (even-numbered lanes). Samples were analyzed by SDS-PAGE and radioimaging. BiP becomes defective when kar2-159 (RSY579) microsomes are incubated at 37 °C for 30 min in the presence of ATP mix (gray box). The asterisks indicate O-mannosylated forms. {Delta}Gp, p{alpha}F mutants without N-linked glycan; m{Delta}Gp, O-mannosylated p{alpha}F mutants without N-linked glycan; pp, pp{alpha}F.

 
Aggregation states of N-glycosylation site mutants of p{alpha}F and its O-mannosylated forms in the microsomes incubated at restrictive temperature (37 °C) were analyzed by sucrose density gradient centrifugation after solubilization of the microsomes with Triton X-100 (schematized in Fig. 3A). After translocation of N-glycosylation site mutants of p{alpha}F into wild-type and kar2-159 microsomes at 23 °C ("translocation"), incubation of the microsomes at 23 °C with ATP mix and cytosol is required to generate O-mannosylated forms ("O-mannosylation"). These microsomes were then incubated at 37 °C (second incubation). {Delta}G(QQQ)p{alpha}F solubilized from the wild-type microsomes were fractionated with a molecular mass corresponding to 70 kDa, but its mannosylated form m{Delta}G(QQQ)p{alpha}F was recovered exclusively in the fractions corresponding to <30 kDa (Fig. 3B). Therefore, m{Delta}G(QQQ)p{alpha}F and {Delta}G(QQQ)p{alpha}F are soluble as monomers and probably as a small oligomer, respectively. In kar2-159 microsomes, p{alpha}F mutants that lack two or three N-glycosylation sites (1G(NQQ)p{alpha}F, 1G(QNQ)p{alpha}F, and {Delta}G(QQQ)p{alpha}F) were aggregated and recovered only at the bottom of the tube after incubation at restrictive temperature (37 °C; BiP is not functional) (Fig. 3C). On the other hand, their O-mannosylated forms (m1G(NQQ)p{alpha}F, m1G(QNQ)p{alpha}F, and m{Delta}G(QQQ)p{alpha}F) were recovered in the fractions corresponding to <30 kDa after incubation at restrictive temperature (37 °C) (Fig. 3D). This result suggests that the O-mannosylated forms are soluble as a monomer without the assistance of BiP. The high solubilities of O-mannosylated p{alpha}F mutants may be compared with those of 3G(NNN)p{alpha}F, 2G(NQN)p{alpha}F, and 2G(QNN)p{alpha}F. In the kar2-159 microsomes, 3G(NNN)p{alpha}F, 2G(NQN)p{alpha}F, and 2G(QNN)p{alpha}F were recovered in the fractions for 30–70 kDa after incubation at restrictive temperature (37 °C) (Fig. 3E), suggesting that they also remained soluble, but probably in oligomeric forms.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 3.
O-Mannosylated p{alpha}F mutants are soluble independently of the function of BiP. A, strategies for analyzing the aggregation states of N-glycosylation mutants of p{alpha}F and their O-mannosylated forms in B–E are schematically outlined. Both in wild-type and kar2-159 microsomes, p{alpha}F mutants that lack two N-glycosylation sites and {Delta}G(QQQ)p{alpha}F receive O-mannosylation during the incubation at 23 °C for 50 min in the presence of ATP mix and the yeast cytosol. BiP becomes defective when kar2-159 (RSY579) microsomes are subsequently incubated at 37 °C for 30 min in the presence of ATP mix (gray box). B, radiolabeled p(QQQ)p{alpha}F was translocated into wild-type (RSY607) microsomes at 23 °C for 60 min. The reaction mixtures were split in half, and the {Delta}G(QQQ)p{alpha}F-loaded membranes were isolated. One sample was subjected to incubation with ATP mix at 37 °C for 30 min. The remaining sample was incubated with ATP mix and the yeast cytosol fraction at 23 °C for 50 min to generate the O-mannosylated form of {Delta}G(QQQ)p{alpha}F(m{Delta}G(QQQ)p{alpha}F), and subsequently, the membranes were isolated and incubated with ATP mix at 37 °C for 30 min. Samples were solubilized with 1% Triton X-100 and subjected to sucrose density gradient centrifugation. Fractions were collected, and proteins were analyzed by SDS-PAGE and radioimaging. Relative amounts of {Delta}G(QQQ)p{alpha}F and m{Delta}G(QQQ)p{alpha}F were quantified. C and D, radiolabeled p(NQQ)p{alpha}F, p(QNQ)p{alpha}F, and p(QQQ)p{alpha}F were translocated into kar2-159 (RSY579) microsomes at 23 °C for 60 min. The reaction mixtures were subsequently divided into halves, and the 1G(NQQ)p{alpha}F-, 1G(QNQ)p{alpha}F-, and {Delta}G(QQQ)p{alpha}F-loaded membranes were isolated. One aliquot was subjected to incubation with ATP mix at 37 °C for 30 min (C). The other aliquot was incubated with ATP mix and yeast cytosol at 23 °C for 50 min to generate O-mannosylated forms of 1G(NQQ)p{alpha}F, 1G(QNQ)p{alpha}F, and {Delta}G(QQQ)p{alpha}F (m1G(NQQ)p{alpha}F, m1G(QNQ)p{alpha}F, and m{Delta}G(QQQ)p{alpha}F). The membranes were subsequently isolated and incubated with ATP mix at 37 °C for 30 min (D). Samples were subjected tosucrose density gradient centrifugation and analyzed as in B. Relative amounts of 1G(NQQ)p{alpha}F, 1G(QNQ)p{alpha}F, {Delta}G(QQQ)p{alpha}F(C), m1G(NQQ)p{alpha}F, m1G(QNQ)p{alpha}F, and m{Delta}G(QQQ)p{alpha}F (D) were quantified. E, radiolabeled p(NNN)p{alpha}F, p(NQN)p{alpha}F, and p(QNN)p{alpha}F were translocated into kar2-159 (RSY579) microsomes at 23 °C for 60 min. The 3G(NNN)p{alpha}F-, 2G(NQN)p{alpha}F-, and 2G(QNN)p{alpha}F-loaded membranes were isolated and incubated with ATP mix at 37 °C for 30 min. Samples were subjected to sucrose density gradient centrifugation and analyzed as in B. Relative amounts of 3G(NNN)p{alpha}F, 2G(NQN)p{alpha}F, and 2G(QNN)p{alpha}F were quantified. The numbers indicate fractions (from top to bottom). The vertical arrowheads show the positions of apoferritin (440 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (70 kDa), and carbonic anhydrase (30 kDa). {Delta}Gp, 1Gp, 2Gp, and 3Gp, p{alpha}F mutants with 0, 1, 2, or 3 N-linked glycan(s), respectively. m{Delta}Gp and m1Gp, O-mannosylated p{alpha}F mutants with 0 or 1 N-linked glycan, respectively. pp, pp{alpha}F; p, p{alpha}F.

 
These results indicate that the p{alpha}F mutants that lack two or three N-glycosylation sites are prone to aggregate if BiP is inactivated. In other words, BiP is required for these p{alpha}F mutants to remain soluble in the ER lumen. However, a significant fraction of these N-glycosylation site mutants of p{alpha}F become O-mannosylated forms in the ER lumen, which are, in contrast to the nonglycosylated forms, highly soluble independent of the BiP function.

Pmt2p Facilitates Secretion of p{alpha}F Mutants—Although the previous in vitro study indicated that O-mannosylated {Delta}G(QQQ)p{alpha}F failed to be secreted from the ER (23), the in vitro ERAD assay system with yeast microsomes did not reconstitute downstream membrane trafficking along the secretory pathway. To analyze these downstream events, we followed the fate of O-mannosylated p{alpha}F mutants or their secretion by using an in vivo halo assay.

The halo assay relies on the ability of secreted {alpha}-factor to block growth of {alpha}-factor-sensitive yeast mutant cells and a zone of growth inhibition that manifests as a "halo" (46). The amount of the secretion of {alpha}-factor can be estimated semiquantitatively. Since N- and O-glycosylation sites of p{alpha}F are present in the pro-region, but not in the mature region, changes in glycosylation will not affect the {alpha}-factor activity of the mature peptide pheromone itself. p(NNN)p{alpha}F, p(NQQ)p{alpha}F, and p(QQQ)p{alpha}F were expressed from a single copy (CEN) plasmid in PMT2 and pmt2{Delta} cells and subjected to the halo assay (Fig. 4). In wild-type cells, the amount of {alpha}-factor secreted by p(NQQ)p{alpha}F and p(QQQ)p{alpha}F were ~36 and ~8% as compared with p(NNN)p{alpha}F, respectively, indicating that elimination of N-glycosylation sites led to reduction in {alpha}-factor secretion. Larger effects were observed for p(QQQ)p{alpha}F with all three glycosylation sites eliminated than for p(NQQ)p{alpha}F with two sites eliminated (Fig. 4), a result that is consistent with previous data (45). In pmt2{Delta} cells, the amount of {alpha}-factor secreted by p(NQQ)p{alpha}F was ~15% as compared with p(NNN)p{alpha}F, and no halo was observed for p(QQQ)p{alpha}F, indicating that elimination of the N-linked glycosylation sites on p{alpha}F mutant secretion became more prominent in pmt2{Delta} cells (Fig. 4), in which O-mannosylation of 1G(NQQ)p{alpha}F and {Delta}G(QQQ)p{alpha}F was suppressed. This result strongly suggested that the O-mannosylated forms of p{alpha}F mutants, which were highly soluble in the ER lumen, became competent for transport from the ER to the Golgi and were finally secreted out of the cell.



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 4.
Deletion of PMT2 affects the secretion of p{alpha}F mutants. Shown are halo assays for wild-type (SNY9) and pmt2{Delta} (KNY1) cells expressing p(NNN)p{alpha}F, p(NQQ)p{alpha}F, or p(QQQ)p{alpha}F from a single copy (CEN) plasmid. The plates were incubated at 30 °C for 2 days.

 
{Delta}pro Is O-Mannosylated When ERAD Is Blocked in Vivo—Is solubilization of aberrant proteins by O-mannosylation in the ER specific to p{alpha}F mutants? Harty et al. (23) observed that, in addition to authentic substrates for O-mannosylation, many proteins isolated from tunicamycin-treated yeast cells can be O-mannosylated. As a test for the generalization of our model for the role of O-mannosylation, we analyzed another substrate, {Delta}pro, a pro-region-deleted derivative of RNAP-I, for possible O-mannosylation. RNAP-I does not have potential N-glycosylation sites and the N-terminal pro-region is essential for its folding and secretion (47). Upon expression in yeast, {Delta}pro does not exit from the ER but is instead degraded irrespective of the block of the ER-to-Golgi transport (38). When {Delta}pro was expressed in wild-type cells under the control of the GAL1 promoter and analyzed by pulse-chase experiments, the amount of {Delta}pro decreased rapidly (Fig. 5A, wild-type) (38). We found that a weak smeared band, which escaped detection in the previous study (38), appeared above the band for {Delta}pro and shifted upward on SDS-polyacrylamide gels with increasing time of incubation (Fig. 5A, wild-type). Intensities of the smeared bands were markedly enhanced when analyzed with der1{Delta} cells and cue1{Delta} cells, in which the gene for Der1p, an integral ER membrane protein involved in the ERAD (31), or Cue1p, which recruits the ubiquitin-conjugating enzyme to the ER membrane for the ERAD (32), was deleted, respectively (Fig. 5A, der1{Delta} and cue1{Delta}). The smeared bands disappeared in pmt2{Delta}, pmt2{Delta}der1{Delta}, and pmt2{Delta}cue1{Delta} cells, in which the PMT2 gene or both the PMT2 and DER1 or CUE1 genes were deleted, respectively (Fig. 5B, pmt2{Delta}, pmt2{Delta}der1{Delta}, and pmt2{Delta}cue1{Delta}); hence, they reflect O-mannosylation of {Delta}pro.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 5.
{Delta}pro is partly degraded via the ubiquitin-proteasome pathway and receives O-mannosylation when its degradation is inhibited. A and B, wild-type (KUY90), der1{Delta} (KNY13), cue1{Delta} (KNY14) (A), pmt2{Delta} (KNY40), pmt2{Delta}der1{Delta} (KNY41), and pmt2{Delta}cue1{Delta} (KNY42) (B) cells expressing {Delta}pro were metabolically labeled at 30 °C for 5 min and chased for indicated times. {Delta}pro was recovered from cell extracts by immunoprecipitation with anti-RNAP-I antibodies and analyzed by SDS-PAGE and radioimaging. Relative amounts of {Delta}pro (including its O-mannosylated form) were quantified. The amounts of {Delta}pro at 0-min chase were set to 100%. C, erg6{Delta} (KNY43; left gel) or doa4{Delta} (KNY44; right gel) cells expressing {Delta}pro were metabolically labeled at 30 °C for 5 min and chased for the indicated times with (+MG132) or without (–MG132, doa4{Delta}) 200 µM MG132. {Delta}pro was immunoprecipitated and analyzed by SDS-PAGE and radioimaging as in A. Relative amounts of {Delta}pro (including its O-mannosylated form) were quantified. The amounts of {Delta}pro at 0-min chase were set to 100%.

 
The sum of the amounts of {Delta}pro and its O-mannosylated form (m{Delta}pro) decreased with half-lives of 23, 60, and 65 min in wild-type, der1{Delta}, and cue1{Delta} cells, respectively (Fig. 5A, der1{Delta} and cue1{Delta}). The retardation of degradation of the {Delta}pro species ({Delta}pro and m{Delta}pro) in der1{Delta} and cue1{Delta} cells suggests that they were at least partly degraded via the ERAD pathway. This is in contrast to the suggestion in the previous report (38). We thus reevaluated the degradation of the total {Delta}pro species by compromising the activity of the ubiquitin-proteasome pathway with MG132, an inhibitor of the 26 S proteasome (48), or depletion of Doa4p, a deubiquitination enzyme (49). In both cases, {Delta}pro species ({Delta}pro and m{Delta}pro) were partially stabilized; {Delta}pro and m{Delta}pro were degraded now with half-lives of 50 and 60 min, respectively (Fig. 5C, +MG132 and doa4{Delta}). O-Mannosylation of {Delta}pro was hardly observed, probably due to the weak inhibition of degradation. It should be noted that, in pmt2{Delta} cells, {Delta}pro was degraded efficiently with a half-life of 25 min (Fig. 5B, pmt2{Delta}), suggesting that O-mannosylation takes place for a fraction of {Delta}pro that already escaped degradation, as in the case of the p{alpha}F mutants.

{Delta}pro Is O-Mannosylated in Vitro—We next tested the possibility that {Delta}pro was also O-mannosylated in vitro. Radiolabeled p{Delta}pro synthesized in vitro could be co-translationally but not posttranlationally translcoated into yeast microsomes (Fig. 6). p{Delta}pro received signal sequence cleavage and formed {Delta}pro, which is resistant against digestion with externally added trypsin (Fig. 6, upper panel, trypsin (+), Triton X-100 (–)) but, after solubilization of the membrane with Triton X-100, became susceptible to trypsin digestion (Fig. 6, upper panel, trypsin (+), Triton X-100 (+)). Upon incubation of {Delta}pro-loaded microsomes with ATP mix and yeast cytosol (the ERAD conditions; see Refs. 5 and 34), the amount of {Delta}pro decreased with increasing time of incubation, but a smeared band (Fig. 6, upper panel, m{Delta}pro) concomitantly appeared above the band for {Delta}pro and increased its intensity. m{Delta}pro responsible for the smeared band was precipitated with ConA-Sepharose beads (Fig. 6, upper panel, ConA). When we used microsomes prepared from the pmt2{Delta} strain, that lacks the PMT2 gene, one of the seven PMT genes for mannosyl transferases in yeast, we did not observe the smeared band above the band for translocated {Delta}pro (Fig. 6, lower panel). This confirms that {Delta}pro is O-mannosylated by Pmt2p in wild-type microsomes in vitro. We did not observe degradation of {Delta}pro during incubation of the microsomes under the present ERAD conditions, and this absence of degradation may have facilitated O-mannosylation of {Delta}pro in microsomes.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 6.
{Delta}pro is translocated into isolated yeast microsomes and O-mannosylated. Radiolabeled p{Delta}pro was co-translationally translocated into wild-type (SEY6210; upper panel) and pmt2{Delta} (KNY2; lower panel) microsomes at 20 °C for 60 min. After reisolation, the {Delta}pro-loaded membranes were subjected to incubation with ATP mix and yeast cytosol at 30 °C for the indicated times. The membranes were treated with or without 0.5 mg/ml trypsin and 1% Triton X-100 on ice for 30 min. The O-mannosylated form of {Delta}pro was precipitated with ConA-Sepharose after solubilization with 1% SDS (upper panel, ConA{downarrow}). m{Delta}pro, O-mannosylated form of {Delta}pro.

 
O-Mannosylation Partly Restores the Normal Secretory Pathway Transport of {Delta}pro—Since O-mannosylation increased the solubility of p{alpha}F mutants, we analyzed the solubility of m{Delta}pro formed in vivo. Proteins extracted from wild-type, der1{Delta}, and cue1{Delta} cells that express {Delta}pro were analyzed by sucrose density gradient centrifugation (5) followed by immunoblotting with anti-RNAP-I antibodies. After centrifugation, {Delta}pro was mainly recovered in the pellet (Fig. 7A) (42), whereas m{Delta}pro in der1{Delta} and cue1{Delta} cells were recovered in the fractions corresponding to 30–60 kDa (Fig. 7, B and C). This result indicates that O-mannosylation renders {Delta}pro soluble.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 7.
O-Mannosylation increases the solubility of {Delta}pro. A–C, wild-type (KUY90) (A), der1{Delta} (KNY13) (B), and cue1{Delta} (KNY14) (C) cells expressing {Delta}pro were grown in SCGal medium. Cell lysates were prepared and subjected to sucrose density gradient centrifugation as described previously (5). Fractions were collected and analyzed by SDS-PAGE. {Delta}pro and m{Delta}pro (O-mannosylated form of {Delta}pro) were detected by immunoblotting with anti-RNAP-I antibodies. Relative amounts of {Delta}pro and m{Delta}pro were quantified.

 
We next followed the fate of m{Delta}pro in various mutant strains that have defects in Der1p or Cue1p. When cell extracts were analyzed for {Delta}pro in der1{Delta} and cue1{Delta} cells by immunoblotting with anti-RNAP-I antibodies, smeared bands arising from O-mannosylation of {Delta}pro were observed (Fig. 8A, lanes 2 and 3). In pmt2{Delta}der1{Delta} and pmt2{Delta}cue1{Delta} cells, only {Delta}pro without O-mannosylation was detected (Fig. 8A, lanes 5 and 6). Since Pmt2p is not required for degradation of {Delta}pro (Fig. 5B), {Delta}pro did not significantly accumulate in pmt2{Delta} cells (Fig. 8A, lane 4) but did accumulate in pmt2{Delta}der1{Delta} and pmt2{Delta}cue1{Delta} cells (Fig. 8A, lanes 5 and 6). These results are consistent with our interpretation that {Delta}pro is at least partly degraded via the ubiquitin-proteasome pathway and is O-mannosylated when this pathway is blocked.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 8.
A part of O-mannosylated {Delta}pro is secreted to the extracellular medium. A, wild-type (KUY90), der1{Delta} (KNY13), cue1{Delta} (KNY14), pmt2{Delta} (KNY40), pmt2{Delta}der1{Delta} (KNY41), and pmt2{Delta}cue1{Delta} (KNY42) expressing {Delta}pro were grown in YPGal medium to the early logarithmic phase. 0.2 A600 eq of proteins in the cell lysate (cell) and 1.3 A600 eq of proteins in the extracellular medium (medium) were analyzed by SDS-PAGE with 6 M urea and immunoblotting with anti-RNAP-I antibodies. B, glycoproteins in the cell lysate (cell) and extracellular medium (medium) of wild-type (KUY90), der1{Delta} (KNY13), and cue1{Delta} (KNY14) cells expressing {Delta}pro were precipitated with ConA-Sepharose beads (ConA{downarrow}). The precipitated proteins were analyzed by SDS-PAGE with 6 M urea and immunoblotting with anti-RNAP-I antibodies. C, proteins in the cell lysate (cell) and extracellular medium (medium) of wild-type (KUY90), der1{Delta} (KNY13), and cue1{Delta} (KNY14) cells expressing {Delta}pro were analyzed by SDS-PAGE and immunoblotting with anti-BiP and anti-Ssb1p antibodies. D, der1{Delta} (KNY13) and cue1{Delta} (KNY14) cells expressing {Delta}pro were metabolically labeled for 5 min and chased for the indicated times. {Delta}pro was recovered from 2.0 A600 eq of cells by immunoprecipitation with anti-RNAP-I antibodies (1st antibody). The immunoprecipitates were solubilized, and aliquots were subjected to the second round of immunoprecipitation with anti-{alpha}1,3-mannose antibodies (2nd antibody). The immunoprecipitates were analyzed by SDS-PAGE and radioimaging. A small fraction of {Delta}pro was precipitated irrespective of the presence of anti-{alpha}1,3-mannose antibodies (data not shown) due to aggregate formation of {Delta}pro (42).

 
When extracellular medium was analyzed for {Delta}pro with anti-RNAP-I antibodies, m{Delta}pro, but not unmodified {Delta}pro, was detected in the medium for der1{Delta} and cue1{Delta} cells (Fig. 8A, lanes 8 and 9). m{Delta}pro in the medium bound to ConA-Sepharose beads, confirming that m{Delta}pro indeed contained O-linked mannose (Fig. 8B, lanes 5 and 6). As a control, when {Delta}pro was expressed in pmt2{Delta}der1{Delta} and pmt2{Delta}cue1{Delta} cells, m{Delta}pro was not detected in the cell extract or in the medium (Fig. 8A, lanes 11 and 12). An ER chaperone BiP and a cytosolic chaperone Ssb1p were not detected in the medium for der1{Delta} and cue1{Delta} cells (Fig. 8C), ruling out the trivial possibility that m{Delta}pro detected in the medium was due to cell lysis.

In parallel, we confirmed the ER-to-Golgi transport of m{Delta}pro by analyzing its Golgi-specific glycosylation by using anti-{alpha}1,3-mannose antibodies. der1{Delta} and cue1{Delta} cells expressing {Delta}pro under the control of the GAL1 promoter were pulse-labeled, chased, and subjected to immunoprecipitation with anti-RNAP-I antibodies to precipitate {Delta}pro and m{Delta}pro and subsequently with the anti-{alpha}1,3-mannose antibodies to precipitate proteins with the Golgi-specific addition of {alpha}1,3-mannose. m{Delta}pro was immunoprecipitated with anti-{alpha}1,3-mannose antibodies (Fig. 8D, lanes 4 and 8), suggesting that m{Delta}pro was indeed modified by the late Golgi enzymes. Therefore, m{Delta}pro was secreted to the extracellular medium via the normal secretory pathway through the Golgi complex.

Mutations Affecting ERAD and O-Mannosylation Cause Synthetic Phenotypes—Partial recovery of the normal transport of aberrant proteins by O-mannosylation reflects their disengagement from interactions with BiP or other ER chaperones, which usually prevents further secretion through the organelles of the secretory pathway. However, although they are released from ER chaperones, a large fraction of the O-mannosylated forms appear to remain in the ER (data not shown). Nevertheless, since the O-mannosylated proteins remain soluble irrespective of functional BiP, O-mannosylation may reduce the load for ER chaperones significantly. We tested this possibility by monitoring the effects of O-mannosylation on the UPR. Under normal growth conditions, defects in the ERAD lead to an increase in the amounts of nonnative proteins in the ER, which results in constitutive and moderate activation of the UPR (50, 51). If O-mannosylation contributes to the ER quality control by solubilization of aberrant proteins in the ER when ERAD is blocked, deletion of genes involved both in O-mannosylation and in ERAD should further activate the UPR. We thus monitored the UPR using a {beta}-galactosidase reporter under the control of the promoter containing the UPR-response element (Fig. 9A). Whereas deletion of the DER1 or PMT2 gene alone led to moderate induction of the {beta}-galactosidase activity, pmt2{Delta}der1{Delta} and pmt2{Delta}cue1{Delta} mutants displayed significant increase in the {beta}-galactosidase activity. This result suggested that simultaneous deletion of the DER1/CUE1 and PMT2 genes caused synergistic activation of the UPR.



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 9.
Impairment of O-mannosylation and ERAD causes synthetic phenotypes. A, wild-type (R27-7C-1C), der1{Delta} (KNY48), cue1{Delta} (KNY49), pmt2{Delta} (KNY50), pmt2{Delta}der1{Delta} (KNY51), and pmt2{Delta}cue1{Delta} (KNY52) cells were transformed with pSCZ-Y (UPR-reporter construct) and grown in SD medium to the early logarithmic phase and {beta}-galactosidase activity was measured. The values are means ± S.D. B, subcellular localization of BiP and {Delta}pro by indirect immunofluorescence. Cells of wild-type (KUY90; a, e, and i), der1{Delta} (KNY13; b, f, and j), pmt2{Delta} (KNY40; c, g, and k), and pmt2{Delta}der1{Delta} (KNY41; d, h, and l) expressing {Delta}pro from the chromosome under the GAL1 promoter were fixed and subjected to immunofluorescence microscopy using anti-RNAP-I (a–d), anti-BiP (e–h), and anti-PDI (i–l) antibodies. The arrowheads show a dotlike distribution along the perinuclear structure. These structures probably represent aggregates formed by {Delta}pro with BiP, which was neither degraded nor O-mannosylated but accumulated in the ER lumen. Bar, 2 µm.

 
Umebayashi et al. (42) reported previously that {Delta}pro, which was overexpressed from a multicopy plasmid under the GAL1 promoter, formed aggregates containing BiP but not PDI. In addition, immunoelectron microscopic analyses revealed that the aggregates of {Delta}pro were visible as electron-dense regions in the ER and nuclear envelope. In this study, although {Delta}pro is expressed from the chromosome under the GAL1 promoter, we reasoned that {Delta}pro, which was neither degraded nor O-mannosylated in pmt2{Delta}der1{Delta} and pmt2{Delta}cue1{Delta} cells, became insoluble and accumulated in the ER lumen. When wild-type cells expressing {Delta}pro were stained with anti-RNAP-I, anti-BiP, and anti-PDI antibodies, indirect immunofluorescence microscopy showed perinuclear staining with several extensions in the cytoplasm (Fig. 9B, a, e, and i). This staining is typical for yeast ER proteins and suggests that {Delta}pro is distributed uniformly in the ER lumen at this expression level of {Delta}pro. However, 35% of der1{Delta} cells (b), 35% of pmt2{Delta} cells (c), and 76% of pmt2{Delta}der1{Delta} cells (d) showed a dotlike distribution along the perinuclear structure, when stained with anti-RNAP-I antibodies (arrowhead in d). When stained with anti-BiP antibodies, 25% of der1{Delta} cells (f), 33% of pmt2{Delta} cells (g), and 90% of pmt2{Delta}der1{Delta} cells (h) showed similar distribution patterns (arrowhead in h). Since anti-PDI antibodies exhibited normal staining of ER proteins (j–l), the dotlike subcellular distribution is specific for {Delta}pro and the ER chaperone BiP, but not for PDI. These structures probably represent aggregates formed by {Delta}pro with BiP, which was neither degraded nor O-mannosylated but accumulated in the ER lumen, as observed in the previous study (42).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we analyzed the consequence of the ER O-mannosylation of model nonnative proteins in the ERAD. We found, by using N-glycosylation site mutants of p{alpha}F as aberrant proteins, that removal of N-glycosylation sites decreased the solubility of p{alpha}F, but in turn, subsequent O-mannosylation regained the solubility. Aberrant substrate proteins have to be soluble, hence require BiP or ER chaperones, for receiving O-mannosylation. However, once they have become O-mannosylated, they do not require BiP to remain soluble. We also demonstrated that {Delta}pro, an aberrant secretory protein derivative that lacks its pro-sequence, was O-mannosylated in the ER both in vivo and in vitro and became soluble.

Since "early aggregate" of misfolded proteins are often toxic rather than simply nonfunctional (52), the cell possesses a protein quality control system to remove aggregation-prone proteins (1, 8, 53). Avoidance of early aggregate is achieved by association with molecular chaperones that shield the exposed hydrophobic surfaces from one another or by degradation of the proteins into small peptides. Attachment of hydrophilic glycans to incompletely folded or misfolded proteins would also contribute to the avoidance of aggregate formation by increasing their solubilities, as shown in the present study. In the case of p{alpha}F mutants, serine and threonine residues, potential O-mannosylation acceptor sites, are clustered in its pro-region, where N-glycosylation sites are also localized in wild-type p{alpha}F (54), so that O-mannosylation may well compensate the lowered solubility due to failure of N-glycosylation. Reduced solubility of {Delta}pro due to deletion of the pro-region of RNAP-I could be also cured by simply attaching highly hydrophilic mannosyl chains. In this connection, it is interesting to note that O-mannosylation by Pmt proteins has a preference for unfolded substrates but not for fully folded proteins (18, 23). This raises an attractive hypothesis that Pmt proteins may be, like glucosyl transferase in the ER lumen (55), directly involved in recognition of unfolded and therefore aggregation-prone substrate proteins. On the other hand, whereas N-linked glycosylation (56) and presumably O-mannosylation of authentic substrate proteins are compatible with correct folding process of substrate proteins, O-mannosylation of aberrant proteins may interfere with their achievement of native conformations and/or assembly in the ER. Therefore, the role of O-mannosylation of aberrant proteins would be to increase solubility of the proteins, irrespective of their abilities to fold into compact native conformations.

Although the O-mannosylated form of N-glycosylation site mutants of p{alpha}F and {Delta}pro was partly secreted to the extracellular medium, the secreted fraction was not so large. It is thus unclear whether clearance of aberrant proteins from the ER along the secretory pathway is the major biological consequence of O-mannosylation. Rather, a more likely scenario could be that O-mannosylation functions as a mechanism to avoid accumulation and subsequent aggregate formation of aggregation-prone proteins in the ER, which could be otherwise detrimental to cell survival. The aberrant proteins that failed to proceed to degradation and were retained in the ER are instead O-mannosylated and remain soluble without the assistance of ER molecular chaperones, which are up-regulated by UPR in response to accumulation of nonnative proteins. Although molecular chaperones are major components that prevent aggregation of aberrant proteins and the ERAD is the major pathway to eliminate them, O-mannosylation of aggregation-prone proteins could contribute to reduction of the load of the ER stress for the ER protein quality control systems. Indeed, deletions of the ERAD-related genes, DER1 and CUE1, and the PMT2 gene led to aggregate formation of {Delta}pro involving BiP when {Delta}pro was overexpressed and showed synergic activation of the UPR (Fig. 9B). In addition, PMT1-4 genes are transcriptionally up-regulated by the UPR (51). These observations suggest that O-mannosylation as well as the ERAD eliminates nonnative misfolded proteins whose accumulation increases the load for ER chaperones and triggers the UPR. In other words, O-mannosylation may 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.

Is our observation of solubilization of aberrant proteins by O-mannosylation biologically relevant to normal cell functions? Although we analyzed O-mannosylation of only two proteins, p{alpha}F and {Delta}pro, under conditions where the ERAD was more or less compromised, many endogenous proteins were reported to be O-mannosylated when N-glycosylation was inhibited by tunicamycin treatment (23). Besides, we found that impairment of O-mannosylation by deletion of the PMT2 gene alone led to activation of the UPR even under normal growth conditions (Fig. 9A). This result suggests that stochastically generated misfolded proteins in the ER of wild-type cells may be handled with partly by O-mannosylation. Alternatively, the inactivation of Pmt2p could prevent O-mannosylation of authentic substrate proteins for Pmt2p, which may lead to an increase in the load for the ER quality control system. Nevertheless, the latter interpretation raises the possibility that the role of O-mannosylation of authentic substrate proteins is also to enhance their activity and efficient secretion by preventing their aggregation. This hypothesis can be tested in future studies.


   FOOTNOTES
 
* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology and by a grant from the Japan Science and Technology Agency (to T. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ A Research Fellow of the Japan Society for the Promotion of Science. Present address: Dept. of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260. Back

§§ To whom correspondence should be addressed: Dept. of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. Tel.: 81-52-789-2490; Fax: 81-52-789-2947; E-mail: endo{at}biochem.chem.nagoya-u.ac.jp.

1 The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; UPR, unfolded protein response; pp{alpha}F, prepro-{alpha}-factor; p{alpha}F, pro-{alpha}-factor; RNAP-I, R. niveus aspartic proteinase-I; {Delta}pro, pro-region-deleted derivative of RNAP-I; p{Delta}pro, the precursor form of {Delta}pro; PDI, protein-disulfide isomerase; GDPMan, GDP-mannose; ConA, concanavalin A. Back

2 S. Nishikawa, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank the members of the Endo laboratory for valuable discussions. We are grateful to J. L. Brodsky for the plasmid for in vitro translation of p(QQQ)p{alpha}F, A. Nakano for anti-pp{alpha}F antibodies and anti-{alpha}-1,3-mannose antibodies, H. Tachikawa for anti-PDI antibodies, and K. Mori for the plasmid for the UPR reporter assay (pSCZ-Y).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ellgaard, L., and Helenius, A. (2003) Nat. Rev. Mol. Cell Biol. 4, 181–191[CrossRef][Medline] [Order article via Infotrieve]
  2. Brodsky, J. L., and McCracken, A. A. (1997) Trends Cell Biol. 7, 151–156[Medline] [Order article via Infotrieve]
  3. Plemper, R. K., and Wolf, D. H. (1999) Trends Biochem. Sci. 24, 266–270[CrossRef][Medline] [Order article via Infotrieve]
  4. Fewell, S. W., Travers, K. J., Weissman, J. S., and Brodsky, J. L. (2001) Annu. Rev. Genet. 35, 149–1915[CrossRef][Medline] [Order article via Infotrieve]
  5. Nishikawa, S., Fewell, S. W., Kato, Y., Brodsky, J. L., and Endo, T. (2001) J. Cell Biol. 153, 1061–1069[Abstract/Free Full Text]
  6. Silberstein, S., Schlenstedt, G., Silver, P. A., and Gilmore, R. (1998) J. Cell Biol. 143, 921–933[Abstract/Free Full Text]
  7. Helenius, A., and Aebi, M. (2001) Science 291, 2364–2369[Abstract/Free Full Text]
  8. Kostova, Z., and Wolf, D. H. (2003) EMBO J. 22, 2309–2317[CrossRef][Medline] [Order article via Infotrieve]
  9. Nakatsukasa, K., Nishikawa, S., Hosokawa, N., Nagata, K., and Endo, T. (2001) J. Biol. Chem. 276, 8635–8638[Abstract/Free Full Text]
  10. Jakob, C. A., Bodmer, D., Spirig, U., Bättig, P., Marcil, A., Dignard, D., Bergeron, J. J., Thomas, D. Y., and Aebi, M. (2001) EMBO Rep. 2, 423–430[Medline] [Order article via Infotrieve]
  11. Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A., and Nagata, K. (2001) EMBO Rep. 2, 415–422[Medline] [Order article via Infotrieve]
  12. Gillece, P., Luz, J. M., Lennarz, W. J., de la Cruz, F. J., and Römisch, K. (1999) J. Cell Biol. 147, 1443–1456[Abstract/Free Full Text]
  13. Tsai, B., Ye, Y., and Rapoport, T. A. (2002) Nat. Rev. Mol. Cell Biol. 3, 246–255[CrossRef][Medline] [Order article via Infotrieve]
  14. Liley, B. N., and Ploegh, H. L. (2004) Nature 429, 834–840[CrossRef][Medline] [Order article via Infotrieve]
  15. Ye, Y., Shibata, Y., Yun, C., Ron, D., and Rapoport, T. A. (2004) Nature 429, 841–847[CrossRef][Medline] [Order article via Infotrieve]
  16. Ye, Y., Meyer, H. H., and Rapoport, T. A. (2001) Nature 414, 652–656[CrossRef][Medline] [Order article via Infotrieve]
  17. Kaufman, R. J., Scheuner, D., Schroder, M., Shen, X., Lee, K., Liu, C. Y., and Arnold, S. M. (2002) Nat. Rev. Mol. Cell Biol. 3, 411–421[CrossRef][Medline] [Order article via Infotrieve]
  18. Strahl-Bolsinger, S., Gentzsch, M., and Tanner, W. (1999) Biochim. Biophys. Acta. 1426, 297–307[Medline] [Order article via Infotrieve]
  19. Proszynski, T. J., Simons, K., and Bagnat, M. (2004) Mol. Biol. Cell. 15, 1533–1543[Abstract/Free Full Text]
  20. Gentzsch, M., and Tanner, W. (1996) EMBO J. 15, 5752–5759[Medline] [Order article via Infotrieve]
  21. Sanders, S. L., Gentzsch, M., Tanner, W., and Herskowitz, I. (1999) J. Cell Biol. 145, 1177–1188[Abstract/Free Full Text]
  22. Lommel, M., Bagnat, M., and Strahl, S. (2004) Mol. Cell Biol. 24, 46–57[Abstract/Free Full Text]
  23. Harty, C., Strahl, S., and Römisch, K. (2001) Mol. Biol. Cell 12, 1093–1101[Abstract/Free Full Text]
  24. Coughlan, C. M., Walker, J. L., Cochran, J. C., Wittrup, K. D., and Brodsky, J. L (2004) J. Biol. Chem. 279, 15289–15297[Abstract/Free Full Text]
  25. Vashist, S., Kim, W., Belden, W. J., Spear, E. D., Barlowe, C., and Ng, D. T. (2001) J. Cell Biol. 155, 355–368[Abstract/Free Full Text]
  26. Schülke, N., and Schmid, F. X. (1988) J. Biol. Chem. 263, 8832–8837[Abstract/Free Full Text]
  27. Wojczyk, B. S., Stwora-Wojczyk, M., Shakin-Eshleman, S., Wunner, W. H., and Spitalnik, S. L. (1998) Glycobiology 8, 121–130[Abstract/Free Full Text]
  28. Ioannou, Y. A., Zeidner, K. M., Grace, M. E., and Desnick, R. J. (1998) Biochem. J. 332, 789–797[Medline] [Order article via Infotrieve]
  29. Jones, J. S., and Prakash, L. (1990) Yeast 6, 363–366[CrossRef][Medline] [Order article via Infotrieve]
  30. Brachman, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998) Yeast 14, 115–132[CrossRef][Medline] [Order article via Infotrieve]
  31. Knop, M., Finger, A., Braun, T., Hellmuth, K., and Wolf, D. H. (1996) EMBO J. 15, 753–763[Medline] [Order article via Infotrieve]
  32. Biederer, T., Volkwein, C., and Sommer, T. (1997) Science 278, 1806–1809[Abstract/Free Full Text]
  33. Hansen, W., Garcia, P. D., and Walter, P. (1986) Cell 45, 397–406[CrossRef][Medline] [Order article via Infotrieve]
  34. McCracken, A. A., and Brodsky, J. L. (1996) J. Cell Biol. 132, 291–298[Abstract/Free Full Text]
  35. Sikorski, R. S., and Hieter, P. (1989) Genetics 122, 19–27[Abstract/Free Full Text]
  36. Nishikawa, S., and Nakano, A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8179–8183[Abstract/Free Full Text]
  37. Nishikawa, S., and Nakano, A. (1991) Biochim. Biophys. Acta 1093, 135–143[Medline] [Order article via Infotrieve]
  38. Umebayashi, K., Fukuda, R., Hirata, A., Horiuchi, H., Nakano, A., Ohta, A., and Takagi, M. (2001) J. Biol. Chem. 276, 41444–41454[Abstract/Free Full Text]
  39. Kaiser, C., Michaelis, S., and Mitchell, A. (1994) Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  40. Nishikawa, S., Hirata, A., and Nakano, A. (1994) Mol. Biol. Cell 5, 1129–1143[Abstract]
  41. Nishikawa, S., and Endo, T. (1997) J. Biol. Chem. 272, 12889–12892[Abstract/Free Full Text]
  42. Umebayashi, K., Hirata, A., Fukuda, R., Horiuchi, H., Ohta, A., and Takagi, M. (1997) Yeast 13, 1009–1020[CrossRef][Medline] [Order article via Infotrieve]
  43. Pilon, M., Schekman, R., and Römisch, K. (1997) EMBO J. 16, 4540–4548[CrossRef][Medline] [Order article via Infotrieve]
  44. Lee, R. J., Liu, C., Harty, C., McCracken, A. A., Latterich, M., Römisch, K., DeMartino, G. N., Thomas, P. J., and Brodsky, J. L. (2004) EMBO J. 23, 2206–2215[CrossRef][Medline] [Order article via Infotrieve]
  45. Caplan, S., Green, R., Rocco, J., and Kurjan, J. (1991) J. Bacteriol. 173, 627–635[Abstract/Free Full Text]
  46. Sprague, G. F. Jr (1991) Methods Enzymol. 194, 77–93[Medline] [Order article via Infotrieve]
  47. Fukuda, R., Umebayashi, K., Horiuchi, H., Ohta, A., and Takagi, M. (1996) J. Biol. Chem. 271, 14252–14255[Abstract/Free Full Text]
  48. Lee, D. H., and Goldberg, A. L. (1996) J. Biol. Chem. 271, 27280–27284[Abstract/Free Full Text]
  49. Papa, F. R., and Hochstrasser, M. (1993) Nature 366, 313–319[CrossRef][Medline] [Order article via Infotrieve]
  50. Friedlander, R., Jarosch, E., Urban, J., Volkwein, C., and Sommer, T. (2000) Nat. Cell Biol. 2, 379–384[CrossRef][Medline] [Order article via Infotrieve]
  51. Travers, K. J., Patil, C. K., Wodicka, L., Lockhart, D. J., Weissman, J. S., and Walter, P. (2000) Cell 101, 249–258[CrossRef][Medline] [Order article via Infotrieve]
  52. Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., and Stefani, M. (2002) Nature 416, 507–511[CrossRef][Medline] [Order article via Infotrieve]
  53. Trombetta, E. S., and Parodi, A. J. (2003) Annu. Rev. Cell Dev. Biol. 19, 649–676[CrossRef][Medline] [Order article via Infotrieve]
  54. Kurjan, J., and Herskowitz, I. (1982) Cell 30, 933–943[CrossRef][Medline] [Order article via Infotrieve]
  55. Ritter, C., and Helenius, A. (2000) Nat. Struct. Biol. 7, 278–280[CrossRef][Medline] [Order article via Infotrieve]
  56. Imperiali, B., and O'Connor, S. E. (1999) Curr. Opin. Chem. Biol. 3, 643–649[CrossRef][Medline] [Order article via Infotrieve]
  57. Horiuchi, H., Ashikari, T., Amachi, T., Yoshizumi, H., Takagi, M., and Yano, K. (1990) Agric. Biol. Chem. 54, 1771–1779[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 GlycobiologyHome page
M. Lommel and S. Strahl
Protein O-mannosylation: Conserved from bacteria to humans
Glycobiology, August 1, 2009; 19(8): 816 - 828.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
A. Murakami-Sekimata, K. Sato, K. Sato, A. Takashima, and A. Nakano
O-Mannosylation is required for the solubilization of heterologously expressed human beta-amyloid precursor protein in Saccharomyces cerevisiae.
Genes Cells, February 1, 2009; 14(2): 205 - 215.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
T. Makio, S.-i. Nishikawa, T. Nakayama, H. Nagai, and T. Endo
Identification and characterization of a Jem1p ortholog of Candida albicans: dissection of Jem1p functions in karyogamy and protein quality control in Saccharomyces cerevisiae
Genes Cells, October 1, 2008; 13(10): 1015 - 1026.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
H. Hirayama, M. Fujita, T. Yoko-o, and Y. Jigami
O-Mannosylation is Required for Degradation of the Endoplasmic Reticulum-associated Degradation Substrate Gas1*p via the Ubiquitin/Proteasome Pathway in Saccharomyces cerevisiae
J. Biochem., April 1, 2008; 143(4): 555 - 567.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
K. Kuroda, K. Kobayashi, Y. Kitagawa, T. Nakagawa, H. Tsumura, T. Komeda, D. Shinmi, E. Mori, K. Motoki, K. Fuju, et al.
Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta
Appl. Envir. Microbiol., January 15, 2008; 74(2): 446 - 453.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Manya, T. Suzuki, K. Akasaka-Manya, H.-K. Ishida, M. Mizuno, Y. Suzuki, T. Inazu, N. Dohmae, and T. Endo
Regulation of Mammalian Protein O-Mannosylation: PREFERENTIAL AMINO ACID SEQUENCE FOR O-MANNOSE MODIFICATION
J. Biol. Chem., July 13, 2007; 282(28): 20200 - 20206.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
S. Ito-Harashima, K. Kuroha, T. Tatematsu, and T. Inada
Translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization by proteasome in yeast
Genes & Dev., March 1, 2007; 21(5): 519 - 524.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
M. Takeuchi, Y. Kimata, A. Hirata, M. Oka, and K. Kohno
Saccharomyces cerevisiae Rot1p Is an ER-Localized Membrane Protein That May Function with BiP/Kar2p in Protein Folding.
J. Biochem., March 1, 2006; 139(3): 597 - 605.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
M. Fujita, T. Yoko-o, and Y. Jigami
Inositol Deacylation by Bst1p Is Required for the Quality Control of Glycosylphosphatidylinositol-anchored Proteins
Mol. Biol. Cell, February 1, 2006; 17(2): 834 - 850.
[Abstract] [Full Text] [PDF]

Home page
 MicrobiologyHome page
T. Oka, Y. Sameshima, T. Koga, H. Kim, M. Goto, and K. Furukawa
Protein O-mannosyltransferase A of Aspergillus awamori is involved in O-mannosylation of glucoamylase I
Microbiology, November 1, 2005; 151(11): 3657 - 3667.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
S.-i. Nishikawa, J. L. Brodsky, and K. Nakatsukasa
Roles of Molecular Chaperones in Endoplasmic Reticulum (ER) Quality Control and ER-Associated Degradation (ERAD)
J. Biochem., May 1, 2005; 137(5): 551 - 555.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/48/49762    most recent
M403234200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakatsukasa, K.
Right arrow Articles by Endo, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakatsukasa, K.
Right arrow Articles by Endo, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement