Translation Rate of Human Tyrosinase Determines ItsN-Linked Glycosylation Level
2001; Elsevier BV; Volume: 276; Issue: 8 Linguagem: Inglês
10.1074/jbc.m009203200
ISSN1083-351X
AutoresAndrea Újvári, Rebecca Aron, Thomas Eisenhaure, Elaine Cheng, H.A. Parag, Yoel Smicun, Ruth Halaban, Daniel N. Hebert,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoTyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or sevenN-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2–3-fold faster (7.7–10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinaseN-linked glycosylation, as well as other potential cotranslational maturation events. Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or sevenN-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2–3-fold faster (7.7–10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinaseN-linked glycosylation, as well as other potential cotranslational maturation events. endoplasmic reticulum azetidine-2-carboxylic acid cycloheximide deoxynojirimycin green fluorescent protein murine major histocompatibility complex class I molecule Kbsignal sequence oligosaccharyl transferase polyacrylamide gel electrophoresis rough ER rabbit reticulocyte lysate human tyrosinase tyrosinase with 6 glycans tyrosinase with 7 glycans untranslocated tyrosinase wheat germ 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Whereas prokaryotic proteins fold posttranslationally due to their rapid rate of translation, the maturation of nascent proteins in eukaryotic cells often begins cotranslationally as a vectorial process and continues posttranslationally after the release of the protein from the ribosome (1Chen W. Helenius J. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. 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Because key protein maturation events for eukaryotic cells occur cotranslationally and have a large impact on the fidelity of the overall maturation process, it is important to fully understand these cotranslational processes. For proteins that traverse the secretory pathway, the cotranslational processes include the translocation of the protein across the endoplasmic reticulum (ER)1membrane, the site of entry into the secretory pathway. In this case, protein folding commences upon emergence of the polypeptide chain into the lumen of the ER. The ER is an organelle that specializes in the efficient folding, modification, and assembly of proteins to their native structures prior to their packaging into transport vesicles. The milieu of the ER is topologically equivalent to the extracellular space, with oxidizing conditions permitting the cotranslational and posttranslational formation of disulfide bonds (1Chen W. Helenius J. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. 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The biosynthesis of melanin takes place in post-Golgi endomembranous compartments called melanosomes or pigmented granules. Mutational analysis of tyrosinase-positive albinism has identified AP-3 as an important protein involved in the sorting of tyrosinase in thetrans-Golgi to melanosomes (26–28, reviewed in Ref. 29Spritz R.A. Trends Genet. 1999; 15: 337-340Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, additional key sorting decisions for tyrosinase are made in the early secretory pathway that are critical for pigmentation (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar, 31Halaban R. Svedine S. Cheng E. Aron R. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5889-5894Crossref PubMed Scopus (156) Google Scholar, 32Petrescu S.M. Branza-Nichita N. Negroiu G. Petrescu A.J. Dwek R.A. Biochemistry. 2000; 39: 5229-5237Crossref PubMed Scopus (46) Google Scholar). Tyrosinase is a membrane glycoprotein with an N-terminal signal sequence that targets the protein to the ER (Fig. 1 A) (33Kwon B.S. Haq A.K. Pomerantz S.H. Halaban R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7473-7477Crossref PubMed Scopus (396) Google Scholar, 34Ruppert S. Muller G. Kwon B. Schutz G. EMBO J. 1988; 7: 2715-2722Crossref PubMed Scopus (141) Google Scholar, 35Bouchard B. Fuller B.B. Vijayasaradhi S. Houghton A.N. J. Exp. Med. 1989; 169: 2029-2042Crossref PubMed Scopus (158) Google Scholar, 36Yamamoto H. Takeuchi S. Kudo T. Sato C. Takeuchi T. Jpn. J. Genet. 1989; 64: 121-135Crossref PubMed Scopus (94) Google Scholar). The human protein possesses 7 putative N-linked glycosylation sites and 15 lumenal Cys residues that can participate in disulfide bond formation. Mutations in tyrosinase are the cause of tyrosinase-negative oculocutaneous albinism 1, an autosomal recessive genetic disorder characterized by the absence of melanin (reviewed in Refs. 37Oetting W.S. King R.A. Hum. Mutat. 1999; 13: 99-115Crossref PubMed Scopus (280) Google Scholar and 38King R.A. Nordlund J.J. Boissy R. Hearing V.J. King R.A. Ortonne J.-P. The Pigmentary System. Physiology and Pathophysiology. 1st Ed. Oxford University Press, New York1998Google Scholar). The mutant protein in several tyrosinase-negative albino melanocytes of human and mouse origin is retained in the ER (31Halaban R. Svedine S. Cheng E. Aron R. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5889-5894Crossref PubMed Scopus (156) Google Scholar,39Berson J.F. Frank D.W. Calvo P.A. Bieler B.M. Marks M.S. J. Biol. Chem. 2000; 275: 12281-12289Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The essential role of the ER in the regulation of tyrosinase has also been demonstrated in amelanotic melanoma cells in which ER retention of wild type tyrosinase leads to subsequent degradation by the 26 S proteasome (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). Here, we demonstrate that the faster rate of translation of tyrosinase in melanoma cells than that of normal melanocytes hampered glycosylation at the inefficient Asn-290 site. This was determined by studying tyrosinase glycosylation and maturation under conditions that altered the rate of translation in normal melanocytes and melanoma cells, as well as in a cell-free system that recapitulated the ER processes. Rabbit reticulocyte lysate (RRL), wheat germ (WG), dithiothreitol, and RNasin were from Promega Corp. (Madison, WI). Canine pancreas microsomes were a generous gift from Dr. R. Gilmore (Worcester, MA). [35S]Methionine/cysteine (EasyTag) and CHAPS were from PerkinElmer Life Sciences and Pierce, respectively. Restriction endonucleases and ribonucleotide triphosphates were from New England Biolabs, Inc. (Beverly, MA). mMessage mMachine and T7 transcription kits were from Ambion (Austin, TX), and the QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). Zysorbin (fixed and killedStaphylococcus aureus) was obtained from Zymed Laboratories Inc. (San Francisco, CA). All other reagents, including the anti-FLAG M2 monoclonal antibody, were from Sigma. The plasmid pcTYR carrying the human tyrosinase gene (GenBankTM accession number Y00819) was a gift from Dr. R. Spritz (Denver, CO). The EcoRI tyrosinase fragment excised from pcTYR was subcloned into pGEM 7Zf (Promega). To improvein vitro translation/translocation, the original signal sequence of tyrosinase was exchanged with the murine major histocompatibility complex class I molecule Kbsignal sequence as follows. A XbaI restriction site was introduced at the end of the 18-amino acid signal sequence in pGEM 7Zf-TYR plasmid by site-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene). The tyrosinase XbaI fragment (encoding the full sequence of tyrosinase minus the signal peptide) was inserted into the XbaI restriction site of pSP72/KbSS-CD3δ plasmid (from Drs. J. Huppa and H. Ploegh, Boston, MA) (40Huppa J.B. Ploegh H.L. J. Exp. Med. 1997; 186: 393-403Crossref PubMed Scopus (82) Google Scholar). This created a hybrid molecule comprising the class I heavy chain H2-Kb signal peptide in frame with the tyrosinase protein downstream of the T7 promoter, termed pSP72/KbSS-TYR. To generate single-site glycosylation deletion mutant proteins, the consensus N-linked glycosylation sites Asn-X-Thr/Ser in pSP72/KbSS-TYR were eliminated or modified in the cDNA by changing threonine or serine to an alanine, except for Thr-373, which was changed to the albino mutation T373K. The site at Asn-290 was modified by exchanging the proline at position 293 to alanine (P293A). The P293A and T292A mutations were also introduced to tyrosinase cDNA in the enhanced green fluorescent protein (enhanced GFP) vector (31Halaban R. Svedine S. Cheng E. Aron R. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5889-5894Crossref PubMed Scopus (156) Google Scholar). In addition, the wild type and mutant cDNAs were subcloned from the enhanced GFP plasmids into theKpnI/HindII cloning sites of p3XFLAG-CMV-14 expression vector (Sigma) to generate FLAG-tagged tyrosinase proteins. In all cases, DNA sequencing of the entire tyrosinase gene verified the inserted mutations. Transfection of plasmids into mouse melanocytes was done as described (31Halaban R. Svedine S. Cheng E. Aron R. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5889-5894Crossref PubMed Scopus (156) Google Scholar). Messenger RNA was prepared by in vitro run-off transcription of the cDNA that was linearized with NdeI or HaeII, according to the manufacturer's instructions. Radioactive35S-labeled tyrosinase was translated for 1 h at 27 °C in RRL (14Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar) or in WG and translocated cotranslationally into canine pancreas microsomes. In the latter case, WG lysate (40 μl) was mixed with ribonuclease-treated rough ER (RER) microsomes (6 μl), an amino acid mixture lacking methionine and cysteine (6 μl of a 1 mm solution of each), [35S]Met/Cys (63 μCi), RNase-free water (18.4 μl), RNase inhibitor (1.6 μl), and mRNA (1 μg/μl, 3.5 μl). Samples were alkylated withN-ethylmaleimide (20 mm) to block free sulfhydryls (7Braakman I. Hoover-Litty H. Wagner K.R. Helenius A. J. Cell Biol. 1991; 114: 401-411Crossref PubMed Scopus (252) Google Scholar). Alkylated samples were either analyzed directly by SDS-PAGE or immunoprecipitated with anti-tyrosinase antibodies (α-TYR) prior to electrophoresis. Deoxynojirimycin (DNJ) (0.5 mm) was used to inhibit ER glucosidases when indicated. Half of each sample was subjected to nonreducing SDS-PAGE, and the other half was reduced by the addition of dithiothreitol (100 mm) and resolved by reducing SDS-PAGE. Semipermeabilized cells were prepared from subconfluent mouse B10BR melanocytes (41Bennett D.C. Cooper P.J. Dexter T.J. Devlin L.M. Heasman J. Nester B. Development. 1989; 105: 379-385Crossref PubMed Google Scholar) permeabilized with 20 μg/ml digitonin using a method described previously by Wilson et al. (42Wilson R. Allen A.J. Oliver J. Brookman J.L. High S. Bulleid N.J. Biochem. J. 1995; 307: 679-687Crossref PubMed Scopus (133) Google Scholar). Radioactive35S-labeled tyrosinase was translated for 1 h at 27 °C in RRL with semipermeabilized cells (1.3 × 104 cells/μl) replacing the RER microsomes. To separate glycosylated (TYR) from nonglycosylated (untranslocated (UTYR)) proteins, 10 μl of the translation mixture was solubilized in 200 μl of 2% CHAPS buffer (2% CHAPS, 50 mm HEPES, 200 mm sodium chloride, pH 7.5), and glycoproteins were captured on wheat germ agglutinin-bound beads at 4 °C for 2 h under constant rotation. The beads were pelleted by centrifugation at 2500 × g and washed once with 0.5% CHAPS buffer. The bound proteins were then eluted in SDS-sample buffer at 95 °C for 5 min. Alternatively, untranslocated tyrosinase was separated from the translocated protein by centrifugation of the translation products (10 μl) through a sucrose cushion (100 μl, 0.5m sucrose, 50 mm triethanolamine, 1 mm dithiothreitol, pH 7.4) in a Beckman Airfuge ultracentrifuge for 10 min. For protease protection, translation products were digested with proteinase K (0.35 μg/μl) in the absence or presence of 1% Triton X-100 for 1 h on ice. Protease digestion was stopped with 10 mm phenylmethylsulfonyl fluoride. Samples were added to a 100-μl solution of 0.1m Tris, 1% SDS (pH 8) and heated at 95 °C for 10 min. Translation mixtures were centrifuged through a sucrose cushion, and pellets were solubilized in a solution containing 0.2% SDS, 100 mm sodium phosphate, 25 mm EDTA (pH 6.9) at 95 °C for 5 min. The samples were cooled to room temperature, diluted with 2% Triton X-100 in 100 mm sodium phosphate, 25 mm EDTA (pH 6.9), and digested with 0.1–1 μl of 1 units/μl PNGase F at 37 °C for the indicated times. 35S-Labeled tyrosinase was immunoprecipitated with anti-tyrosinase antibodies (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). To determine radioactivity in total proteins, lysates were blotted on filter paper and treated with 10% trichloroacetic acid on ice for 30 min. Samples were washed with ice-cold 100% ethanol. The radioactivity of the blots was determined in a liquid scintillation counter. The approximate concentration of TYR in the in vitro translation reactions was determined as described below. Radioactive samples labeled with [35S]Met/Cys were fully resolved by SDS-PAGE to quantify the intensity of the translocated tyrosinase band by phosphorimaging. Equivalent samples were also loaded on the same gel after the run was 70% completed. Therefore, the samples were not resolved and the intensity of the total free label in the reaction could be determined. Because tyrosinase has a total of 28 Met and Cys, its approximate concentration is the following: [Tyr] = (T/F) × [Met/Cys]/28, where Tis the intensity of the tyrosinase band and F is the intensity of the total free label. [Met/Cys] is the sum of the concentrations of endogenous reticulocyte lysate (3.9 μm) and the radioactive [35S]Met/Cys (0.9 μm). Normal human melanocytes were cultured from newborn foreskins in Ham's F-10 medium supplemented with 7% fetal bovine serum and several ingredients required for their proliferation, including 12-O-tetradecanoylphorbol-13-acetate, 3-isobutyl-1-methylxanthine, dbcAMP (N6, 2′-O-dibutyryladenosine 3, 5-cyclic monophosphate) and cholera toxin (43Böhm M. Moellmann G. Cheng E. Alvarez-Franco M. Wagner S. Sassone-Corsi P. Halaban R. Cell Growth Differ. 1995; 6: 291-302PubMed Google Scholar). Melanoma cells strains 501 mel and YUSIT1 were grown in Ham's F-10 medium plus serum (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). Cells were starved for 2 h in Met/Cys free-RPMI 1640 medium (supplemented with 3% dialyzed calf serum, 1% glutamine, 0.1 mm3-isobutyl-1-methylxanthine, and 50 μg/ml 12-O-tetradecanoylphorbol-13-acetate), were then incubated with [35S]Met/Cys (0.7 mCi/ml) for 10 min, scraped into phosphate-buffered saline supplemented with 20 mm N-ethylmaleimide, and lysed in 2% CHAPS buffer. Cell extracts were immunoprecipitated with anti-tyrosinase antibodies (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). Newly synthesized tyrosinase from normal melanocytes migrated as a 70-kDa doublet band separated by ∼3 kDa (Fig. 1 B, lane 1) (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). An [35S]TYR doublet of similar mobility also accumulated in melanoma cells, but the intensities of the individual doublet bands varied. The ratio of the larger TYR to the smaller TYR in melanoma cells was half of that in normal melanocytes (0.4–0.5 compared with 0.8, respectively), indicating that the faster migrating form (TYRS) accumulated more predominantly in the melanoma cells. To identify the cause of the TYR doublet, TYR was expressed in a cell-free system that facilitated experimental manipulation. The translation system consisted of rabbit reticulocyte lysate and mRNA encoding human TYR in the presence and absence of isolated RER membranes (Fig. 2). The native tyrosinase mRNA or a modified message termed KbSS-TYR, in which the TYR signal sequence was exchanged with the corresponding signal sequence from murine major histocompatibility complex class I molecule Kb (KbSS) (40Huppa J.B. Ploegh H.L. J. Exp. Med. 1997; 186: 393-403Crossref PubMed Scopus (82) Google Scholar, 44Bijlmakers M.J. Neefjes J.J. Wojcik-Jacobs E.H.M. Ploegh H. Eur. J. Immunol. 1993; 23: 1305-1313Crossref PubMed Scopus (26) Google Scholar), was used. A 60-kDa protein, recognized by anti-tyrosinase antibodies, was generated in the absence of microsomes that corresponded to untranslocated and unglycosylated TYR (Fig. 2, A, lanes 1 and 5, U TYR). However, a 70-kDa doublet identical to that observed previously in intact melanocytes (30Halaban R. Chang E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar) (Fig. 1 B) accumulated in the presence of RER microsomes. This band represented translocated tyrosinase that had received multiple N-linked carbohydrates (Fig. 2 A, lanes 3 and 7). The KbSS-TYR chimera was translated and translocated 4–6-fold more efficiently than native tyrosinase (Fig. 2 A, comparelane 3 to lane 4). The untranslocated KbSS-TYR migrated slightly slower than wild type TYR (Fig.2 A, compare lane 1 to lane 2 andlane 5 to lane 6, U TYR) due to the different sizes of signal sequence (24 amino acids in KbSS and 18 amino acids in TYR). However, the mobility of the translocated products was identical (Fig. 2 A, comparelane 3 to lane 4 and lane 7 tolane 8, TYR) and was similar to newly synthesized tyrosinase produced in intact melanocytes, justifying the use of the KbSS-TYR in all subsequent cell-free experiments. The protein doublet constituted the translocated and glycosylated tyrosinase because it was precipitated with wheat germ agglutinin-bound beads (Fig. 2 B, lane 4, TYR), and it sedimented through a sucrose cushion designed to isolate only membranes decorated with ribosomes (Fig. 2 B, lane 6, TYR). Digestion with proteinase K degraded UTYR but not the TYR form, unless the microsomes were first solubilized by detergent (Fig.2 B, lanes 7–9). The downward mobility shift caused by proteinase K cleavage of the 29-amino acid cytosolic tail of the translocated form is consistent with correct insertion of the type I membrane protein into the microsomal membrane bilayer (Fig.2 B, compare lane 6 to lane 8, TYR). Taken together, the data supported the conclusion that the in vitro translated 70-kDa doublet represented ER translocated and glycosylated forms of tyrosinase. The two translocated ER isoforms of tyrosinase could be generated by heterogeneous glucose or mannose trimming of N-linked glycans in the ER or through differential recognition by the OST generating differences in the total number of attachedN-linked glycans. Differential trimming of glucose residues as the source for this variability was ruled out by the persistent production of doublet protein in the presence of DNJ, although the translated and processed products displayed slower mobility due to inhibition of glucosidases I and II activities (Fig.3 A, lanes 3 and 5). Likewise, differential ER carbohydrate trimming was excluded by the persistence of the doublet in the presence of the mannosidase inhibitor deoxymannojirimycin (data not shown). Heterogeneity in the carbohydrate side chains as the source of TYR doublet was indicated after digestion with the bacterial endoglycosidase PNGase F. This endoglycosidase removed allN-linked side chains coalescing the doublet into a single band that migrated slightly faster than the untranslocated form (Fig.3 A, lanes 6 and 7). The faster mobility of the PNGase F digested over the untranslocated form of tyrosinase (Fig.3 A, compare lanes 1 and 2 tolanes 6 and 7) was due to the cleavage of the signal sequence during RER processing. Therefore, we concluded that the doublet represented tyrosinase glycoforms with different numbers ofN-linked glycans. The number of glycans was then resolved by creating a ladder of partially cleaved PNGase F glycosidase products (Fig. 3 B). Tyrosinase was produced in the presence of DNJ to eliminate variation due to glucose trimming. Complete digestion with high concentration of PNGase F generated an unglycosylated protein (Fig. 3 B, lanes 4–6, band 0). However, PNGase F at lower concentrations produced a discrete ladder of eight bands corresponding to proteins with seven to zero glycans (Fig. 3 B, lane 3; top andbottom bands, respectively). This analysis revealed that the doublet represented tyrosinase with seven (TYR7) and six (TYR6) N-linked glycans. The partial digestion by PNGase F suggested that the doublet was generated by inefficient glycosylation of one of the consensus sites. The smaller glycoform (TYRS) could be the product of site-specific inefficient glycosylation or random vacancy of any one of the sites. Glycosylation of a consensus site could be suppressed by inefficient transfer of truncated dolichol pyrophosphate precursors, competition with disulfide bond formation, or the presence of inefficient sites (45Turco S.J. Stetson B. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4411-4414Crossref PubMed Scopus (130) Google Scholar, 46Gavel Y. von Heijne G. Protein Eng. 1990; 3: 433-442Crossref PubMed Scopus (635) Google Scholar, 47Allen S. Naim H.Y. Bulleid N.J. J. Biol. Chem. 1995; 270: 4797-4804Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 48Holst B. Bruun A.W. Kielland-Brandt M.C. Winther J.R. EMBO J. 1996; 15: 3538-3546Crossref PubMed Scopus (79) Google Scholar). The involvement of truncated dolichol pyrophosphate sugar precursor was excluded because complete glycosylation of a variety of multiglycosylated substrates has been reported with this cell-free system (14Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 49Cannon K.S. Hebert D.N. Helenius A. J. Biol. Chem. 1996; 271: 14280-14284Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 50Hebert D.N. Zhang J.-X. Chen W. Foellmer B. Helenius A. J. Cell Biol. 1997; 139: 613-623Crossref PubMed Scopus (221) Google Scholar), and the doublet appeared in cells. Furthermore, we ruled out competition by disulfide bond formation, because the doublet persisted even when tyrosinase was synthesized in the presence of the reducing agent dithiothreitol (Fig. 2 and data not shown). A clue that a proline residue had a role in the hypoglycosylation came from experiments using the proline analogue azet
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