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Ribophorin I Regulates Substrate Delivery to the Oligosaccharyltransferase Core

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Ribophorin I Regulates Substrate Delivery to the Oligosaccharyltransferase Core Ribophorin I regulates substrate delivery to the oligosaccharyltransferase core Cornelia M. Wilson, Quentin Roebuck, and Stephen High* Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom Edited by Stuart A. Kornfeld, Washington University School of Medicine, St. Louis, MO, and approved April 29, 2008 (received for review December 19, 2007) Protein N-glycosylation is widespread among biological systems, Mammalian ribophorin I and ribophorin II are abundant ER and the fundamental process of transferring a lipid-linked glycan membrane proteins (14, 15) that were coisolated as part of an to suitable asparagine residues of newly synthesized proteins enzyme complex showing in vitro OST activity (16). Although occurs in both prokaryotes and eukaryotes. The core reaction is subsequent studies suggested that ribophorin I and its S. cerevisiae mediated by Stt3p family members, and in many organisms this equivalent (Ost1p) could form part or all of the active site of the component alone is sufficient to constitute the so called oligosac- OST complex (17, 18), the case for STT3 playing this role is now charyltransferase (OST). However, eukaryotes typically have a compelling (11). In a previous study, we showed that a subset of more elaborate OST with several additional subunits of poorly newly synthesized membrane proteins transiently associated with defined function. In the mammalian OST complex one such sub- ribophorin I immediately after their departure from the Sec61 unit, ribophorin I, is proposed to facilitate the N-glycosylation of translocon (19). Based on these data, we proposed that ribophorin certain precursors during their biogenesis at the endoplasmic I may function to retain potential substrates in close proximity to reticulum. Here, we use cell culture models to show that ribophorin the catalytic subunit of the OST, thereby improving the efficiency I depletion results in substrate-specific defects in N-glycosylation, of their N-glycosylation (19). clearly establishing a defined physiological role for ribophorin I. To Our subsequent in vitro analysis of ribophorin I function showed address the molecular mechanism of ribophorin I function, a that this OST subunit dramatically enhances the N-glycosylation of cross-linking approach was used to explore the environment of selected substrates but is apparently dispensable for the efficient nascent glycoproteins during the N-glycosylation reaction. We N-glycosylation of many others (10). Here, we show for the first show for the first time that ribophorin I can regulate the delivery time that ribophorin I depletion has a selective effect upon the of precursor proteins to the OST complex by capturing substrates N-glycosylation of endogenous substrates expressed in cultured and presenting them to the catalytic core. mammalian cells. By using a novel combination of siRNA-mediated depletion and in vitro cross-linking, we find that ribophorin I acts to cross-linking ͉ endoplasmic reticulum ͉ N-glycosylation ͉ RNAi ͉ STT3 selectively regulate the delivery of substrates to the catalytic core of the OST complex. n eukaryotic cells, N-glycosylation is typically the most common Results and Discussion protein modification that occurs in the ER lumen. N- I Ribophorin I Function in Cultured Cells. HepG cells express a number glycosylation is facilitated by a large heterologous protein complex 2 of secretory glycoproteins (20) and hence were selected to study the called the oligosaccharyltransferase (OST), which catalyses the effect of ribophorin I depletion by siRNA. Importantly, our pre- attachment of a high mannose oligosaccharide en bloc onto suitable vious work has established that the integrity of the remaining OST free asparagine residues of newly synthesized nascent chains during complex is not disrupted after ribophorin I depletion (10), mini- translocation into the ER lumen (1). The addition of an N-glycan mizing the possibility of indirect or pleiotropic effects after ribo- provides a molecular tag that can promote the folding, maturation, phorin I knockdown. For comparison, the two mammalian STT3 and quality control of a precursor. Defects in the process of isoforms were also depleted [supporting information (SI) Fig. S1A], N-glycosylation, for example, congenital disorders, can result in and cells were treated with the antibiotic tunicamycin that inhibits devastating physiological consequences (2). assembly of the lipid-linked oligosaccharide precursor necessary for Our current understanding of the OST stems from research that N-glycosylation to occur. Both of these treatments were proven to utilizes a number of systems, including simple models such as be effective at inhibiting protein N-glycosylation in vitro (10). prokaryotes and more complex and diverse eukaryotes. The mam- ␣1-antitrypsin (␣1AT) is a secretory protein with three sites for malian equivalents of many, if not all Saccharomyces cerevisiae (S. N-glycosylation. Whole cell extracts were analyzed after metabolic cerevisiae) OST subunits, are known and comprise: ribophorin I labeling and immunoprecipitation; two major intracellular forms of (Ost1p), ribophorin II (Swp1p), OST48 (Wbp1p), STT3A and ␣1AT were observed (Fig. 1A). The faster migrating species has a STT3B (Stt3), N33 and IAP (Ost3p and Ost6p), and Dad1 (Ost2p) high mannose form of N-linked glycan added [Fig. 1A, lane 6, (3–5). In addition, two other putative mammalian OST subunits, ϩ3CHO ␣1AT(H)] and is sensitive to Endo-H treatment (Fig. DC2 and KCP2, have been identified but to date have no known S1B). The second, slower migrating species has more complex function (5). N-linked glycan typically acquired during transit through the Golgi There is now compelling evidence that the catalytic Center of the OST complex lies with the STT3 subunit(s) (6–9). In mammals, two STT3 isoforms exist, STT3A and STT3B; these share 70% se- Author contributions: C.M.W. and S.H. designed research; C.M.W. and Q.R. performed quence similarity and are expressed at varying levels depending research; C.M.W. and Q.R. analyzed data; and C.M.W. and S.H. wrote the paper. upon tissue or cell-type (4). Despite their similarity, these two STT3 The authors declare no conflict of interest. isoforms are not functionally equivalent when analyzed in vitro (10). This article is a PNAS Direct Submission. In many prokaryotes, a single STT3 homologue is sufficient to Freely available online through the PNAS open access option. mediate the N-glycosylation reaction (11–13). This observation *To whom correspondence should be addressed. E-mail: [email protected]. raises the interesting question of why most eukaryotic OSTs contain This article contains supporting information online at www.pnas.org/cgi/content/full/ multiple distinct subunits and what precise role these other subunits 0711846105/DCSupplemental. play during N-glycosylation. © 2008 by The National Academy of Sciences of the USA 9534–9539 ͉ PNAS ͉ July 15, 2008 ͉ vol. 105 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711846105 Downloaded by guest on September 24, 2021 lane 1), although Western blot analysis confirmed a substantial A anti-α1 antitrypsin B anti-Transferrin reduction of Ͼ80% in the cellular levels of the target protein after B B I siRNA treatment (Fig. S1A) (cf. ref. 10). A second endogenous siRib siSTT3AsiSTT3 siRF Tunic Mock siRibI siSTT3AsiSTT3 siRF Tunic Mock HepG2 secretory glycoprotein, transferrin, was also analyzed for the 220 effects of OST subunit depletion. Once again, tunicamycin treat- 220 97.4 ment and siRNA-mediated depletion of STT3A or STT3B resulted 97.4 66 +3CHO α1AT(C) +2CHO Tr(H) in a complete loss of N-glycosylated transferrin (Fig. 1B, lanes 2, 3, 66 Tr 5, and 6, see Tr), whereas ribophorin I knockdown did not affect the +3CHO α1AT(H) 46 α1AT N-glycosylation status of transferrin. However, less total material 46 was recovered from the ribophorin I knockdown compared with the control cells [Fig. 1B, lanes 1, 5, and 6, see ϩ2CHOTr(H)]. 30 Our previous in vitro studies (10) suggested that the N- 30 glycosylation of secretory proteins was unaffected by ribophorin I depletion, and our analysis of endogenously expressed glycopro- teins in HepG2 cells is entirely consistent with this notion. We next 654321 654321 HepG2 HepG2 addressed the substrate specificity of ribophorin I by using Mel Juso cells, because these express the invariant chain of the MHC class II C anti-Tyrosinase D anti-αIi complex (Ii) (21), a glycoprotein that we identified as a ribophorin I dependent substrate in vitro (10). We observed that cellular levels I of ribophorin I, STT3A and STT3B, in Mel Juso cells were ck STT3A o siRib siSTT3AsiSTT3B siRF Tunic Mock siRibI siRibI scramsi siSTT3B siRF Tunic M specifically and efficiently knocked down by using siRNA (Fig. 220 S1D). We also analyzed the effect on the N-glycosylation of two 220 endogenously expressed membrane proteins, tyrosinase and Ii. 97.4 After metabolic labeling and immunoprecipitation, tyrosinase was 97.4 66 ] +6/7CHO TYR(H) found to migrate as a 70-kDa doublet corresponding to products 66 TYR with 6 and 7 N-linked glycans as previously described (22) (Fig. 1C, 46 46 o lane 6). Both products are sensitive to Endo-H treatment (Fig. S1E). Depletion of STT3A and STT3B results in the complete loss +2CHO Ii(C) +2CHO Ii(H) of tyrosinase N-glycosylation (Fig. 1C, lanes 2, 3, 5, and 6), whereas 30 Ii 30 ribophorin I knockdown has no effect (Fig. 1C, lanes 1, 5, and 6). 123456 123456 7 Mel Juso Mel Juso When we examined the Ii expressed in Mel Juso cells by using the same approach, two distinct species of ϳ35 kDa were observed Fig. 1. Effect of OST subunit knockdown on the N-glycosylation of endog- after immunoprecipitation (Fig. 1D, lane 7). The faster migrating enously expressed proteins in HepG2 and Mel Juso cells. (A and B) HepG2 cells product has high mannose type N-linked glycans [Fig.
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