Ribophorin I regulates substrate delivery to the oligosaccharyltransferase core

Abstract

Protein N-glycosylation is widespread among biological systems, and the fundamental process of transferring a lipid-linked glycan to suitable asparagine residues of newly synthesized proteins occurs in both prokaryotes and eukaryotes. The core reaction is mediated by Stt3p family members, and in many organisms this component alone is sufficient to constitute the so called oligosaccharyltransferase (OST). However, eukaryotes typically have a more elaborate OST with several additional subunits of poorly defined function. In the mammalian OST complex one such subunit, ribophorin I, is proposed to facilitate the N-glycosylation of certain precursors during their biogenesis at the endoplasmic reticulum. Here, we use cell culture models to show that ribophorin I depletion results in substrate-specific defects in N-glycosylation, clearly establishing a defined physiological role for ribophorin I. To address the molecular mechanism of ribophorin I function, a cross-linking approach was used to explore the environment of nascent glycoproteins during the N-glycosylation reaction. We show for the first time that ribophorin I can regulate the delivery of precursor proteins to the OST complex by capturing substrates and presenting them to the catalytic core.

Fig. 1.

Effect of OST subunit knockdown on the N-glycosylation of endogenously expressed proteins in HepG₂ and Mel Juso cells. (A and B) HepG₂ cells were transfected with siRNAs targeting ribophorin I (lane 1), STT3A (lane 2), STT3B (lane 3), a nonfunctional control siRNA (siRF) (lane 4), or mock treated (lane 6). To control for loss of N-glycosylation, HepG₂ cells were incubated with 2 μg/ml tunicamycin for 12 h before isolation on day 2 (lane 5). Cells were pulse labeled with [³⁵S] methionine/cysteine for 45 min, solubilized in IP buffer and specific products recovered by immunoprecipitating with antisera recognizing α1-antitrypsin (α1AT, see A) or transferrin (Tr, see B). In all cases, the presence and number (x) of N-linked glycans present on a particular protein is indicated by the suffix xCHO. For α1AT (A), two populations of glycoproteins reflecting products with both high mannose type N-linked glycans [+3CHOα1AT(H)] and complex N-glycans [+3CHOα1AT(C)] were detected. With Tr (B), only high mannose type glycans were observed. (C and D) Mel Juso cells were treated with siRNAs or tunicamycin as above. Additionally, a scrambled ribophorin I duplex (siRibI scram) was used during the analysis of the invariant chain (D, lane 2). Cells were labeled with [³⁵S] methionine/cysteine for 30 min, solubilized in IP buffer and immunoprecipitated with antisera recognizing tyrosinase (TYR, see C) or invariant chain (Ii, see D). Two closely migrating forms of tyrosinase were detected representing chains with 6 or 7 high mannose type N-glycans attached [+6/7 CHO TYR(H)]. Two quite distinct forms of N-glycosylated Ii were detected; these reflect discrete populations of proteins with high mannose [+2CHO Ii(H)] or complex type [+2CHO Ii(C)] N-linked glycans as described above for α1AT. The appearance of higher molecular weight species after inhibiting N-glycosylation of α1AT and Ii (A and D, filled circles) may reflect aberrant dimerization of the nonglycosylated proteins (compare with ref. 34). In the case of TYR, Mel Juso cells express a cross-reacting glycoprotein of ∼46 kDa (C, open circle) that behaves in a similar fashion to authentic TYR after treatment with the various siRNAs or tunicamycin. This product is most likely a splice variant of tyrosinase (35).

Fig. 2.

Analyzing nascent chain OST interactions. (A) PPLNKT translocation intermediates of increasing chain length (aa) were synthesized in rabbit reticulocyte lysate (RRL) supplemented with semipermeabilized HeLa cells and cross-linked by using DSS. The resulting glycosylated (+CHO PPL) and nonglycosylated PPL (PPL) chains are indicated together with two major DSS-dependent adducts that are visible in the total products (filled circles and diamonds). (B) Specific cross-linking products of the PPLNKT chains with Sec61α (filled diamonds) and STT3A (filled circles) were identified by immunoprecipitation.

Fig. 3.

Molecular analysis of a ribophorin I−independent substrate. (A) Full-length PPLNKT was synthesized in vitro by using semipermeabilized HeLa cells prepared after siRNA mediated depletion of ribophorin I (lane 1), STT3A (lane 2), STT3B (lane 3), a nonfunctional control (siRF) (lane 4) or mock treatment (lane 6). Tunicamycin treatment served as a control (compare to Fig. 1). The resulting products, glycosylated (PL.CHO) and nonglycosylated (PL) prolactin and the precursor with intact signal sequence (PPL) are shown after SDS/PAGE. The relative proportion of glycosylated polypeptide was calculated for each sample and expressed as a percentage of the total protein recovered. The values below the lanes are the mean ± SEM of three independent experiments. Levels of N-glycosylation that differ from the mock treated control with a significance of P < 0.02 are indicated by asterisks. (B) A 170-residue PPLNKT translocation intermediate encoded by an mRNA lacking a stop codon was synthesized as before (compare to Fig. 2) and analyzed as described for A. (C) A proportion of the PPLNKT-170 translocation intermediates shown in B were treated with DSS and the resulting adducts with STT3A were recovered by immunoprecipitation. The amount of STT3A-PPLNKT-170 adduct for the mock sample was quantified and set to a nominal value of 100%. Other values are the mean of three independent experiments where the amount of adduct obtained after the different treatments is expressed relative to the mock treated sample. Values that differ from the mock treated control with a significance of P < 0.02 are indicated by asterisks.

Fig. 4.

Molecular analysis of ribophorin I dependent substrate. (A) Full-length invariant chain (Ii) was synthesized as above (Fig. 3) to determine the extent of its N-glycosylation. The resulting glycosylated (+2CHOIi) and nonglycosylated (Ii) polypeptides and the proportion of N-glycosylated products obtained after various treatments are indicated as described for Fig. 3. (B) A 214-residue integration intermediate of Ii was synthesized by using a truncated mRNA lacking a stop codon and analyzed as described for A. (C) A proportion of the 214-residue Ii–integration intermediates were treated with DSS and the resulting STT3A adducts recovered by immunoprecipitation. The relative percentage of STT3A adduct formation under the various experimental conditions was calculated as before (compare to Fig. 3). Symbols are as defined in the legend to Fig. 3.