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First published online 30 January 2007
doi: 10.1242/jcs.000729

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Ribophorin I acts as a substrate-specific facilitator of N-glycosylation

Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (63K): [in this window] [in a new window]   Fig. 1. Consequences of siRNA-mediated knockdown of ribophorin I, STT3A and STT3B. (A) Lysates of cells 48 hours after transfection with ribophorin I, STT3A and STT3B siRNA duplexes (lanes 1-6), a non-functional siRisc-free control (siRF) (lane 7), or mock-transfected cells (lane 8) were probed with antibodies specific for ribophorin I (RibI), STT3A, STT3B or -tubulin (Tub). Mature forms of these proteins are indicated by arrows and the location of unglycosylated forms of ribophorin I and STT3A are shown by asterisks. For STT3B, the signal is relatively weak, and two non-specific products are also detected (filled circles). (B) Lysates of cells were prepared as in A except the RibIB siRNA duplex was omitted. Samples were probed using antibodies recognising OST48, ribophorin II (RibII), Dad1 or -tubulin.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Glycerol gradient analysis of OST complexes after siRNA treatment. HeLa cells were mock treated (A) or transfected with siRNAs against ribophorin I (B), STT3A (C) or STT3B (D) and subsequently solubilised in homogenisation buffer containing 1.5% digitonin. The resulting supernatant was loaded onto 8-30% glycerol gradients containing 0.125% digitonin and after centrifugation, thirteen 1-ml fractions were collected and analysed by immunoblotting for the presence of ribophorin I (RibI), OST48, STT3A and STT3B. The data are expressed graphically, and represent the relative band intensity for each component detected following correction of any background. Signals were quantified using Aida software (Fuji), and several different exposures times were used to ensure that the film gave a linear response.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effect of OST subunit knockdown on the N-glycosylation of type I membrane proteins. Transmembrane topology and number of N-linked glycans (branched structures) for the two proteins studied. (A) APP-C99'.1CHO was synthesised as a radiolabelled polypeptide using rabbit reticulocyte lysate supplemented with semi-permeabilised HeLa cells prepared 48 hours after transfection with siRNAs specific for the mRNAs encoding ribophorin I (lane 1), STT3A (lane 2) or STT3B (lane 3), a scrambled form of the ribophorin I siRNA (siScram. RibI) (lane 4), a non-functional control siRNA (siRF) (lane 5) or following mock transfection (lane 7). As a positive control for loss of N-glycosylation, HeLa cells were incubated with 2 µg/ml tunicamycin for 12 hours before isolation on day 2 (lane 6). The resulting glycosylated (+ CHO) and non-glycosylated (– CHO) polypeptides are shown following SDS-PAGE. The relative proportion of glycosylated polypeptide was calculated for each sample and expressed as a percentage of the total protein recovered. The numbers below the lanes are the mean ± s.e.m. of three independent experiments. Levels of N-glycosylation that differ from the mock-treated control by a significance of at least 0.02 are indicated (*). (B) Human glycophorin C was synthesised as in A except that the scrambled ribophorin I siRNA control was omitted. The proportion of N-glycosylated chains was calculated as for A and symbols are as previously described.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Effect of OST subunit knockdown on the N-glycosylation of type II membrane proteins. Transmembrane topology and number of N-linked glycans (branched structures) for the two proteins studied. (A) Human invariant chain was synthesised as previously described to determine the extent of its N-glycosylation (see Fig. 3A). The resulting glycosylated (+ 2CHO) and non-glycosylated (– CHO) polypeptides are shown with the proportion of N-glycosylated products calculated as before (see Fig. 3A), and symbols are as previously described. (B) Human asialoglycoprotein receptor (ASGPR) was synthesised and products analysed as in A. The numbers below the lanes are the mean ± s.e.m. of three independent experiments. Levels of N-glycosylation that differ from the mock-treated control by a significance of at least 0.02 are indicated (*).

View larger version (24K): [in this window] [in a new window]   Fig. 5. Effect of OST subunit knockdown on the N-glycosylation of polytopic membrane proteins. (A) The extent of bovine opsin N-glycosylation was determined and quantified as before (Fig. 3A). (B) Rat neurotensin receptor N-glycosylation was determined as for A. The numbers below the lanes are the mean ± s.e.m. of three independent experiments. Levels of N-glycosylation that differ from the mock-treated control by a significance of at least 0.02 are indicated (*).

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effect of OST subunit knockdown on the N-glycosylation of secretory proteins. (A) Yeast prepro factor was synthesised as before to determine the extent of its N-glycosylation (cf. Fig. 3A). The resulting fully glycosylated (+ 3CHO) and non-glycosylated (– CHO) polypeptides are shown. In this case any untranslocated prepro factor where the signal sequence has not been cleaved co-migrates with the translocated signal sequence cleaved form that has not been glycosylated (see products labelled CHO/ppf). This means that the measured proportion of correctly translocated, N-glycosylated, chains is an underestimate of the true value (cf. Fig. 6B). The proportion of N-glycosylated pro factor was calculated as before (Fig. 3A) with symbols as previously defined. (B) Human -interferon was synthesised as in A, four forms of the protein can be resolved, the doubly glycosylated form (+ 2CHO), a singly glycosylated form (+ 1CHO), the non-glycosylated form (– CHO) and an untranslocated form where the pre-sequence has not been cleaved (pIF). Data analysis of -interferon was performed as in A. Numbers below the lanes are the mean ± s.e.m. of three independent experiments. Levels of N-glycosylation that differ from the mock-treated control by a significance of at least 0.02 are indicated (*).

View larger version (25K): [in this window] [in a new window]   Fig. 7. Model for the role of ribophorin I during N-glycosylation at the OST. During translocation of the nascent polypeptide through the Sec61 translocon, the catalytic subunit of the oligosaccharyltransferase, STT3 scans the nascent chain for potential N-glycosylation sites. (A) If the nascent polypeptide is efficiently N-glycosylated or retention at the OST complex is mediated by a different OST subunit, then an interaction with ribophorin I is not required for efficient N-glycosylation. (B) Where N-glycosylation is inefficient, binding of the substrate to ribophorin I increases the time available for the STT3 subunit to recognise and modify the newly synthesised protein even after it has exited the ER translocon (Wilson et al., 2005).

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