Abstract
The biosynthesis of membrane proteins at the endoplasmic reticulum (ER) involves the integration of the polypeptide at the Sec61 translocon together with a number of maturation events, such as N-glycosylation and signal sequence cleavage, that can occur both during and after synthesis. To better understand the events occurring after the release of the nascent chain from the ER translocon, we investigated the ER components adjacent to the transmembrane-spanning domain of a well characterized fragment of the amyloid precursor protein. Using individual cysteine residues as site-specific cross-linking targets, we found that several ER components can be cross-linked to the fully integrated polypeptide. We identified strong adducts with both the ribophorin I subunit of the oligosaccharyltransferase complex and the 25-kDa subunit of the signal peptidase complex. Focusing on the association with ribophorin I, we found that adduct formation occurred exclusively after the exit of the nascent chain from the Sec61 translocon and was unaffected by the N-glycosylation status of the associated precursor. Only a subset of newly made membrane proteins associated with ribophorin I in vitro, and we could recapitulate a specific association between the amyloid precursor protein fragment and ribophorin I in vivo. Taken together, our data suggest a model where ribophorin I may function to retain potential substrates in close proximity to the catalytic subunit of the oligosaccharyltransferase and thereby stochastically improve the efficiency of the N-glycosylation reaction in vivo. Alternatively ribophorin I may be multifunctional and facilitate additional processes, for example, ER quality control.
Highlights
Most newly synthesized membrane and secretory proteins arrive at the ER1 via the signal recognition particle-dependent targeting pathway and are delivered to the Sec61 translocon that mediates their integration into the lipid bilayer [1, 2]
During its integration at the endoplasmic reticulum (ER) membrane, we observed cross-linking of ribosome-bound amyloid precursor protein (APP)-C99Ј integration intermediates to the translocon components Sec61␣ and Sec61 and found that these adducts were lost upon puromycin treatment, which promotes the lateral exit of the protein from the ER translocon into the lipid bilayer
The nature of these cross-linking partners was dependent upon the precursor studied, and in the case of the model protein APP-C99Ј[Cys59], ribophorin I and SPC25 were identified as major adducts
Summary
Most newly synthesized membrane and secretory proteins arrive at the ER1 via the signal recognition particle-dependent targeting pathway and are delivered to the Sec translocon that mediates their integration into the lipid bilayer [1, 2]. In the first study to link ribophorin I with the Nglycosylation at the ER, it was suggested that its transmembrane domain might function as a dolichol binding site [11] and thereby act to bring the lipid-linked high mannose form of the glycan in close proximity to the OST complex and any potential protein substrates. During the maturation of the amyloid precursor protein (APP), specific proteolytic cleavage events generate peptides that are strongly implicated in the pathology of Alzheimer’s disease [16]. In this case, we used the biologically relevant APP-C99 fragment as a model to study mem-. APP-C99 is generated in vivo by the -secretase (-site APP-cleaving enzyme)-mediated proteolytic cleavage of full-length APP, and it is this fragment that is the substrate for the ␥-secretase, which cleaves it within its transmembrane region to generate the A peptides that are a key feature of Alzheimer’s disease [17]
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