Dumax-Vorzet A, Roboti P, High S

The eukaryotic oligosaccharyltransferase (OST) is a membrane-embedded protein complex that catalyses N-glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum (ER), a highly conserved biosynthetic process that enriches protein structure and function. All OSTs contain a homologue of the catalytic STT3 subunit, although in many cases this is ...

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assembled with several additional components that influence function. In S. cerevisiae, one such component is Ost4p, an extremely small membrane protein that appears to stabilise interactions between subunits of assembled OST complexes. OST4 has been identified as a putative human homologue, but to date neither its relationship to the OST complex, nor its role in protein N-glycosylation, have been directly addressed. Here, we establish that OST4 is assembled into native OST complexes containing either the catalytic STT3A or STT3B isoforms. Co-immunoprecipitation studies suggest that OST4 associates with both STT3 isoforms and ribophorin I, an accessory subunit of mammalian OSTs. These presumptive interactions are perturbed by a single amino acid change to the transmembrane region of OST4. Using siRNA knockdowns and native gel analysis, we show that OST4 plays an important role in maintaining native OST complexes stability. Hence, upon OST4 depletion well defined OST complexes are partially destabilised and a novel ribophorin I-containing subcomplex is detected. Strikingly, cells depleted of either OST4 or STT3A show a remarkably similar defect in the N-glycosylation of endogenous prosaposin, and we conclude that OST4 most likely promotes co-translational N-glycosylation by stabilising STT3A-containing OST isoforms.

Date: Apr. 19, 2013

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