Interactions among Subunits of the Oligosaccharyltransferase Complex

Jie Fu,Mindong Ren,Gert Kreibich

doi:10.1074/jbc.272.47.29687

Abstract

The mammalian oligosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and Dad1 (defender against apoptotic death) suggests that Dad1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (RIL and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of Dad1 and the short cytoplasmic tail of OST48, thus placing Dad1 firmly into the OST complex. In addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery.

Highlights

Gradients and by ion exchange chromatography [4]
Using the yeast two-hybrid approach in conjunction with in vitro binding studies, we demonstrated that the luminal domain of OST48 interacts with the luminal domains of ribophorin I (RI) and ribophorin II (RII), but we detected no direct interactions between RIL and RIIL
Interactions between OST48L and RI or RII resulted in blue yeast colonies, indicating that OST48L interacts with both RIL and RIIL

Summary

Interactions among Subunits of the Oligosaccharyltransferase Complex*

Vol 272, No 47, Issue of November 21, pp. 29687–29692, 1997 Printed in U.S.A. Jie Fu, Mindong Ren, and Gert Kreibich‡ From the Department of Cell Biology, New York Medical Center, New York, New York 10016. We found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery During their translocation into the lumen of the rough endoplasmic reticulum, polypeptides made on membrane-bound polysomes may be cotranslationally modified by N-glycosylation [1, 2]. Site-specific antibodies directed against the cytoplasmic domain of RI, but not those against the luminal domain of the protein, inhibited the targeting to microsomal membranes of ribosome-nascent chain complexes formed in a cell-free translation mixture [20] These results suggest that the ribophorins are adjacent to the polypeptide translocation site. The Sec61p complex, which is composed of an ␣, ␤, and ␥ subunit, is a key component of the translocation apparatus since it forms the translocation pore but has ribosome binding activity [21, 22]

It may be expected that the enzymatic activity of the OST

EXPERIMENTAL PROCEDURES

RESULTS

RIL RIIL RIL

RIL RIL RIL

DISCUSSION