Retention of Subunits of the Oligosaccharyltransferase Complex in the Endoplasmic Reticulum

Abstract

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.

Unraveling the Mechanism of Protein N-Glycosylation

“…However, if it is co-expressed with ribophorin I, ribophorin II, or chimeras that contain domains of ribophorin I, ribophorin II, and the Tac antigen (interleukin-2 receptor ␣ chain precursor), which by themselves do not exit from the ER, OST48ss could be retained in the ER. Similar observations were found with ribophorin II (24,25). Therefore, it is possible that ribophorin I, ribophorin II, and OST48 may play a critical role in maintaining the mammalian OT complex in the ER.…”

“…However, deletion of this segment did not lead to a significant growth defect in yeast cells, suggesting that it does not function to retain the OT complex in the ER (20,23). In contrast, in the mammalian system it has been shown (24,25) that when one of the components of OT complex was mislocated by mutations in its own retention signal, it could be retained in the ER via its interaction with other subunits. Three mammalian OT subunits, ribophorin I, ribophorin II, and OST48 (mammalian homologs of yeast Ost1p, Swp1p, and Wbp1p, respectively), were found to contain specific ER localization signals.…”

The Double Lysine Motif of Tapasin Is a Retrieval Signal for Retention of Unstable MHC Class I Molecules in the Endoplasmic Reticulum

“…Studies from a number of KKXX motif-containing ER molecules indicate that how far down the secretory pathway KKXX mutants escape varies widely in the range from plasma membrane expression to limited increase in post-ER compartments (18,36). This variation is largely due to differences in interaction with other ER retention proteins and the maturation process in its own, e.g., glycosylation (37). However, most of the studied ER proteins with KKXX mutations have been shown to increase their distribution in post-ER compartments (18,37).…”

“…This variation is largely due to differences in interaction with other ER retention proteins and the maturation process in its own, e.g., glycosylation (37). However, most of the studied ER proteins with KKXX mutations have been shown to increase their distribution in post-ER compartments (18,37). Although tpn is an ER residential molecule, a proportion of tpn is detected in the Golgi (4) and tpn interacts with an ER export receptor (38), supporting the notion that tpn could escape from the ER.…”

Alternative Splicing Regulates the Endoplasmic Reticulum Localization or Secretion of Soluble Secreted Endopeptidase

“…Because no differences were observed in the localization and/or retention of the membrane-bound SEP/SEP ⌬ , only the findings with SEP ⌬ are presented. Several reports have shown that the cytoplasmic domain of type II membrane proteins determine ER localization (15)(16)(17). Thus, we first analyzed a chimeric protein S/S/E (SEP ⌬ cytoplasmic, SEP ⌬ transmembrane, and bECE-1b luminal, Fig.…”