Fu J, Kreibich G
Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.
J Biol Chem. 2000 Feb 11;275(6):3984-90. doi: 10.1074/jbc.275.6.3984.
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.
内质网（ER）的膜蛋白可能通过涉及保留、回收或两者结合的机制定位于该细胞器。对于含有KDEL结构域的内质网腔蛋白以及携带双赖氨酸基序的I型跨膜蛋白，已经确定了特定的回收机制。然而，大多数内质网膜蛋白并不含有易于识别的回收基序。内质网定位信息已在细胞质、跨膜或腔结构域中发现。在本研究中，我们在三种I型跨膜蛋白核糖体结合蛋白I（RI）、核糖体结合蛋白II（RII）和OST48中鉴定出了内质网定位结构域。这些膜蛋白与DAD1一起形成具有寡糖基转移酶（OST）活性的寡聚复合物。我们之前已经表明，内质网保留信息独立存在于RII的跨膜和细胞质结构域中，就RI而言，由腔结构域组成的截短形式保留在内质网中。为了确定RI的其他结构域是否携带额外的保留信息，我们通过将Tac抗原的各个结构域与RI的相应结构域交换生成了嵌合体。我们在此证明，只有RI的腔结构域含有内质网保留信息。我们还表明，OST48中的双赖氨酸基序作为内质网定位基序发挥作用，因为两个赖氨酸残基被丝氨酸取代的OST48（OST48ss）不再保留在内质网中，而是也存在于质膜上。然而，当与RI、RII或本身不会从内质网中输出的嵌合体共表达时，OST48ss保留在内质网中，这表明它们可能通过与OST48的腔结构域相互作用形成部分寡聚复合物。就仅含有RII腔结构域的Tac嵌合体而言，其本身会从内质网中输出并迅速降解，当与OST48共表达时，它保留在内质网中并变得稳定。
