Hardt B, Aparicio R, Breuer W, Bause E
Institut für Physiologische Chemie, Universität Bonn, Germany.
Biol Chem. 2001 Jul;382(7):1039-47. doi: 10.1515/BC.2001.130.
Pig liver oligosaccharyltransferase (OST) is a heterooligomeric protein complex responsible for the co-translational transfer of GlcNAc2-Man9-Glc3 from Dol-PP onto specific asparagine residues in the nascent polypeptide. OST48, one of the catalytic subunits in this complex, exerts a typical type I membrane topology, containing a large luminal domain, a hydrophobic transmembrane domain and a short cytosolic peptide tail. Because OST48 is found within the endoplasmic reticulum (ER) when overexpressed in COS-1 cells, we carried out experiments to identify structural signals potentially capable of directing ER-targeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man9-mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the C-terminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48AK) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ER-residency on OST48. The double-lysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48AK-3, when co-expressed in COS-1 cells together with myc-tagged ribophorin 1, was quantitatively retained in the ER. By contrast, co-expression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMOdeltaK-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the C-terminus of the Man9-mannosidase luminal domain. Thus ER-localisation of OST48 is probably brought about by complex formation with ribophorin I and this most likely involves the luminal domains of both proteins. Consequently, the double-lysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in re-capture of molecules which have escaped from the ER.
猪肝寡糖基转移酶（OST）是一种异源寡聚蛋白复合物，负责将来自多萜醇焦磷酸（Dol-PP）的GlcNAc2-Man9-Glc3共翻译转移至新生多肽链上特定的天冬酰胺残基。OST48是该复合物中的催化亚基之一，具有典型的I型膜拓扑结构，包含一个大的腔结构域、一个疏水跨膜结构域和一个短的胞质肽尾。由于在COS-1细胞中过表达时OST48定位于内质网（ER），我们利用OST48突变体以及由单个OST48结构域和Man9-甘露糖苷酶组成的杂合蛋白开展实验，以鉴定可能指导内质网靶向的结构信号。免疫荧光显微镜检查显示，C末端赖氨酸-3或赖氨酸-5（而非赖氨酸-7）被亮氨酸取代的OST48突变体（OST48AK）可在细胞表面检测到。这表明这两个赖氨酸残基足以使OST48在内质网驻留。双赖氨酸基序仅在暴露于胞质溶胶时起作用，此时它作为重新定位信号而非滞留信号。当OST48AK-3与带有myc标签的核糖体结合蛋白1在COS-1细胞中共表达时，它被定量保留在内质网中。相比之下，在核糖体结合蛋白I存在的情况下共表达，对于含有连接到Man9-甘露糖苷酶腔结构域C末端的OST48胞质和跨膜结构域的OMOdeltaK-5嵌合体，细胞表面荧光没有降低。因此，OST48的内质网定位可能是通过与核糖体结合蛋白I形成复合物实现的，这很可能涉及两种蛋白的腔结构域。因此，OST48胞质结构域中的双赖氨酸基序除了参与重新捕获从内质网逃逸的分子外，不太可能具有主要功能。
