Kim P K, Janiak-Spens F, Trimble W S, Leber B, Andrews D W
Department of Biochemistry, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada.
Biochemistry. 1997 Jul 22;36(29):8873-82. doi: 10.1021/bi970090t.
Subcellular localization of proteins with carboxyl-terminal insertion sequences requires the molecule be both targeted to and integrated into the correct membrane. The mechanism of membrane integration of cytochrome b5 has been shown to be promiscuous, spontaneous, nonsaturable, and independent of membrane proteins. Thus endoplasmic reticulum localization for cytochrome b5 depends primarily on accurate targeting to the appropriate membrane. Here direct comparison of this mechanism with that of three other proteins integrated into membranes via carboxyl-terminal insertion sequences [vesicle-associated membrane protein 1(Vamp1), polyomavirus middle-T antigen, and Bcl-2] revealed that, unlike cytochrome b5, membrane selectivity for these molecules is conferred at least in part by the mechanisms of membrane integration. Bcl-2 membrane integration was similar to that of cytochrome b5 except that insertion into lipid vesicles was inefficient. Unlike cytochrome b5 and Bcl-2, Vamp1 binding to canine pancreatic microsomes was saturable, ATP-dependent, and abolished by mild trypsin treatment of microsomes. Surprisingly, although the insertion sequence of polyomavirus middle-T antigen was sufficient to mediate electrostatic binding to membranes, binding did not lead to integration into the bilayer. Together these results demonstrate that there are at least two different mechanisms for correct membrane integration of proteins with insertion sequences, one mediated primarily by targeting and one relying on factors in the target membrane to mediate selective integration. Our results also demonstrate that, contrary to expectation, hydrophobicity is not sufficient for insertion sequence-mediated membrane integration. We suggest that the structure of the insertion sequence determines whether or not specific membrane-bound receptor proteins are required for membrane integration.
具有羧基末端插入序列的蛋白质的亚细胞定位要求该分子既要被靶向到正确的膜上，又要整合到该膜中。细胞色素b5的膜整合机制已被证明是混杂的、自发的、不饱和的，且不依赖于膜蛋白。因此，细胞色素b5在内质网的定位主要取决于准确地靶向到合适的膜上。在此，将这种机制与其他三种通过羧基末端插入序列整合到膜中的蛋白质[囊泡相关膜蛋白1（Vamp1）、多瘤病毒中T抗原和Bcl-2]的机制进行直接比较，结果显示，与细胞色素b5不同，这些分子的膜选择性至少部分是由膜整合机制赋予的。Bcl-2的膜整合与细胞色素b5相似，只是插入脂质囊泡的效率较低。与细胞色素b5和Bcl-2不同，Vamp1与犬胰腺微粒体的结合是饱和的、ATP依赖性的，并且通过对微粒体进行温和的胰蛋白酶处理可消除这种结合。令人惊讶的是，尽管多瘤病毒中T抗原的插入序列足以介导与膜的静电结合，但这种结合并未导致其整合到脂双层中。这些结果共同表明，对于具有插入序列的蛋白质，至少存在两种不同的正确膜整合机制，一种主要由靶向介导，另一种则依赖于靶膜中的因子来介导选择性整合。我们的结果还表明，与预期相反，疏水性不足以实现插入序列介导的膜整合。我们认为，插入序列的结构决定了膜整合是否需要特定的膜结合受体蛋白。
