Kim P K, Hollerbach C, Trimble W S, Leber B, Andrews D W
Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
J Biol Chem. 1999 Dec 24;274(52):36876-82. doi: 10.1074/jbc.274.52.36876.
The vesicle-associated membrane proteins (Vamp(s)) function as soluble N-ethylmaleimide-sensitive factor attachment receptor proteins in the intracellular trafficking of vesicles. The membrane attachment of Vamps requires a carboxyl-terminal hydrophobic sequence termed an insertion sequence. Unlike other insertion sequence-containing proteins, targeting of the highly homologous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP and a membrane-bound receptor. To determine if this mechanism of targeting to the endoplasmic reticulum extends to other Vamps, we compared the membrane binding of Vamp1 and Vamp2 with the distantly related Vamp8. Similar to the other Vamps, Vamp8 requires both ATP and a membrane component to target to the endoplasmic reticulum. Furthermore, binding curves for the three Vamps overlap, suggesting a common receptor-mediated process. We identified a minimal endoplasmic reticulum targeting domain that is both necessary and sufficient to confer receptor-mediated, ATP-dependent, binding of a heterologous protein to microsomes. Surprisingly, this conserved sequence includes four positively charged amino acids spaced along an amphipathic sequence, which unlike the carboxyl-terminal targeting sequence in mitochondrial Vamp isoforms, is amino-terminal to the insertion sequence. Because Vamps do not bind to phospholipid vesicles, it is likely that these residues mediate an interaction with a protein, rather than bind to acidic phospholipids. Therefore, we suggest that a bipartite motif is required for the specific targeting and integration of Vamps into the endoplasmic reticulum with receptor-mediated recognition of specifically configured positive residues leading to the insertion of the hydrophobic tail into the membrane.
囊泡相关膜蛋白（Vamp(s)）在囊泡的细胞内运输过程中作为可溶性N - 乙基马来酰亚胺敏感因子附着受体蛋白发挥作用。Vamps的膜附着需要一个称为插入序列的羧基末端疏水序列。与其他含有插入序列的蛋白不同，高度同源的Vamp1和Vamp2靶向内质网需要ATP和一种膜结合受体。为了确定这种靶向内质网的机制是否扩展到其他Vamps，我们将Vamp1和Vamp2的膜结合与远亲的Vamp8进行了比较。与其他Vamps类似，Vamp8靶向内质网既需要ATP也需要一种膜成分。此外，这三种Vamps的结合曲线重叠，表明存在一个共同的受体介导过程。我们鉴定出一个最小的内质网靶向结构域，它对于赋予异源蛋白受体介导的、ATP依赖的与微粒体的结合既必要又充分。令人惊讶的是，这个保守序列包括沿着一个两亲序列间隔排列的四个带正电荷的氨基酸，与线粒体Vamp异构体中的羧基末端靶向序列不同，它位于插入序列的氨基末端。由于Vamps不与磷脂囊泡结合，这些残基可能介导与一种蛋白的相互作用，而不是与酸性磷脂结合。因此，我们认为Vamps特异性靶向内质网并整合到内质网中需要一个二分基序，通过受体介导识别特定构型的正性残基，导致疏水尾部插入膜中。
