The secretory carrier membrane protein family: Structure and membrane topology
The secretory carrier membrane protein family: Structure and membrane topology
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DOI：
10.1091/mbc.11.9.2933
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发表时间：
2000-09-01
影响因子：
3.3
通讯作者：
Castle, D
中科院分区：
文献类型：
--
作者：
Hubbard, C;Singleton, D;Castle, D
分泌性载体膜蛋白（SCAMPs）是在分泌性和内吞性载体中发现的与膜运输有关的整合膜蛋白。使用表达序列标签数据库和文库筛选和DNA测序，我们已经表征了几个新的SCAMP跨越植物和动物王国，并定义了一个广泛保守的蛋白质家族。然而，没有发现明显的真菌同源物。我们已经发现SCAMPs共享几个结构基序。这些包括NPF重复序列，富含带电残基的亮氨酸七肽重复序列，以及N-末端结构域中富含脯氨酸的SH 3样和/或WW结构域结合位点，随后是含有四个推定的跨膜跨度和三个最高度保守的结构元件的两亲性片段的膜核心。除了哺乳动物SCAMP 4之外，所有SCAMP都是32-38 kDa，哺乳动物SCAMP 4与25 kDa相似，并且缺乏其他SCAMP的大部分N-末端亲水结构域。通过北方和Western印迹确定SCAMP 4是真实的，表明较大SCAMP的该部分编码功能结构域。专注于SCAMP 1，我们已经通过有限的蛋白水解和Western印迹进一步表征了其结构，使用分离的分泌颗粒作为抗原的均匀定向来源，并通过在大肠杆菌中表达碱性磷酸酶基因融合体进行拓扑映射。结果显示SCAMP 1从N末端然后C末端依次降解，产生类似于20-kDa的包含四个跨膜跨度的膜核心。使用合成肽对应于膜核心的三个保守的两亲性片段，我们已经证明了它们与磷脂膜的结合，并通过圆二色性光谱显示，连接跨膜跨度2和3的中心两亲性片段是α-螺旋的。在完整的蛋白质中，这些片段可能位于面向细胞质的膜界面。SCAMP 1的当前模型表明，N和C末端形成覆盖膜核心的蛋白质的细胞质表面，其含有位于细胞质界面处的功能结构域，其中蛋白质在胞外域上几乎没有暴露。
Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is similar to 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an similar to 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.