GENETIC-ANALYSIS OF THE MEMBRANE INSERTION AND TOPOLOGY OF MALF, A CYTOPLASMIC MEMBRANE-PROTEIN OF ESCHERICHIA-COLI
GENETIC-ANALYSIS OF THE MEMBRANE INSERTION AND TOPOLOGY OF MALF, A CYTOPLASMIC MEMBRANE-PROTEIN OF ESCHERICHIA-COLI
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DOI：
10.1016/0022-2836(88)90539-6
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发表时间：
1988-04-05
影响因子：
5.6
通讯作者：
BECKWITH, J
中科院分区：
文献类型：
--
作者：
FROSHAUER, S;GREEN, GN;BECKWITH, J
MalF是大肠杆菌麦芽糖转运系统的一种必需质膜蛋白。我们已经开发了一种通用方法，用于通过表征一系列β-葡聚糖的融合物来分析膜蛋白的整合机制及其膜拓扑结构。半乳糖苷酶转化为MalF。融合蛋白的性质表明如下。(1)MalF的前两个假定的跨膜区段足以锚β-半乳糖苷酶牢固地附着在内膜上。(2)具有β-的杂合蛋白质与MalF的假定胞质结构域融合的半乳糖苷酶具有高β-半乳糖苷酶活性。半乳糖苷酶特异性活性;与周质结构域的融合物具有低活性。因此，我们建议，周质和胞质结构域的完整膜蛋白可以区分这种杂交蛋白的酶性质。一般而言，当连接到β-半乳糖苷酶时，似乎切割或未切割的信号序列半乳糖苷酶使其嵌入膜中，这导致杂合蛋白不能组装成活性酶。这些融合蛋白的其他特性有助于我们理解MalF合成的调节。MalF蛋白作为大肠杆菌malEFG操纵子的一部分合成。大肠杆菌中的麦芽糖蛋白在细胞中的丰度比MalE蛋白（麦芽糖结合蛋白）低约30倍。融合蛋白的差异量表明malF基因内发生调节信号，其负责从malE基因到malF基因的表达逐步下降。
MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of .beta.-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor .beta.-galactosidase firmly to the inner membrane. (2) Hybrid proteins with .beta.-galactosidase fused to a presumed cytoplasmic domain of MalF have high .beta.-galactosidase specific activty; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to the .beta.-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.