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首页> 外文期刊>Biochemistry >Insertion of the polytopic membrane protein lactose permease occurs by multiple mechanisms.
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Insertion of the polytopic membrane protein lactose permease occurs by multiple mechanisms.

机译：多靶膜蛋白乳糖通透酶的插入通过多种机制发生。

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摘要

The lactose permease of Escherichia coli has 12 transmembrane hydrophobic domains in probable alpha-helical conformation connected by hydrophilic loops. Previous studies [Consler, T. G., Persson, B., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6934-6938] demonstrate that a peptide fragment (the XB domain) containing a factor Xa protease site immediately upstream of a biotin acceptor domain can be engineered into the permease, thereby allowing rapid purification to a high state of purity. Here we describe the use of the XB domain to probe topology and insertion. Cells expressing permease with the XB domain at the N terminus, at the C terminus, or in loop 6 or 10 on the cytoplasmic face of the membrane catalyze active transport, although only the chimeras with the XB domain at the C terminus or in loop 6 are biotinylated. In contrast, chimeras with the XB domain in periplasmic loop 3 or 7 are inactive, but strikingly, both constructs are biotinylated. Furthermore, the XB domain in all the constructs,particularly in the loop 3 and loop 7 chimeras, is accessible from the cytoplasmic face of the membrane, as evidenced by factor Xa proteolysis or avidin binding studies with spheroplasts and disrupted membrane preparations. Finally, alkaline phosphatase fusions one loop downstream from each periplasmic XB domain exhibit high phosphatase activity. Thus, the presence of the XB domain in a periplasmic loop apparently blocks translocation of a discrete segment of the permease consisting of the loop and the two adjoining helices without altering insertion of the remainder of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
机译：大肠杆菌的乳糖通透酶具有12个跨膜疏水域，其可能的α-螺旋构象由亲水环连接。先前的研究[Consler，T.G.，Persson，B。，等。 （1993）美国国家科学院院刊。 Natl。学院科学美国专利号90，6934-6938]证明，可以将含有紧接生物素受体域上游Xa因子Xa蛋白酶位点的肽片段（XB域）改造成通透酶，从而可以快速纯化至高纯度。在这里，我们描述了使用XB域探测拓扑和插入。在膜的细胞质面上，在N末端，C末端或环6或10中表达XB结构域的通透酶的细胞可催化主动转运，尽管只有在C末端或环6中具有XB结构域的嵌合体。被生物素化。相反，在周质环3或7中具有XB结构域的嵌合体是无活性的，但引人注目的是，两种构建体均被生物素化。此外，在所有构建体中，特别是在环3和环7嵌合体中的XB结构域，都可以从膜的细胞质表面进入，如Xa因子蛋白水解或亲和素结合原生质球和破坏的膜制备所证明的。最后，碱性磷酸酶融合体在每个周质XB结构域下游的一个环上均表现出高磷酸酶活性。因此，周质环中XB结构域的存在显然阻止了由环和两个相邻的螺旋组成的通透酶离散段的移位，而没有改变蛋白质的其余部分的插入。（摘录于250字）

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