Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains.
Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains.
复制标题
乳糖通透酶在连续片段中的表达作为跨膜结构域的探针。
作者：
K. Zen;E. McKenna;E. Bibi;D. Hardy;H. Kaback
大肠杆菌乳糖通透酶是一种膜转运蛋白，含有12个由亲水环连接的跨膜疏水结构域。编码对应于通透酶的第一和第二半的连续多肽的lacY基因片段的共表达[Bibi，E.，& Kapack，H. R.等人（1990）Proc. Acad. Sci. U.S.A.87，4325-4329]或分子的前两个跨膜结构域和剩余部分[Wrubel，W.，Stochaj，U.，美国索内瓦尔德，There，C. & Ehring，R.等人（1990）J. Bacteriol. 172，5374-5381]导致主动乳糖转运。它在这里示出，连续的通透酶片段与环1（周质），环6（细胞质），或环7（周质）中的不连续表现出运输活性，然而，跨膜结构域III或VII中的不连续的片段未能做到这一点。结果是一致的解释，即连续的通透酶片段与亲水性环中的不连续性形成功能性双链体，而跨膜α-螺旋结构域中的不连续性的片段不。在此基础上的概念，一系列连续的，不重叠的通透酶片段在环6，推定的螺旋VII，和环7的不同位置的不连续性共表达近似推定的跨膜结构域VII的边界。在Leu 222和Trp 223之间或Gly 254和Glu 255之间具有不连续性的连续体片段是有功能的，但在Cys 234和Thr 235之间、Gln 241和Gln 242之间或Phe 247和Thr 248之间具有不连续性的片段是无活性的。因此，Leu 222和Gly 254可能分别位于亲水环6和7中，而Cys 234、Gln 241和Phe 247可能位于跨膜结构域VII内。（250字处删节）
The lactose permease of Escherichia coli is a membrane transport protein containing 12 transmembrane hydrophobic domains connected by hydrophilic loops. Coexpression of lacY gene fragments encoding contiguous polypeptides corresponding to the first and second halves of the permease [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325-4329] or the first two transmembrane domains and the remainder of the molecule [Wrubel, W., Stochaj, U., Sonnewald, U., Theres, C., & Ehring, R. (1990) J. Bacteriol. 172, 5374-5381] leads to active lactose transport. It is shown here that contiguous permease fragments with discontinuities in loop 1 (periplasmic), loop 6 (cytoplasmic), or loop 7 (periplasmic) exhibit transport activity; however, fragments with discontinuities in transmembrane domains III or VII fail to do so. The results are consistent with the interpretation that contiguous permease fragments with discontinuities in hydrophilic loops form functional duplexes, while fragments with discontinuities in transmembrane alpha-helical domains do not. On the basis of this notion, a series of contiguous, nonoverlapping permease fragments with discontinuities at various positions in loop 6, putative helix VII, and loop 7 were coexpressed to approximate the boundaries of putative transmembrane domain VII. Contiguous fragments with a discontinuity between Leu222 and Trp223 or between Gly254 and Glu255 are functional, but fragments with a discontinuity between Cys234 and Thr235, between Gln241 and Gln242, or between Phe247 and Thr248 are inactive. Therefore, it is likely that Leu222 and Gly254 are located in hydrophilic loops 6 and 7, respectively, while Cys234, Gln241, and Phe247 are probably located within transmembrane domain VII.(ABSTRACT TRUNCATED AT 250 WORDS)