EXPRESSION OF LACTOSE PERMEASE IN CONTIGUOUS FRAGMENTS AS A PROBE FORMEMBRANE-SPANNING DOMAINS

The lactose permease of Escherichia coli is a membrane transport prote in containing 12 transmembrane hydrophobic domains connected by hydrop hilic loops. Coexpression of lacY gene fragments encoding contiguous p olypeptides corresponding to the first and second halves of the permea se [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 o f the molecule [Wrubel, W., Stochaj, U., Sonnewald, U., Theres, C., & Ehring, R. (1990) J. Bacteriol. 172, 5374-5381] leads to active lactos e 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 d iscontinuities in transmembrane domains III or VII fail to do so. The results are consistent with the interpretation that contiguous permeas e 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 conti guous, nonoverlapping permease fragments with discontinuities at vario us positions in loop 6, putative helix VII, and loop 7 were coexpresse d 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 discon tinuity between Cys234 and Thr235, between Gln241 and Gln242, or betwe en Phe247 and Thr248 are inactive. Therefore, it is likely that Leu222 and Gly254 are located in hydrophilic loops 6 and 7, respectively, wh ile Cys234, Gln241, and Phe247 are probably located within transmembra ne domain VII. These and other results are consistent with a modified secondary-structure model of lactose permease in which Asp237 and Asp2 40 are contained within transmembrane domain VII rather than periplasm ic loop 7.