Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains.
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)
Howard Hughes Medical Institute, Department of Physiology, University of California at Los Angeles 90024-1662.
K H
KH
Zen
E
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McKenna
E
E
Bibi
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Hardy
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Kaback
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology*; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D017433:Protein Structure, Secondary; D027981:Symporters; D014169:Transformation, Bacterial
D003062:Codon; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D027981:Symporters; C021499:lactose permease; D007785:Lactose