Cysteine-scanning mutagenesis of transmembrane domain XII and the flanking periplasmic loop in the lactose permease of EScherichia coli.
Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain XII and the periplasmic loop between putative helices XI and XII (loop XI/XII) was replaced individually with Cys. Out of 34 mutants, 31 exhibit 60-100% or more of C-less activity, mutants Gly377-->Cys and Leu385-->Cys exhibit lower rates of transport but accumulate lactose about 60-70% as well as C-less, and mutant Leu400-->Cys exhibits < 20% of C-less activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to that of C-less with the exception of mutants Gly377-->Cys and Leu385-->Cys which are expressed about 40% as well as C-less and mutant Leu400-->Cys which is hardly detectable. When transferred to the wild-type background, however, mutant Leu400-->Cys is expressed normally and exhibits highly significant transport activity. Finally, each active Cys-replacement mutant was assayed for sensitivity to N-ethylmaleimide, and with three exceptions, the mutants are essentially unaffected by the alkylating agent. Mutants Val367-->Cys, Gly370-->Cys, and Tyr373-->Cys which are predicted to be immediately distal to helix XI in loop XI/XII are significantly inactivated. The periodicity observed suggests that the periplasmic end of transmembrane domain XI may extend to position 373. In the following paper [Voss, J., He, M. M., Hubbell, W. L., & Kaback, H. R. (1996) Biochemistry 35, 12915-12918], site-directed spin labeling of single-Cys mutants at positions 387-402 is used to demonstrate that transmembrane domain XII is in an alpha-helical conformation.
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Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Sun
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J
Kaback
H R
HR
D000595:Amino Acid Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D007785:Lactose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose; D003545:Cysteine; D005033:Ethylmaleimide