Evidence that the final turn of the last transmembrane helix in the lactose permease is required for folding.
Although truncation of the hydrophilic C-terminal tail of the lactose (lac) permease of Escherichia coli (residues 401-417) has no significant effect on membrane insertion, stability, or transport activity, sequential substitution of stop codons for amino acid codons 398-401 leads to a progressive increase in transport activity and in the lifetime of the permease in the membrane (McKenna, E., Hardy, D., Pastore, J. C., and Kaback, H. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969-2973). Thus, either the last turn of putative helix XII or the region immediately distal to helix XII is important for proper folding, and hence, activity and resistance to proteolysis. In an effort to determine whether this 3-4-amino acid sequence comprises the final turn of the last transmembrane helix of the permease or the beginning of the hydrophilic C-terminal tail, we deleted residues 401-417 and replaced amino acid residues 397-400 with either 4 Leu residues ("helix making") or Gly-Pro-Gly-Pro ("helix breaking"). Permease with 4 Leu residues at positions 397-400 is fully functional with respect to transport and completely stable, as judged by [35S]methionine labeling experiments. In marked contrast, permease with Gly-Pro-Gly-Pro at the same positions exhibits minimal activity and is unstable. The results imply that the amino acid sequence ... Val397Phe398Thr399 Leu400 ... in lac permease may comprise the last turn of transmembrane helix XII, rather than the beginning of the C-terminal tail.
McKenna
E
E
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1662.
Hardy
D
D
Kaback
H R
HR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D007785:Lactose / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010957:Plasmids; D011487:Protein Conformation; D027981:Symporters
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose