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 significa nt effect on membrane insertion, stability, or transport activity, seq uential substitution of stop codons for amino acid codons 398-401 lead s 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, 2969-297 3). Thus, either the last turn of putative helix XII or the region imm ediately distal to helix XII is important for proper folding, and henc e, activity and resistance to proteolysis. In an effort to determine w hether this 3-4-amino acid sequence comprises the final turn of the la st transmembrane helix of the permease or the beginning of the hydroph ilic C-terminal tail, we deleted residues 401-417 and replaced amino a cid residues 397-400 with either 4 Leu residues ("helix making") or Gl y-Pro-Gly-Pro ("helix breaking"). Permease with 4 Leu residues at posi tions 397-400 is fully functional with respect to transport and comple tely stable, as judged by [S-35]methionine labeling experiments. In ma rked contrast, permease with Gly-Pro-Gly-Pro at the same positions exh ibits minimal activity and is unstable. The results imply that the ami no acid sequence ...Val397Phe398Thr399 Leu400... in lac permease may c omprise the last turn of transmembrane helix XII, rather than the begi nning of the C-terminal tail.