LYSINE-319 INTERACTS WITH BOTH GLUTAMIC ACID-269 AND ASPARTIC ACID-240 IN THE LACTOSE CARRIER OF ESCHERICHIA-COLI

It is believed that there are several charged amino acid residues in m embrane-spanning alpha-helices of the lactose carrier of Escherichia c oli. Evidence has previously been presented for two different salt bri dges in membrane-spanning regions of the lactose carrier. One of these involves an interaction between Asp-237 and Lys-358; another involves interaction between Asp-240 and Lys-319. Additional studies of Lys-31 9 suggest that it may intereact with Glu-269 as well as Asp-240. A cel l containing the LacY gene with the mutation Lys-319 --> Asn failed to ferment melibiose and after several days melibiose-positive mutants a rose on indicator plates. These revertants showed second site mutation s which replaced Asp-240 by neutral amino acids (Val or Gly). In addit ion, a second site mutation showed Glu-269 changed to Asn. Cells conta ining the mutation Lys-319 --> Leu also failed to ferment melibiose an d melibiose-positive revertants showed Asp-240 --> Ala and Asp-240 --> Tyr as well as Tyr-236 --> Phe and His-322 --> Arg. Second site rever tants were also sought from the mutant Glu-269 --> Asn which grew poor ly on melibiose minimal plates. Melibiose-positive revertants included the double mutant Gln-269/Asn-319. All of the Glu-269 --> Asn mutants were extremely defective in transport. It was concluded thet Lys-319 interacts with Glu-269 and Asp-240 probably as salt bridges.