EVIDENCE THAT TRANSMEMBRANE SEGMENT-2 OF THE LACTOSE PERMEASE IS PARTOF A CONFORMATIONALLY SENSITIVE INTERFACE BETWEEN THE 2 HALVES OF THEPROTEIN

A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), is found in a large gro up of evolutionarily related membrane proteins involved in the transpo rt of small molecules across the membrane, This motif is located withi n the cytoplasmic side of transmembrane domain 2 (TM-2) and extends th rough the hydrophilic loop that connects transmembrane domains 2 and 3 , The motif is repeated again in the second half of the protein, In a previous study concerning the loop 2/3 motif (Jessen-Marshall, A, E,, Paul, N, J,, and Brooker, R, J, (1995) J, Biol, Chem, 270, 16251-16257 ), it was shown that the conserved aspartate at the fifth position in the motif is critical for transport activity since a variety of site-d irected mutations were found to greatly diminish the rate of transport , In the current study, two of these mutations, in which the conserved aspartate was changed to threonine or serine, were used as parental s trains to isolate second site suppressor mutations that restore transp ort function, A total of 10 different second site mutations were ident ified among a screen of 19 independent mutants, One of the suppressors was found within loop 1/2 in which Thr-45 was changed to arginine. Si nce the conserved aspartate and position 45 are at opposite ends of TM -2, these results suggest that the role of the conserved aspartate res idue in loop 2/3 is to influence the topology of TM-2, Surprisingly, t he majority of suppressor mutations were found in the second half of t he permease, All of these are expected to alter helix topology in eith er of two ways, Some of the mutations involved residues within transme mbrane segments 7 and 11 that produced substantial changes in side cha in volume: TM-7 (Cys-234 --> Trp or Phe, Gln-241 --> Leu, and Phe-247 --> Val) and TM-11 (Ser-366 --> Phe), Alternatively, other mutations w ere highly disruptive substitutions at the ends of transmembrane segme nts or within hydrophilic loops (Gly-257 --> Asp, Val-367 --> Glu, Ala -369 --> Pro, and a 5-codon insertion into loop 11/12), It is hypothes ized that the effects of these suppressor mutations are to alter the h elical topologies in the second half of the protein to facilitate a be tter interaction with the first half, Overall, these results are consi stent with a transport model in which TM-2 acts as an important interf ace between the two halves of the lactose permease, According to our t ertiary model, this interaction occurs between TM-2 and TM-11.