The yeast mitochondrial citrate transport protein - Probing the secondary structure of transmembrane domain IV and identification of residues that likely comprise a portion of the citrate translocation pathway

The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine. A cysteine-less CTP retaining wild-type functional pro perties served as the starting template. The single Cys CTP variants were o verexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo firs t order rate constants for inhibition of CTP function, These rate constants varied by seven orders of magnitude. With three independent data sets we o bserved peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193, Ba sed on the pattern of accessibility we conclude that residues 177-193 exist as an alpha-helix. Furthermore, a water-accessible face of the helix has b een defined consisting of Pro-177, Val-178, Arg-181, Gln-182, Asn-185, Gln- 186, Arg-189, Leu-190, and Tyr-193, and a water-inaccessible face has been delineated consisting of Ser-179, Met-180, Ala-183, Ala-184, Ala-187, Val-1 88, Gly-191, and Ser-192. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer.