Abstract
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 properties served as the starting template. The single Cys CTP variants were overexpressed 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 first order rate constants for inhibition of CTP function. These rate constants varied by seven orders of magnitude. With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193. Based 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 been 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-188, 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.
Highlights
The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine
An advantage afforded by the yeast mitochondrial CTP is that following overexpression in Escherichia coli and subsequent isolation, CTP function can be reconstituted with high specific activity
We have previously described this methodology in detail [10, 11]2 and have shown that it results in abundant quantities of both the wildtype and Cys-less CTPs, which are highly functional when incorporated into phospholipid vesicles
Summary
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. The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function. We measured the accessibility of each mutant to hydrophilic cysteine-specific reagents (i.e. MTSES, MTSET, and MTSEA) by determining the pseudo first order rate constant for inhibition of citrate transport. Both the water-accessible and -inaccessible faces of this helix have been clearly defined We infer from these studies that the water-accessible face represents a portion of the substrate translocation pathway through the CTP, and the water-inaccessible surface likely faces the lipid bilayer
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