The yeast mitochondrial citrate transport protein - Probing the roles of cysteines, Arg(181), and Arg(189) in transporter function

Utilizing site-directed mutagenesis in combination with chemical modificati on of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharo myces cerevisiae. Our strategy consisted of the sequential replacement of e ach of the four endogenous cysteine residues with Ser or in the case of Cys (73) with Val. Wild-type and mutated forms of the CTP were overexpressed in Escherichia coli, purified, and reconstituted in phospholipid vesicles. Du ring the sequential replacement of each Cys, the effects of both hydrophili c and hydrophobic sulfhydryl reagents were examined. The data indicate that Cys(73) and Cys(256) are primarily responsible for inhibition of the wild- type CTP by hydrophilic sulfhydryl reagents. Experiments conducted with tri ple Cys replacement mutants (i.e. Cys(192) being the only remaining Cys) in dicated that sulfhydryl reagents no longer inhibit but in fact stimulate CT P function 2-3-fold. Following the simultaneous replacement of all four end ogenous Cys, the functional properties of the resulting Cys-less CTP were s hown to be quite similar to those of the wild-type protein. Finally, utiliz ing the Cys-less CTP as a template, the roles of Arg(181) and Arg(189), two positively charged residues located within transmembrane domain IV, in CTP function were examined. Replacement of either residue with a Cys abolishes function, whereas replacement with a Lys or a Cys that is subsequently cov alently modified with (2-aminoethyl) methanethiosulfonate hydrobromide, a r eagent that restores positive charge at this site, supports CTP function. T he results clearly show that positive charge at these two positions is esse ntial for CTP function, although the chemistry of the guanidinium residue i s not. Finally, these studies: (i) definitely demonstrate that Cys residues do not play an important role in the mechanism of the CTP; (ii) prove the utility of the Cys-less CTP for studying structure/function relationships w ithin this metabolically important protein; and (iii) have led to the hypot hesis that the polar face of alpha-helical transmembrane domain IV, within which Arg(181), Arg(189), and Cys(192) are located, constitutes an essentia l portion of the citrate translocation pathway through the membrane.