Abstract
Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae. Our strategy consisted of the sequential replacement of each 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. During the sequential replacement of each Cys, the effects of both hydrophilic 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 triple Cys replacement mutants (i.e. Cys(192) being the only remaining Cys) indicated that sulfhydryl reagents no longer inhibit but in fact stimulate CTP function 2-3-fold. Following the simultaneous replacement of all four endogenous Cys, the functional properties of the resulting Cys-less CTP were shown to be quite similar to those of the wild-type protein. Finally, utilizing 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 covalently modified with (2-aminoethyl)methanethiosulfonate hydrobromide, a reagent that restores positive charge at this site, supports CTP function. The results clearly show that positive charge at these two positions is essential for CTP function, although the chemistry of the guanidinium residue is 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 within this metabolically important protein; and (iii) have led to the hypothesis that the polar face of alpha-helical transmembrane domain IV, within which Arg(181), Arg(189), and Cys(192) are located, constitutes an essential portion of the citrate translocation pathway through the membrane.
Highlights
Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae
This work demonstrates that cysteines are not essential to the CTP translocation mechanism but importantly: (i) provides insight as to which cysteines are responsible for sulfhydryl reagent-mediated inhibition; (ii) demonstrates the suitability of the Cys-less CTP for a variety of structure/function analyses; and (iii) provides important new information regarding the role of positive charge and Cys192 within transmembrane domain IV of the CTP
The main goals of the present investigation were 3-fold, namely: (i) a determination of whether any of the four endogenous cysteine residues are critical to the CTP transport mechanism, as well as which residues are responsible for the well documented sulfhydryl reagent-mediated inhibition of CTP function; (ii) the construction of a cysteine-less CTP that displays wild-type functional properties; and (iii) the subsequent use of the Cys-less construct to probe the roles of Arg181 and Arg189 in CTP function
Summary
Site-directed Mutagenesis, Overexpression, and Isolation of the Citrate Transport Protein Mutants—Mutant CTP genes were prepared utilizing the PCR Site-Directed Mutagenesis System (Life Technologies, Inc.) This system combines the use of PCR to introduce the desired mutation followed by uracil DNA glycosylase cloning. Reconstituted BTC-sensitive citrate transport and a determination of the substrate specificity of the wild-type and mutant CTPs were carried out exactly as described previously [6, 11]. Proteoliposomes (45–50 l) were preincubated with 3.5– 4.0 l of either buffer [6] (experimental tube) or 200 mM BTC (control tube) for 3– 4.5 min and were further incubated with 3.5– 4.0 l of either a given inhibitor or deionized water for 3–5 min immediately prior to the transport triggering addition of 1.1–1.3 mM [14C]citrate. Miscellaneous Procedures—Protein incorporation into phospholipid vesicles was quantified following centrifugation of the Dowex eluate at approximately 314,000 ϫ g (maximum) for 1.5 h and subsequent assay of the resulting supernatant and pellet fractions via the method of Kaplan and Pedersen [13] modified as previously detailed [6]
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