Oligomeric state of wild-type and cysteine-less yeast mitochondrial citrate transport proteins

Experiments have been conducted to determine the oligomeric state of the mi tochondrial citrate transport protein (CTP) from the yeast Saccharomyces ce revisiae. Both wild-type and cysteine-less (Cys-less) CTPs were overexpress ed in E. coli and solubilized with sarkosyl. The purity of the solubilized material is approximately 75%. Upon incorporation into phospholipid vesicle s, a high specific transport activity is obtained with both the wild-type a nd Cys-less CTPs, thereby demonstrating the structural and functional integ rity of the preparations. Two independent approaches were utilized to deter mine native molecular weight. First, CTP molecular weight was determined vi a nondenaturing size-exclusion chromatography. With this methodology we obt ained molecular weight values of 70,961 and 70,118 for the wild-type and Cy s-less CTPs, respectively. Second, charge-shift native gel electrophoresis was carried out utilizing a low concentration of the negatively charged det ergent sarkosyl, which served to both impart a charge shift to the CTP and the protein standards, as well as to promote protein solubility. Via the se cond method, we obtained molecular weight values of 69,122 and 74,911 for t he wild-type and Cys-less CTPs, respectively. Both methods clearly indicate that following solubilization, the wild-type and the Cys-less CTPs exist e xclusively as dimers. Furthermore, disulfide bonds are not required for eit her dimer formation or stabilization. The dimeric state of the CTP has impo rtant implications for the structural basis underlying the CTP translocatio n mechanism.