Oligomeric state of wild-type and cysteine-less yeast mitochondrial citrate transport proteins.
Experiments have been conducted to determine the oligomeric state of the mitochondrial citrate transport protein (CTP) from the yeast Saccharomyces cerevisiae. Both wild-type and cysteine-less (Cys-less) CTPs were overexpressed in E. coli and solubilized with sarkosyl. The purity of the solubilized material is approximately 75%. Upon incorporation into phospholipid vesicles, a high specific transport activity is obtained with both the wild-type and Cys-less CTPs, thereby demonstrating the structural and functional integrity of the preparations. Two independent approaches were utilized to determine native molecular weight. First, CTP molecular weight was determined via nondenaturing size-exclusion chromatography. With this methodology we obtained molecular weight values of 70,961 and 70,118 for the wild-type and Cys-less CTPs, respectively. Second, charge-shift native gel electrophoresis was carried out utilizing a low concentration of the negatively charged detergent 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 second method, we obtained molecular weight values of 69,122 and 74,911 for the wild-type and Cys-less CTPs, respectively. Both methods clearly indicate that following solubilization, the wild-type and the Cys-less CTPs exist exclusively as dimers. Furthermore, disulfide bonds are not required for either dimer formation or stabilization. The dimeric state of the CTP has important implications for the structural basis underlying the CTP translocation mechanism.
Kotaria
R
R
Department of Biochemistry and Molecular Biology, FUHS/Chicago Medical School, Illinois 60064, USA.
Mayor
J A
JA
Walters
D E
DE
Kaplan
R S
RS
D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D002850:Chromatography, Gel; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004586:Electrophoresis; D008928:Mitochondria / Q000737:chemistry*; D008958:Models, Molecular; D008970:Molecular Weight; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D002352:Carrier Proteins; C020723:citrate-binding transport protein; D003545:Cysteine