ISSN:
1573-6881
Keywords:
Mitochondria
;
transporter
;
dimer
;
overexpression
;
membrane protein
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Physics
Notes:
Abstract Experiments have been conducted to determine the oligomeric state of the mitochondrial citratetransport protein (CTP) from the yeast Saccharomyces cerevisiae. Both wild-type andcysteine-less (Cys-less) CTPs were overexpressed in E. coli and solubilized with sarkosyl. The purity ofthe solubilized material is approximately 75%. Upon incorporation into phospholipid vesicles, ahigh specific transport activity is obtained with both the wild-type and Cys-less CTPs, therebydemonstrating the structural and functional integrity of the preparations. Two independentapproaches were utilized to determine native molecular weight. First, CTP molecular weightwas determined via nondenaturing size-exclusion chromatography. With this methodology weobtained 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 lowconcentration of the negatively charged detergent sarkosyl, which served to both impart acharge 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 forthe wild-type and Cys-less CTPs, respectively. Both methods clearly indicate that followingsolubilization, 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 dimericstate of the CTP has important implications for the structural basis underlying the CTPtranslocation mechanism.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1005460810527
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