The yeast mitochondrial citrate transport protein: characterization of transmembrane domain III residue involvement in substrate translocation

• Published in: The Journal of biological chemistry Vol. 280; no. 3; pp. 2331 - 2340
• Main Authors: Ma, Chunlong, Kotaria, Rusudan, Mayor, June A, Remani, Sreevidya, Walters, D Eric, Kaplan, Ronald S
• Format: Journal Article
• Language: English
• Published: United States 21.01.2005
• Subjects: Biological Transport; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Kinetics; Mitochondria - metabolism; Models, Molecular; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Temperature
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M411474200

Previous examination of the accessibility of a panel of single-Cys mutants in transmembrane domain III (TMDIII) of the yeast mitochondrial citrate transport protein to hydrophilic, cysteine-specific methanethiosulfonate reagents, enabled identification of the water-accessible surface of this domain and suggested its potential participation in the formation of a portion of the substrate translocation pathway. To evaluate this idea, we conducted a detailed characterization of the functional properties of 20 TMDIII single-Cys substitution mutants. Kinetic studies indicate that the A118C, S123C, and K134C mutants displayed a 3- to 7-fold increase in K(m). Moreover, the A118C mutation caused a doubling of the V(max) value, whereas the S123C, E131C, and K134C mutations caused V(max) to dramatically decrease, resulting in a reduction of the catalytic efficiencies of these three mutants by >97%. Examination of the ability of citrate to protect against the inhibition mediated by sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) indicated that citrate conferred significant protection of cysteines substituted at eight water-accessible locations (i.e. Gly-115, Leu-116, Gly-117, Leu-121, Ser-123, Val-127, Glu-131, and Thr-135), but not at other sites. Importantly, similar levels of protection were observed at both 4 degrees C and 20 degrees C. The temperature independence of the protection indicates that substrate binding and/or occupancy of the transport pathway sterically blocks the access of MTSES to these sites, thereby providing direct protection, without involvement of a major protein conformational change. The significance of these extensive functional investigations is discussed in terms of the three-dimensional CTP homology model that we previously developed and a new model of the dimer interface.