The yeast mitochondrial citrate transport protein: characterization of transmembrane domain III residue involvement in substrate translocation.
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.
Ma
Chunlong
C
Department of Biochemistry & Molecular Biology, Rosalind Franklin University of Medicine and Science/The Chicago Medical School, North Chicago, Illinois 60064, USA.
Kotaria
Rusudan
R
Mayor
June A
JA
Remani
Sreevidya
S
Walters
D Eric
DE
Kaplan
Ronald S
RS
D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008958:Models, Molecular; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013696:Temperature
D002352:Carrier Proteins; C020723:citrate-binding transport protein