The yeast mitochondrial citrate transport protein. Probing the secondary structure of transmembrane domain iv and identification of residues that likely comprise a portion of the citrate translocation pathway.
The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine. A cysteine-less CTP retaining wild-type functional properties served as the starting template. The single Cys CTP variants were overexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function. These rate constants varied by seven orders of magnitude. With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193. Based on the pattern of accessibility we conclude that residues 177-193 exist as an alpha-helix. Furthermore, a water-accessible face of the helix has been defined consisting of Pro-177, Val-178, Arg-181, Gln-182, Asn-185, Gln-186, Arg-189, Leu-190, and Tyr-193, and a water-inaccessible face has been delineated consisting of Ser-179, Met-180, Ala-183, Ala-184, Ala-187, Val-188, Gly-191, and Ser-192. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer.
Kaplan
R S
RS
Department of Biochemistry & Molecular Biology, FUHS/The Chicago Medical School, North Chicago, Illinois 60064, USA. kaplanr@mis.finchcms.edu
Mayor
J A
JA
Brauer
D
D
Kotaria
R
R
Walters
D E
DE
Dean
A M
AM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000737:chemistry*; D003545:Cysteine / Q000032:analysis; D004926:Escherichia coli; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives; D005656:Fungal Proteins / Q000737:chemistry*; D007202:Indicators and Reagents; D008698:Mesylates; D008928:Mitochondria / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary
D002352:Carrier Proteins; D005656:Fungal Proteins; D007202:Indicators and Reagents; D008698:Mesylates; C020723:citrate-binding transport protein; C093171:methanethiosulfonate ethylammonium; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D005020:Ethyl Methanesulfonate; D003545:Cysteine