Characterization, purification and properties of the yeast mitochondrial dicarboxylate carrier (Saccharomyces cerevisiae).
The dicarboxylate carrier has been characterized and purified from mitochondria of wild strain Saccharomyces cerevisiae. The mitochondria were solubilized with Triton X-100 and the detergent extract was chromatographed on hydroxylapatite. SDS-PAGE of the hydroxylapatite pass-through showed five protein bands with M(r)s ranging from 28,000 to 35,000, by silver nitrate staining. The n-butylmalonate-sensitive succinate(out)/malate(in) exchange activity of the hydroxylapatite pass-through reconstituted into liposomes, was increased nine-fold with respect to the activity of the Triton X-100 extract. The exchange activity was inhibited by p-chloromercuriphenylsulfonate (PMPS), 4.4'diisothiocyanostilbene-2.2'-disulfonate (DIDS) and pyridoxal-phosphate, suggesting that one or more thiol groups and basic residues are implicated in the binding mechanism. The purification of the carrier was achieved by affinity chromatography on Sepharose-immobilized malate dehydrogenase. The purified protein presented the same properties as the dicarboxylate carrier in native mitochondria and displayed a single protein band with an M(r) of 28,000 as determined by SDS-PAGE. The specific activity of the purified carrier showed a 53-fold increase compared to that of the initial material. The Km for the reconstituted exchange was 2 mM for succinate with a V of 1.5 mumol min-1 mg-1 protein at 22 degrees C. The high purification state achieved for the yeast dicarboxylate carrier should allow the study of its molecular properties.
Lançar-Benba
J
J
Université de Rouen, Faculté des Sciences, Laboratoire des Transports intracellulaires, URA-CNRS 203, Mont-Saint-Aignan, France.
Foucher
B
B
Saint-Macary
M
M
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000838:Anions / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry*; D008970:Molecular Weight; D011510:Proteolipids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D000838:Anions; D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D002731:4-Chloromercuribenzenesulfonate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid