Topography of the active site of the Saccharomyces cerevisiae plasmalemmal dicarboxylate transporter studied using lipophilic derivatives of its substrates.
2-Alkylmalonates and O-acyl-L-malates have been found to competitively inhibit the dicarboxylate transporter of Saccharomyces cerevisiae cells, and the substrate derivatives chosen did not penetrate across the plasmalemma under the experiment conditions. Probing of the active site of this transporter has revealed a large lipophilic area stretching between the 0.72 to 2.5 nm from the substrate-binding site. Itaconate inhibited the transport fivefold more effectively than L-malate. This suggests the existence of a hydrophobic region immediately near the dicarboxylate-binding site (to 0.72 nm). The yeast plasmalemmal transporter was different from the rat liver mitochondrial dicarboxylate transporter. An area with variable lipophilicity adjoining the substrate-binding site has been revealed in the latter by a similar method. This area is mainly hydrophobic at distances up to 1.76 nm from the binding site and is separated by a hydrophilic region from 0.38 to 0.88 nm. Fumarate but not maleate competitively inhibited succinate transport into the S. cerevisiae cells. It is suggested that the plasmalemmal transporter binds the substrate in the trans-conformation. The prospects of the proposed approach for scanning lipophilic profiles of channels of different transporters are discussed.
Aliverdieva
D A
DA
Caspian Institute of Biological Resources, Dagestan Research Center, Russian Academy of Sciences, Makhachkala, Russia.
Mamaev
D V
DV
Bondarenko
D I
DI
Sholtz
K F
KF
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D029382:Dicarboxylic Acid Transporters / Q000502:physiology*; D003998:Dicarboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008293:Malates / Q000494:pharmacology; D008565:Membrane Proteins / Q000502:physiology; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D011773:Pyruvates / Q000494:pharmacology; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*; D013379:Substrate Specificity; D013386:Succinates / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids; D008055:Lipids; D008293:Malates; D008565:Membrane Proteins; D011773:Pyruvates; D029701:Saccharomyces cerevisiae Proteins; D013386:Succinates; D014475:Uncoupling Agents; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C030298:malic acid; D005947:Glucose; C005229:itaconic acid