Abstract
We have characterized 1,2,3-benzenetricarboxylic acid-sensitive, mersalyl-insensitive citrate uptake by mitochondria from two strains of Saccharomyces cerevisiae by describing the time course, Km and Vmax values, pH dependence, and response to inhibitors. In unloaded mitochondria from PSY142 CS1- cells, a mutant that lacks mitochondrial citrate synthase, both citrate uptake and efflux were reduced 7- and 8-fold, respectively, compared with the parental strain. No malate uptake was detectable in mitochondria from CS1- cells, while in the parental strain, uptake was 5.4 nmol/min/mg of protein. In contrast, mutations in peroxisomal citrate synthase (CS2-) or in other tricarboxylic acid cycle enzymes did not result in changes in mitochondrial citrate transport, suggesting a specific functional role for mitochondrial citrate synthase in citrate transport. More important, liposomes containing protein extracts from CS1- mitochondria showed the same citrate and malate transport rates as liposomes made from protein extracts of parental strain mitochondria. Thus, an apparently normal amount of both the citrate transporter and the dicarboxylate carrier is present in CS1- mitochondria, but both function abnormally in undisrupted mitochondria. We suggest that cooperation between the citrate transporter and mitochondrial citrate synthase is necessary for normal function of the transporter.
Highlights
From the §Research Service, Department of Veterans Affairs Medical Center and the Wepartmentof Biochemistry and Znternal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75216 and the $University Medical School, Institute of Biochemistry, Szigeti ut 12, H-7624, Hungary
1,2,3-benzenetricarboxylic protein.Subsequentsearchesformitochondrialmembraneacid-sensitive,mersalyl-insensitivecitrateuptake by binding proteins using affinity chromatography have identified mitochondriafrom two strainsof Saccharomyces cerevi- the citrate transporter [5]and the dicarboxylate carrier( 6 )as siae by describing the time courseK, and V, values, being capable of being bound to immobilized citrate synthase pH dependence, and response to inhibitoInrs.unloaded and malate dehydrogenase, respectively. These results suggest mitochondriafrom PSY142CS1- cells, a mutantthat that t h e tricarboxylic acid cycle has a vectorial organization lacks mitochondrial citrate synthase, both citrate up- that includes transportof substrates across the mitochondrial take and efflux were reduce7d- and &fold, respectively, membrane
No malate uptake port and metabolism of glucose (71, carnitine [8], and ornithine ttwtdhheiaideanss.penIda(noeCrttecSenor2cnet-ta)astoulrbralilstnesitntrc,aiohnmitanhun,megtruaietptsoittorcainihnkcsoaenmrindbwirotf5aproix.asoec4ymrhlnoiocmxCniSdosal1orcl-mimiadcliaenlllcclmsiyct,icrgtlarweotafehtepeintlrerszoayy-nnimns--efm(9use)rW.ttahebehoralionvnteoatbhneedgi nuthtneear saceci trtriiaeotsneosft rsatnr usopcf toutrhrteee/rf.turTnioccataribococnoxmysltpiucldaisicehisdthtiosc,ypwcrloeeb e port, suggesting a specific functional role for mitochhoavne-measured the effects of mutations in tricarboxylic acid drial citrate synthase in citrate transportM. ore impor- enzymes of Saccharomyces cerevisiae mitochondria on the uptant, liposomes containing protein extracts from CS1- take of citrate and malate into both intact mitochondria and mitochondria showed the same citrate and malate tmriatnocsh-ondrial protein extracts reconstituted into liposomes
Summary
1,2,3-benzenetricarboxylic protein.Subsequentsearchesformitochondrialmembraneacid-sensitive,mersalyl-insensitivecitrateuptake by binding proteins using affinity chromatography have identified mitochondriafrom two strainsof Saccharomyces cerevi- the citrate transporter [5]and the dicarboxylate carrier( 6 )as siae by describing the time courseK, ,,, and V,, values, being capable of being bound to immobilized citrate synthase pH dependence, and response to inhibitoInrs.unloaded and malate dehydrogenase, respectively. Grigorenko et al [4] provided preliminary evidence that t h e rase mutant (FUM-1 was derived from the W303-1A parental strain loss of mitochondrial citrate synthase results ina reduction of (MATa, ade2-1,his, leu2-3,112,trpl-1, ura, cad-100). Suspensions were diluted with 900 plof ice-cold 40 mM BTC, and the tubes were gently vortexed and centrifuged ina n Eppendorf centrifuge (Model 5415) for 1min at 14,000 rpm (800x0 g ) .The supernatanst olution was decantedt,he wall of the tube was wiped to remove residual radioactivity, and the pellet was dissolved i n 100 pl of concentrated formic acid and transferred into. Mitochondria from the PSY142 parental strain were Protein Determination-Mitochondrial extractswere collected by usually loaded for 10 min, while those from the CS1- mutant were centrifugation in a n Eppendorf centrifuge (14,000 rpmfor 1rnin), preloaded for 15 min. Extramitochondrial space was determined with [U-'4Clsucrose as a marker
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