Abstract
In Saccharomyces cerevisiae, alpha-isopropylmalate (alpha-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported alpha-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed alpha-IPM transporter. Transport was saturable with a half-saturation constant of 75 +/- 4 microm for alpha-IPM and 0.31 +/- 0.04 mm for beta-IPM and was inhibited by the substrates of Oac1p. Though not transported, alpha-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, alpha-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an alpha-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with alpha-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant DeltaOAC1DeltaLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of DeltaOAC1DeltaLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates alpha-ketoisocaproate and alpha-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of alpha-IPM, probably in exchange for oxalacetate.
Highlights
The obligatory specific intermediate of leucine biosynthesis in Saccharomyces cerevisiae, ␣-isopropylmalate (␣-IPM),2 is Ricerca (MUR), the Center of Excellence in Genomics (CEGBA) and Apulia Region, a contract from the European Commission (LSHM-CT-2004503116), and University local funds
Tel.: 39-0-805443374; Fax: 39-0-805442770; E-mail: fpalm@ farmbiol.uniba.it. 2 The abbreviations used are: ␣-IPM, ␣-isopropylmalate; Ctp1p, citrate transport protein; Dic1p, dicarboxylate carrier; Fsf1p, fungal sideroflexin 1; -IPM, -isopropylmalate; KIC, ␣-ketoisocaproate; KIV, ␣-ketoisovalerate; KMV, ␣-keto--methylvalerate; Leu3p, transcription factor that regulates produced from KIV in the mitochondrial matrix by the action of two isopropylmalate synthetases, Leu9p3 and Leu4lp, and in the cytosol by the action of Leu4sp. ␣-IPM, which is formed in mitochondria, must be exported to the cytosol because the subsequent two reactions of leucine biosynthesis producing KIC are catalyzed by two enzymes, IPM-isomerase and -IPM dehydrogenase, which are exclusively cytosolic [2, 3]
It has been suggested that Fsf1p (YOR271c), the S. cerevisiae member of the sideroflexin family [4], might be the transporter for ␣-IPM across the mitochondrial membrane, because in all studied genomes the promoter of Fsf1p contains the consensus binding site for a specific regulator of leucine biosynthesis, Leu3p [5]
Summary
Materials—Radioactive compounds were supplied from Amersham Biosciences and PerkinElmer Life Science Products. ␣-IPM was obtained from Sigma and -IPM from Wako. The SLC25A34-pYES2, SLC25A35-pYES2, and SLC25A10-pYES2 plasmids were constructed by cloning the coding sequences of SLC25A34, SLC25A35, and SLC25A10, respectively, into the yeast pYES2 expression vector (Invitrogen) under the control of the constitutive MIR1 promoter. When measuring the activity of carrier proteins catalyzing uniport besides antiport (e.g. Oac1p), external substrate was removed by exclusion chromatography in the presence of a reversible inhibitor (50 M p-hydroxymercuribenzoate) to avoid efflux of internal substrate, and transport was started by adding combined substrate and 0.5 mM DTE. The external substrate was removed, and radioactivity in the liposomes was measured [19]. In forward exchange kinetic measurements, the initial transport rate was calculated from the radioactivity taken up by the proteoliposomes after 1 min in the initial linear range of substrate uptake. An anaerobic Oxoid jar was used to grow yeast strains in anaerobiosis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
