Identification and functional reconstitution of the yeast peroxisomal adenine nucleotide transporter

Abstract

The requirement for small molecule transport systems across the peroxisomal membrane has previously been postulated, but not directly proven. Here we report the identification and functional reconstitution of Ant1p (Ypr128cp), a peroxisomal transporter in the yeast Saccharomyces cerevisiae, which has the characteristic sequence features of the mitochondrial carrier family. Ant1p was found to be an integral protein of the peroxisomal membrane and expression of ANT1 was oleic acid inducible. Targeting of Ant1p to peroxisomes was dependent on Pex3p and Pex19p, two peroxins specifically required for peroxisomal membrane protein insertion. Ant1p was essential for growth on medium-chain fatty acids as the sole carbon source. Upon reconstitution of the overexpressed and purified protein into liposomes, specific transport of adenine nucleotides could be demonstrated. Remarkably, both the substrate and inhibitor specificity differed from those of the mitochondrial ADP/ATP transporter. The physiological role of Ant1p in S.cerevisiae is probably to transport cytoplasmic ATP into the peroxisomal lumen in exchange for AMP generated in the activation of fatty acids.

Fig. 1. Purification and identification of Ant1p. High-salt extracted peroxisomal membranes were solubilized in SDS and proteins contained therein were separated by preparative reversed-phase HPLC. The protein identified by peptide sequencing as Ant1p (Ypr128cp) is indicated. Molecular weight standards are indicated on the left.

Fig. 2. Ant1p is localized to peroxisomes. (A) Immunological detection of Ant1p. Equal amounts of whole-cell lysates of oleic acid-induced wild-type (UTL-7A) as well as ant1Δ cells were separated by SDS–PAGE and blotted onto a nitrocellulose filter. Antibodies directed against Ant1p were applied and immunoreactive complexes were visualized with the ECL system. (B) Subcellular fractionation analysis. Cell-free extracts of oleate-induced wild-type cells (T) were separated by differential centrifugation into a 25 000 g pellet containing mainly peroxisomes and mitochondria (P) and a supernatant fraction (S). Equal portions of each fraction were analyzed by immunoblotting using anti-Ant1p antibodies. (C) Immunological detection of Ant1p in a sucrose density gradient. Cell-free extracts of oleate-induced wild-type cells (UTL-7A) were separated on a continuous sucrose density gradient (20–53%). The resulting fractions were immunologically analyzed for the distribution of Ant1p, the peroxisomal membrane protein Pex11p and mitochondrial Aac2p. The same fractions were also analyzed for the enzymatic activities of peroxisomal catalase (squares) and mitochondrial fumarase (triangles), presented in each case as the percentage of the peak fraction. The fractions’ densities are illustrated as a hatched line in the same diagram.

Fig. 3. Ant1p is a peroxisomal membrane protein. (A) Sub-peroxisomal fractionation analysis. A 25 000 g pellet of an oleic acid-induced wild-type strain (UTL-7A) was divided into three parts and treated with either 10 mM Tris–HCl pH 8 (low salt), 10 mM Tris–HCl pH 8/500 mM KCl (high salt) or 100 mM Na₂CO₃ pH 11 (carbonate). After 30 min incubation, each sample was separated into a pellet (P) and a supernatant (S) fraction by a 200 000 g centrifugation step. Proportionate volumes of the resulting fractions were subjected to immunoblotting using antibodies directed against Ant1p, Pex3p, Fat2p/Pcs60p, Fox3p and Aac2p. (B) Protease protection assay. Organelles isolated from a wild-type strain were split into four parts and incubated for 30 min with increasing concentrations of proteinase K. Reactions were stopped by the addition of 4 mM PMSF and trichloroacetic acid, separated by SDS–PAGE and analyzed by immunoblotting.

Fig. 4. Peroxisomal location of Ant1p depends on the membrane protein targeting route. (A) Localization of an Ant1p–GFP fusion protein. The wild-type UTL-7A and the otherwise isogenic pex13Δ, pex19Δ and pex3Δ strains expressing Ant1p–GFP under oleic acid-induction conditions were examined for GFP fluorescence. Structural integrity of the cells is documented by bright-field microscopy. (B) Stability of the Ant1p–GFP fusion protein in pex mutants. The same strains as in (A) were induced for 14 h in rich oleate-containing medium. Whole-cell extracts of these samples were analyzed for the amount of Ant1p–GFP, mitochondrial Aac2p and cytosolic Pgk1p by immunological detection.

Fig. 5. Ant1p is involved in medium-chain fatty acid utilization. (A) Kinetics of Ant1p expression under oleic acid-induction conditions. Wild-type strain UTL-7A grown in minimal 0.3% glucose-containing medium was transferred to oleate-containing medium and aliquots were removed at the time points indicated. Whole-cell extracts of these samples were analyzed for the amount of Ant1p, the oleic acid-inducible Fox3p and the constitutively expressed Kar2p by immunological detection. (B) Growth behavior of an ant1Δ mutant on various carbon sources. Serial dilutions of wild-type strain FY1679α and the otherwise isogenic ant1Δ mutant were spotted on plates containing ethanol, oleic acid or lauric acid, and incubated for 2–7 days at 30°C. (C) Analysis of an ant1Δ pat1Δ double mutant. The gene deletion strains indicated (in the genetic background of FY1679α) were similarly tested for growth on oleic acid. (D) Complementation test with C.boidinii Pmp47p. Transformants of the ant1Δ mutant, expressing either Pmp47p from C.boidinii [ant1Δ (PMP47)] from plasmid pRS-315-24-47, Ant1p [ant1Δ (ANT1)] or the empty vector [ant1Δ (vector)] were streaked on lauric acid plates and incubated for 7 days at 30°C.

Fig. 6. Purification of the His₆-tagged Ant1p. (A) Proteins were separated by SDS–PAGE and stained with silver nitrate. Lanes 1 and 2, organellar pellet protein (8 µg) from wild-type (lane 1) and YPH499-pHPR178 cells (lane 2). Lane 3, Triton-solubilized extract (3.5 µg protein) of the organellar pellet in lane 2 after incubation with Ni-NTA agarose (flow through). Lane 4, His₆-tagged Ant1p (0.14 µg) purified from the organellar pellet in lane 2. Lane M, molecular weight markers (97.4, 66.2, 45, 31 and 21.5 kDa). The protein identified by MALDI-TOF mass spectrometry as Ant1p (Ypr128cp) is indicated. (B) 30 µg of organellar pellet protein from wild-type (lane 1) and YPH499-pHPR178 cells (lane 2) were separated by SDS–PAGE, transferred to nitrocellulose, and blotted with antibodies directed against Ant1p and Aac2p. Lane 3, 30 µg of Triton-solubilized extract of the organellar pellet in lane 2 after incubation with Ni-NTA–agarose (flow through). Lane 4, 2 µg of tagged Ant1p purified from the organellar pellet in lane 2.

Fig. 7. Ant1p catalyzes the transport of adenine nucleotides. (A) Time-course of [¹⁴C]ATP/ATP exchange in proteoliposomes reconstituted with the recombinant Ant1p. [¹⁴C]ATP (50 µM) was added to proteoliposomes containing 20 mM ATP (filled circles) or 20 mM GTP (open circles). (B) Dependence of Ant1p activity on internal substrate. Proteoliposomes were preloaded internally with various substrates (concentration 20 mM). Transport was started by adding 50 µM [¹⁴C]ATP and stopped after 15 min. The values are means of at least three experiments. (C) Inhibition of [¹⁴C]ATP/ATP exchange by various reagents. Proteoliposomes were preloaded internally with 20 mM ATP. Transport was started by adding 50 µM [¹⁴C]ATP and stopped after 15 min. Inhibitors were added 5 min before the labeled substrate. The final concentration of the inhibitors was 2 mM except for mercurials (0.1 mM), N-ethylmaleimide (1 mM) and carboxyatractyloside and bongkrekate (0.02 mM). The extents of inhibition (%) from a representative experiment are reported. Similar results were obtained in at least three independent experiments. pCMBS, p-chloromercuriphenylsulfonate; NEM, N-ethylmaleimide; CCN, α-cyano-4-hydroxycinnamate; PLP, pyridoxal 5′-phosphate; BAT, bathophenanthroline; CAT, carboxyatractylate, BKA, bongkrekic acid.

Fig. 8. Model for the function of the adenine nucleotide carrier Ant1p in yeast peroxisomal fatty acid metabolism. Long-chain fatty acids (LCFA) are activated in the cytosol and enter the peroxisome as acyl-CoA esters via the heterodimeric ABC transporter Pat1p/Pat2p. Medium-chain fatty acids (MCFA) enter peroxisomes as free fatty acids, which are activated in the peroxisomal lumen via the acyl-CoA synthetase Faa2p. Ant1p catalyzes the exchange of ATP against AMP across the peroxisomal membrane, which is required for the intraperoxisomal activation of medium-chain fatty acids.