Isolation and Characterization of the Tricarboxylate Transporter from Pea Mitochondria

Abstract

The tricarboxylate transporter was solubilized from pea (Pisum sativum) mitochondria with Triton X-1 14, partially purified over a hydroxylapatite column, and reconstituted in phospholipid vesicles.The proteoliposomes exchanged external [14C] (7,9,17,30).Although mitochondrial transporters are found in both plants and animals, there are some differences in the types of transporters present. Plant leaf mitochondria, for example, rapidly exchange glycine and L-senne to support the photorespiratory conversion of glycine to seine (19,27 for the phosphate transporter has been obtained from Arabidopsis (our unpublished data). Several organic acid transporters have been solubilized from mitoplasts, partially purified, and incorporated into liposomes. The monocarboxylate, dicarboxylate (24), glutamate/aspartate (25), and aketoglutarate (10) transporters have been isolated from plant mitochondria and incorporated into proteoliposomes for detailed study. However, sequence information for the organic acid transporters is not yet available from either plants or animals.The tricarboxylate transporter from animal mitochondria has been studied in considerable detail (6,16,(21)(22)(23). This transporter from rat (4) and bovine (6) mitochondria has been isolated and purified 1000-and 400-fold, respectively. The preparation from rat liver showed a protein with an apparent molecular mass of 30 kD, whereas the preparation from bovine liver had a predominant 37-to 38-kD protein on SDS-PAGE gels. No conclusive proof is available that either band is the mammalian tricarboxylate transporter. Both transporters exchanged citrate, isocitrate, malate, malonate, and phosphoenolpyruvate. In this paper, we report the solubilization, partial purification, reconstitution, and analysis of the tricarboxylate transporter from pea (Pisum sativum L.) mitochondria. buffer B containing 6 mg of cardiolipin mL-1 was added and the sample was incubated for 30 min. Solubilized membranes were then centrifuged for 35 min at 37,000g and the supernatant was removed. This crude Triton X-1 14 extract was then chromatographed on cold dry hydroxylapatite (0.5 g per column, 0.5 mL of crude extract per column). Elution was done with buffer B and the first 1.0 mL off each column was collected and pooled. MATERIALS AND METHODS Preparation of ProteoliposomesDried azolectin was resuspended in buffer C (120 mm Hepes, 50 mm KCl, 1 mm EDTA, pH 7.5) under N2 gas at 120 mg/mL and bath sonicated (Laboratory Supplies Co.) at room temperature until the solution was translucent. Six microliters of a 1-M solution of the organic to be loaded into the proteoliposome (citrate, malate, etc.) was added to 540 ,uL of liposomes and then 460 ,L (6.5 ,ug of protein) of the hydroxylapatite eluate (or 200 ML of the crude Triton X-114 extract containing about 4 mg of protein and 260 ,uL of buffer B) was added. The sample was vortexed and frozen at -800C.Immediately prior to the assay, the proteoliposomes were thawed for 15 min in a room-temperature water bath, cooled on ice for 5 mi...

Plant mitochondrial carriers: an overview

“…The phosphate carrier, which requires a pH gradient across the membrane, imports the Pi, the dicarboxylate carrier exchanges this phosphate for dicarboxylate and the tricarboxylate carrier exchanges dicarboxylate for citrate. A tricarboxylate transporter from pea mitochondria has been partially purified from solubilised mitochondrial membrane by hydroxylapatite chromatography [174]. After reconstitution into artificial membranes (table 1), the transporter exchanges citrate against citrate, succinate and malate.…”

Characteristics, partial purification and reconstitution of the vacuolar malate transporter of the CAM plant Kalancho� daigremontiana Hamet et Perrier de la B�thie

“…Identification and characterization of di-and tricarboxylate carriers of the inner mitochondrial membranes of animal and plant cells has also been achieved using hydroxyapatite chromatography for purification and subsequent reconstitution into liposomes (Kaplan and Pedersen 1985;Bisaccia et al 1988Bisaccia et al , 1989Bolli et at. 1989;Indivieri et al 1989;Genchi et al 1991 ;McIntosh and Oliver 1992;Nalecz et al 1992). Using the approaches successful with mitochondrial membranes, an amino-acid transporter (Thume and Dietz 1991) and the malate transporter of the tonoplast of barley mesophyll cells have been partially purified.…”

Identification of a Novel Transporter for Dicarboxylates and Tricarboxylates in Plant Mitochondria

“…for photorespiration), fatty acid metabolism (e.g. lipid mobilization and fatty acid elongation), gluconeogenesis, and isoprenoid biosynthesis (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). However, protein (gene) sequences are known for only a few plant mitochondrial carriers.…”