Expression of the functional mature chloroplast triose phosphate translocator in yeast internal membranes and purification of the histidine-tagged protein by a single metal-affinity chromatography step.

Abstract

The mature part of the chloroplast triose phosphate-phosphate translocator was cloned into the yeast expression vector pEVP11. This construct was used to transform cells from both Saccharomyces cerevisiae and the rmsion yeast Schizosaccharomyces pombe. The chloroplast translocator protein was functionally expressed in the transformed yeast cells and represented about 1-2% of the Sch. pombe cell membrane protein. It was localized to mitochondrial membranes and/or membranes of the rough endoplasmic reticulum. In order to purify the recombinant translocator protein, a sequence encoding a C-terminal tag of six histidine residues was introduced into the corresponding cDNA. The expressed histidine-tagged translocator protein was purified from the transformed yeast cells under nondenaturing conditions to apparent homogeneity by a single-step affinity chromatography using a Ni2+ nitrilotriacetic acid resin. Both the expressed triose phosphate translocator and the recombinant histidinetagged protein possess substrate specificities identical to those of the authentic chloroplast protein, providing definitive evidOnce for its identity as the triose phosphate translocator and further disproving its assignment as the receptor for chloroplast protein import. The yeast expression system in combination with the Ni2+ nitrilotriacetic acid chromatography thus provides a valuable tool for the production of purified membrane proteins in a functional state.During photosynthetic CO2 fixation, the fixed carbon (in the form of triose phosphates and 3-phosphoglycerate) is exported from the chloroplasts into the cytosol, where it is converted into other substances such as sucrose and amino acids. This transport is mediated by the Frankfurt) and the yeast expression vector pEVP11 was kindly provided by N. Sauer (University of Regensburg). All other chemicals were of the highest purity available.Cloning Procedures. The DNA encoding only the mature part of the spinach translocator protein was generated by PCR from the full-length cDNA clone encoding the entire precursor protein (2). As a sense primer, a synthetic oligonucleotide corresponding to the beginning of the mature part of the cTPT with an additional ATG codon was used (P1), and the second oligonucleotide was a (reverse) T7 primer. The resulting DNA fragment was first cloned into EcoRV-cut pBSC to yield clone pBSC-mPtra. This vector was digested with Sal I, the Sal I ends were filled in, and the Sal I-filled/BamHI fragment was then ligated into the Sac I-filled/BamHI-cut yeast expression vector pEVPll containing the S. cerevisiae LEU2+ gene (8) 2155The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

ArabidopsisSAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development

“…The fact that reconstituted solute transporters display slightly higher K m values than those in their authentic environment is commonly observed. For example, the apparent K m of the plastidic triose phosphate/phosphate translocator reconstituted into liposomes is approximately threefold higher than that determined using intact chloroplasts (Gross et al, 1990;Loddenkotter et al, 1993;Fischer et al, 1997). Also, the apparent K m values observed for plastidic dicarboxylate translocators reconstituted into liposomes are higher than those observed in intact, isolated plastids (Renne et al, 2003).…”

Molecular Identification and Functional Characterization of Arabidopsis thaliana Mitochondrial and Chloroplastic NAD+ Carrier Proteins

“…the barley carrier protein HvSUT2 and the Arabidopsis carrier AtSUT4 are located in the plant vacuolar membrane (57), but both carriers mediate sucrose transport across the plasma membrane when heterologously synthesized in bakers' yeast (58,59). Alternatively, the expression of the plastid triose-phosphate carrier from spinach leads to accumulation of the recombinant protein in yeast endomembranes (60). Obviously, the expression of carriers residing in plant membranes which are absent in yeast cells may provide false information on the subcellular localization.…”

The Plastidic Bile Acid Transporter 5 Is Required for the Biosynthesis of Methionine-Derived Glucosinolates inArabidopsis thaliana

“…BAT5 was also expressed in various heterologous and homologous systems, namely, in Escherichia coli, yeast cells, and plant leaves. Membranes isolated from these systems and/or the recombinant transporter purified using the His-Tag system were reconstituted into artificial membranes prepared from acetone-washed soybean (Glycine max) phospholipids to assess transport characteristics (Loddenkö tter et al, 1993). Unfortunately, all these experiments failed, most probably due to the hydrophobicity of the 2-keto acids, which leads to unspecific membrane binding and permeation, thus covering specific transport events.…”