The 2-oxoglutarate/malate translocator of chloroplast envelope membranes: molecular cloning of a transporter containing a 12-helix motif and expression of the functional protein in yeast cells.

摘要

The 2-oxoglutarate/malate translocator of spinach chloroplasts transports carbon skeletons into chloroplasts for net glutamate synthesis. A sequence of a endoprotease Lys-C peptide derived from the purified protein allowed the design of an oligonucleotide which was then used for a hybridization screening of a cDNA library. A 1945 bp insert of 1 of the isolated clones codes for the entire 569 amino acid residues of the precursor protein corresponding to a molecular mass of 60,288 Da. There was no significant homology to the mitochondrial 2-oxoglutarate/malate carrier from bovine heart or to any other known protein. The translocator protein is composed of a hydrophilic N-terminal region (the transit peptide) with a length of about 90-100 amino acid residues which shows, in contrast to presequences of other known envelope membrane proteins, typical features of higher plant chloroplast transit sequences. The mature protein contains 12 putative transmembrane segments in alpha-helical conformation. It is suggested that this translocator, in contrast to other known transporters of organellar origin which are all homodimers with a 6 + 6 helix folding pattern, may function as a monomer. The in vitro synthesized precursor protein is directed to chloroplasts where it is inserted into the chloroplast envelope membrane in a protease-resistant manner. The cDNA coding for the precursor protein was cloned into the yeast expression vector pEVP11, and this construct was used to transform cells from the fission yeast Schizosaccharomyces pombe. The 2-oxoglutarate/malate translocator could be functionally expressed in the transformed yeast cells, and the recombinant protein showed substrate specificities identical to those of the authentic chloroplast protein.