Identification of a novel membrane transporter associated with intracellular membranes by phenotypic complementation in the yeast Saccharomyces cerevisiae

A partial mouse cDNA was isolated by its ability to functionally complement a thymidine transport deficiency in plasma membranes of the yeast, Saccharomyces cerevisiae, The full-length cDNA encoded a previously unidentified 27-kDa protein (mouse transporter protein (MTP)) with four predicted transmembrane-spanning domains, MTP mRNA was detected in cells of several mammalian species, and its predicted protein sequence exhibited near identity (98%) with that of a human cDNA (HUMORF13), MTP and its homologs evidently reside in an intracellular membrane compartment because a protein (about 24 kDa) that was recognized by MTP-specific antibodies was observed in a subcellular fraction of rat hepatocytes enriched for Gels membranes. Deletion of the hydrophilic C terminus of MTP, which encompassed two putative signal motifs for intracellular localization (Tyr-X-X-hydrophobic amino acid), allowed expression of recombinant protein (MTP Delta C) in plasma membranes of Xenopus laevis oocytes, MTP Delta C-expressing oocytes exhibited greater fragility than nonexpressing oocytes, and those that survived the experimental manipulations were capable of mediated uptake of thymidine, uridine, and adenosine, Thymidine uptake by MTP Delta C-expressing oocytes was inhibited by thymine and dTMP. MTP may function in the transport of nucleosides and/or nucleoside derivatives between the cytosol and the lumen of an intracellular membrane-bound compartment.