Identification of a Novel Membrane Transporter Associated with Intracellular Membranes by Phenotypic Complementation in the Yeast Saccharomyces cerevisiae

Abstract

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 transmembranespanning 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 Golgi 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⌬C) in plasma membranes of Xenopus laevis oocytes. MTP⌬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⌬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.Mammalian cells normally possess the capacity for mediated translocation of nucleosides across their plasma membranes (for review see Ref. 1). These nucleoside-selective transport (NT) 1 processes function in the salvage of extracellular nucleosides for energy metabolism and nucleic acid synthesis and play a central role in regulating extracellular levels of adenosine, uridine, and other naturally occurring nucleosides. NT processes also play an important role in cellular uptake of nucleoside drugs. Seven functionally distinct NT processes have been identified in plasma membranes of mammalian cells, and it is likely that more exist. Characterization of the proteins responsible for NT processes has been hindered by their low abundance in plasma membranes. The identities of three transporters (SNST1 of rabbit kidney, cNT1 of rat intestine, and SPNT of rat liver) that mediate functionally distinct, Na ϩ -dependent NT processes have recently been determined by expression cloning in oocytes of Xenopus laevis (2-4). Although cNT1 and SPNT are members of the same transporter family, the absence of any sequence homology between SNST1 and either cNT1 or SPNT suggests that NT proteins of mammalian cells likely comprise a structurally diverse group of transporter proteins.The present study was undertaken to isolate cDNAs encoding equilibrative NT proteins of mammalian cells by functional expression in the yeast, Saccharomyces cerevisiae. Because it shares many cellular pathways with higher eukaryotes, S. cerevisiae has been used for identification of cDNAs encoding a variety of proteins from higher organisms, incl...

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Molecular cloning and characterization of LAPTM4B, a novel gene upregulated in hepatocellular carcinoma

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