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 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 deltaC) in plasma membranes of Xenopus laevis oocytes. MTP deltaC-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 deltaC-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.
Hogue
D L
DL
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Ellison
M J
MJ
Young
J D
JD
Cass
C E
CE
D000241:Adenosine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D005260:Female; D005816:Genetic Complementation Test; D006801:Humans; D007939:Leukemia L1210 / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013936:Thymidine / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000378:metabolism; D014982:Xenopus laevis
D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; C099283:Laptm4a protein, mouse; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D000241:Adenosine; D013936:Thymidine; D014529:Uridine