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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We have examined binding of [3H]nitrobenzylthioinosine (NBMPR) and influx of [3H]thymidine in adherent cultures of human choriocarcinoma (BeWo) cells and, for comparison, cervical-carcinoma (HeLa) cells. Specific association of NBMPR with BeWo cells at 22 degrees C required 1.5 h to reach an equilibrium between free and bound ligand, whereas association with HeLa cells required 20-30 min. Scatchard analysis of NBMPR binding to low-density cultures of BeWo cells revealed a total of 27 x 10(6) sites per cell, consisting of two distinct populations that differed in their affinities for NBMPR. One population bound NBMPR with 'high' affinity (Bmax.1 15.0 pmol/10(6) cells; Kd1 0.6 nM) and the other, larger, population bound NBMPR with 'low' affinity (Bmax.2 29.0 pmol/10(6) cells; Kd2 14.5 nM). By contrast, HeLa cells possessed only 4.1 x 10(5) sites per cell, and these sites all bound NBMPR with the same affinity (Bmax. 0.7 pmol/10(6) cells; Kd 0.5 nM). Interaction of NBMPR with both populations of sites in BeWo cells could be blocked by nitrobenzylthioguanosine (NBTGR), dilazep or dipyridamole. Concentration-effect relationships for dilazep inhibition of binding of 1 nM- and 25 nM-NBMPR to BeWo cells were monophasic, with virtually complete inhibition achieved at 0.1 microM and 1 microM respectively. Plasma-membrane preparations from BeWo cells also had high numbers of NBMPR-binding sites, and u.v. irradiation of site-bound [3H]NBMPR in such preparations labelled polypeptides that migrated in electrophoretograms as a broad band with a peak M(r) of 60,000. The concentration-effect relationship for NBMPR inhibition of thymidine transport by BeWo cells was biphasic, with an IC50 for inhibition of the 'NBMPR-sensitive' component of 1.6 nM and a substantial (15-20%) component of flux that was not inhibited by 10 microM-NBMPR and was thus 'NBMPR-insensitive'. Vmax. values for thymidine transport by BeWo cells were 20-30-fold larger than the corresponding values for transport by HeLa cells. Elimination of the Na+ gradient had no effect on initial rates of thymidine fluxes measured in either the presence or the absence of NBMPR. Our results demonstrate that BeWo cells have an unusually large capacity for NBMPR-sensitive nucleoside transport, apparently resulting from high levels of expression of 'erythrocyte-like' transport elements, identified by their high-affinity interaction with NBMPR. The relationship of the low-affinity binding sites to NBMPR-sensitive transporter elements is uncertain.

Lysosome-associated protein transmembrane 4 alpha (LAPTM4 alpha) and homologues comprise a family of conserved proteins, which are found in mammals, insects and nematodes. LAPTM4 alpha functions to regulate the intracellular compartmentalization of amphipathic solutes and possibly the sensitivity of cells toward anthracyclines, antibiotics, ionophores, nucleobases and organic cations. This is similar to the multidrug-resistance phenotype exhibited by cells synthesizing high levels of P-glycoprotein. Accordingly, it is possible that LAPTM4 alpha may be a suitable target for development of novel chemotherapeutic agents. LAPTM4 alpha contains four putative membrane-spanning domains and a 55 amino acid C-terminal region that faces the cytoplasm. Localization of LAPTM4 alpha to endosomes and lysosomes appears to be tightly controlled as transient high-level expression of LAPTM4 alpha in cultured cells resulted in no detectable protein on the cell surface. Mutagenic analysis of the C-terminus of LAPTM4 alpha indicated that two tandomly arranged tyrosine-containing motifs in the cytoplasmic domain are required for efficient localization of LAPTM4 alpha to vesicles containing the lysosomal marker lysosomal glycoprotein 120. Although a number of membrane proteins that localize to endosomes/lysosomes contain more than one independently functioning sorting signal, to our knowledge, LAPTM4 alpha is the first example of a membrane protein that requires two tandemly arranged tyrosine-based sorting signals for efficient localization in these compartments.

Mouse transporter protein (MTP) is a highly conserved polytopic membrane protein present in mammalian lysosomes and endosomes. The role of MTP in regulating the in vivo subcellular distribution of numerous structurally distinct small molecules has been examined in this study by its expression in a drug-sensitive strain of the yeast Saccharomyces cerevisiae. Surprisingly, the expression of MTP in membranes of an intracellular compartment resulted in a cellular resistance or hypersensitivity to a range of drugs that included nucleoside and nucleobase analogs, antibiotics, anthracyclines, ionophores, and steroid hormones. The intracellular bioavailability of steroid hormones was altered by MTP, as determined using an in vivo glucocorticoid receptordriven reporter assay in yeast, suggesting that the MTPregulated drug sensitivity arose due to a change in the subcellular compartmentalization of steroid hormones and other drugs. MTP-regulated drug sensitivity in yeast was blocked to varying degrees by compounds that inhibit lysosomal function, interfere with intracellular cholesterol transport, or modulate the multidrug resistance phenotype of mammalian cells. These results indicate that MTP is involved in the subcellular compartmentalization of diverse hydrophobic small molecules and contributes to the inherent drug sensitivity or resistance of the mammalian cell.