| Membrane transporter proteins mediate the translocation of various solutes. The natural substrates of some transporter proteins, like mLAPTm4, have not yet been identified. Determining the subcellular localization of such proteins may provide clues as to their functions. Described here is the localization of mLAPTm4. Recombinant epitope-tagged mLAPTm4 was localized to lysosomes in transiently transfected cells by confocal immunofluorescence microscopy.; Nucleoside transporter proteins have been divided into five distinct superfamilies. Two of these superfamilies, the equilibrative and concentrative nucleoside transporters, have human representatives. The equilibrative nucleoside transporters (ENTs) are bi-directional transporters that translocate nucleosides down their concentration gradients and are important in the uptake of anticancer and antiviral nucleoside drugs. A major objective of this study was to generate anti-hENT1 and anti-hENT2 antibodies for use in studies of cellular distribution and regulation. Antibodies were raised against a hENT1-specific synthetic peptide and a hENT2-specific fusion protein, which corresponded to residues in the large intracellular loop regions of either hENT1 or hENT2, respectively. These antibodies recognized recombinant hENT1 and hENT2 by immunoblotting and immunofluorescence. Endogenous hENT1 and hENT2 were detected in human cell lines by immunofluorescence microscopy. The anti-hENT1 antibodies gave mostly plasma membrane staining, whereas the anti-hENT2 gave mostly intracellular staining, which was localized to mitochondria. An immunoreactive species was detected in enriched human liver mitochondria with the anti-hENT2 antibodies by immunoblot analysis.; The relationship between hENT2 and hHNP36 was examined. The hHNP36 protein is a truncated version of hENT2 that is produced by alternate splicing. In the process of confirming the structure of the hENT2 gene, a hENT2 pseudogene was discovered.; Two different pertubation methods were used to probe regulatory mechanisms of the ENT proteins in human cell lines. The first involved treating CEM cells with hydroxyurea, which resulted in an increase in hENT1 levels that correlated with the hydroxyurea-induced increase in cell size. The second involved production of hENT1 or hENT2 antisense mRNA in HEK293 cells, which did not result in the ablation of either transport activity. This study has developed important tools for the field of nucleoside transport and attempted to probe the regulation of nucleoside transport proteins. |
