Mammalian cells obtain
nucleic acid precursors through the de novo synthesis of
nucleotides and the salvage of exogenous nucleobases and
nucleosides. The first step in the salvage pathway is transport across the plasma membrane. Several transport activities, including equilibrative and concentrative mechanisms, have been identified by their functional properties. We report here the functional cloning of a 2.6-kilobase pair human
cDNA encoding the
nitrobenzylmercaptopurine riboside (NBMPR)-insensitive, equilibrative nucleoside transporter ei by functional complementation of the transport deficiency in a subline of CEM human
leukemia cells. Expression of this
cDNA conferred an
NBMPR-insensitive,
sodium-independent
nucleoside transport activity to the cells that exhibited substrate specificity and inhibitor sensitivity characteristic of the ei transporter. The
cDNA contained a single open reading frame that encoded a 456-residue
protein with 11 potential membrane-spanning regions and two consensus sites for N-glycosylation in the first predicted extracellular loop. The predicted
protein was 50% identical to the recently cloned human
NBMPR-sensitive, equilibrative
nucleoside transporter ENT1 and thus was designated ENT2. Surprisingly, the carboxyl-terminal portion of the ENT2
protein was nearly identical to a smaller
protein in the GenBankTM data base (human HNP36, 326 residues) that has been identified as a
growth factor-induced delayed early response gene of unknown function. Comparison of the ENT2 and HNP36 nucleotide sequences suggested that HNP36 was translated from a second
start codon within the ENT2 open reading frame. Transient expression studies with the full-length ENT2 and a 5'-truncated construct that lacks the first
start codon (predicted
protein 99% identical to HNP36) demonstrated that only the full-length construct conferred
uridine transport activity to the cells. These data suggest that the delayed early response gene HNP36 is a truncated form of ENT2 and that the full-length open reading frame of ENT2 is required for production of a functional plasma membrane ei transporter.