Abstract
The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs. We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter. hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7. Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.
Highlights
Introduction of Novel Glycosylation Sites intohENT1—Glycosylation-defective N48Q was used as PCR template to engineer N-glycosylation sites into various regions of hENT1 (Fig. 1)
The topology of hENT1 predicted by hydropathy analysis of the amino acid sequence using the algorithm of Eisenberg et al [26] contains 11 putative transmembrane helices with an amino terminus that is intracellular and a carboxyl terminus that is extracellular (Fig. 1) [10]
The results obtained for the amino terminus, loops A, B, D, and E, loop H, and the carboxyl terminus validated the topology model of hENT1 shown in Fig. 1 and were consistent with antibody studies, which independently established that loop H of native h/rENT1 was cytoplasmic
Summary
Introduction of Novel Glycosylation Sites intohENT1—Glycosylation-defective N48Q was used as PCR template to engineer N-glycosylation sites into various regions of hENT1 (Fig. 1). We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter.
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