The life cycle of human equilibrative nucleoside transporter 1: from ER export to degradation

Abstract

Nucleoside transporters (NTs) play an essential role in the transport of nucleosides across cellular membranes. Equilibrative NTs (ENTs) allow facilitated diffusion of nucleosides and the prototypic ENT, hENT1, is primarily localized to the plasma membrane (PM). hENT1 is responsible for the uptake of nucleoside analog drugs used in treating viral infections and cancer, but despite its clinical importance, virtually nothing is known about the dynamics of the hENT1 life cycle including trafficking to the PM, endocytosis and degradation. Therefore, we followed the life cycle of tagged hENT1 (GFP- or FLAG-) transiently transfected into mammalian cells to gain insight into the sequence of events, timing and underlying mechanisms regulating the hENT1 life cycle. Protein translocation to the PM was examined using fixed and live cell confocal microscopy while endocytosis and degradation were analyzed by cell surface biotinylation and [(35)S] pulse chase analysis respectively. We determined that tagged hENT1 is trafficked to the PM in association with microtubules and incorporated in the plasma membrane where it subsequently undergoes clathrin-mediated endocytosis and recycling. Finally, internalized protein is degraded via the lysosomal pathway and observations suggest the complete life cycle of tagged hENT1 within these cells is approximately 14 hours.