Department of Biology, York University, Toronto, Ontario, Canada.
Exp Cell Res. 2011 Jul 1;317(11):1567-79. doi: 10.1016/j.yexcr.2011.03.008. Epub 2011 Mar 12.
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.
核苷转运体（NTs）在核苷跨细胞膜转运中发挥着重要作用。平衡核苷转运体（ENTs）允许核苷的易化扩散，而原型 ENT，hENT1，主要定位于质膜（PM）。hENT1 负责摄取用于治疗病毒感染和癌症的核苷类似物药物，但尽管其具有重要的临床意义，但实际上人们对 hENT1 生命周期的动态，包括向 PM 的运输、内吞作用和降解，几乎一无所知。因此，我们跟踪了瞬时转染到哺乳动物细胞中的标记 hENT1（GFP-或 FLAG-）的生命周期，以深入了解调节 hENT1 生命周期的事件顺序、时间和潜在机制。使用固定和活细胞共焦显微镜检查蛋白向 PM 的易位，而通过细胞表面生物素化和 [(35)S]脉冲追踪分析分别分析内吞作用和降解。我们确定标记的 hENT1 与微管一起被运送到 PM，并整合到质膜中，随后通过网格蛋白介导的内吞作用和再循环。最后，内化的蛋白通过溶酶体途径降解，观察结果表明，这些细胞内标记的 hENT1 的完整生命周期约为 14 小时。
