Disrupted plasma membrane localization and loss of function reveal regions of human equilibrative nucleoside transporter 1 involved in structural integrity and activity.
Human Equilibrative Nucleoside Transporter 1 (hENT1) is an integral membrane protein that transports nucleosides and analog drugs across cellular membranes. Very little is known about intracellular processing and localization of hENT1. Here we show that disruption of a highly conserved triplet (PWN) near the N-terminus, or the last eight C-terminal residues (two hydrophobic triplets separated by a positive arginine) result in loss of plasma membrane localization and/or transport function. To understand the role of specific residues within these regions, we studied the localization patterns of N- or C-terminal deletion and/or substitution mutants of GFP-hENT1 using confocal microscopy. Quantification of GFP-hENT1 (mutant and wildtype) protein at the plasma membrane was conducted using nitrobenzylthioinosine (NBTI) binding. Functionality of the GFP-hENT1 mutants was determined by heterologous expression in Xenopus laevis oocytes followed by measurement of uridine uptake. Mutation of the proline within the PWN motif disrupts plasma membrane localization. C-terminal mutations (primarily within the hydrophobic triplets) lead to hENT1 retention within the cell (e.g. in the ER). Some mutants still localize to the plasma membrane but show reduced transport activity. These data suggest that these two regions contribute to the structural integrity and thus correct processing and function of hENT1.
Nivillac
Nicole M I
NM
Department of Biology, York University, Toronto Canada M3J 1P3.
Wasal
Karanvir
K
Villani
Daniela F
DF
Naydenova
Zlatina
Z
Hanna
W J Brad
WJ
Coe
Imogen R
IR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D001943:Breast Neoplasms / Q000378:metabolism; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000737:chemistry; D033721:Equilibrative Nucleoside Transporter 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014529:Uridine / Q000378:metabolism; D014982:Xenopus laevis / Q000254:growth & development / Q000378:metabolism
D017931:DNA Primers; D033721:Equilibrative Nucleoside Transporter 1; C438649:SLC29A1 protein, human; D014529:Uridine