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Molecules 2018, 23(4), 732; https://doi.org/10.3390/molecules23040732

Functional Characterization of the Saccharomyces cerevisiae Equilibrative Nucleoside Transporter 1 (ScENT1)

1
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
2
Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
3
Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Derek J. McPhee
Received: 3 December 2017 / Revised: 7 March 2018 / Accepted: 14 March 2018 / Published: 22 March 2018
(This article belongs to the Special Issue Nucleoside and Nucleotide Analogues)
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Abstract

Equilibrative nucleoside transporters (ENTs) are polytopic membrane transporters responsible for the translocation of nucleosides, nucleobases—to a lesser extent—and nucleoside analog therapeutics across cellular membranes. ENTs function in a diffusion controlled bidirectional manner and are thought to utilize an alternating access transport mechanism. However, a detailed understanding of ENT function at the molecular level has remained elusive. ScENT1 (formerly known as Function Unknown Now 26 or FUN26) is the only known ENT ortholog endogenously expressed in S. cerevisiae, and a proteoliposome assay system was used to study homogenously overexpressed and purified ScENT1 (wildtype relative to L390A and F249I mutants). L390 and F249 are highly conserved residues and were found to alter transporter function. L390A produced a reduction of mean transport activity while F249I increased mean substrate translocation relative to wildtype protein. However, both mutations resulted in transport of UTP—a novel gain of function for any ENT. These residues were then mapped onto an ab initio model of FUN26 which suggests they function in substrate translocation (L390) or cytoplasmic gating (F249). Furthermore, wildtype, L390A, and F249I were found to be sensitive to the presence of alcohols. Ethanol attenuated ScENT1-mediated transport of uridine by ~50%. These findings further demonstrate functional similarities between ScENT1 and human ENT isoforms and support identification of FUN26 as ScENT1, the first ENT isoform in S. cerevisiae. View Full-Text
Keywords: nucleoside transport; transporter; nucleoside; nucleotide; membrane protein nucleoside transport; transporter; nucleoside; nucleotide; membrane protein
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Boswell-Casteel, R.C.; Johnson, J.M.; Hays, F.A. Functional Characterization of the Saccharomyces cerevisiae Equilibrative Nucleoside Transporter 1 (ScENT1). Molecules 2018, 23, 732.

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