Sequence Determinants of GLUT1-mediated Accelerated-exchange Transport: ANALYSIS BY HOMOLOGY-SCANNING MUTAGENESIS

Sabrina S Vollers,Anthony Carruthers

doi:10.1074/jbc.m112.369587

Sabrina S Vollers, Anthony Carruthers

Open Access

https://doi.org/10.1074/jbc.m112.369587

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Abstract

The class 1 equilibrative glucose transporters GLUT1 and GLUT4 are structurally similar but catalyze distinct modes of transport. GLUT1 exhibits trans-acceleration, in which the presence of intracellular sugar stimulates the rate of unidirectional sugar uptake. GLUT4-mediated uptake is unaffected by intracellular sugar. Using homology-scanning mutagenesis in which domains of GLUT1 are substituted with equivalent domains from GLUT4 and vice versa, we show that GLUT1 transmembrane domain 6 is both necessary and sufficient for trans-acceleration. This region is not directly involved in GLUT1 binding of substrate or inhibitors. Rather, transmembrane domain 6 is part of two putative scaffold domains, which coordinate membrane-spanning amphipathic helices that form the sugar translocation pore. We propose that GLUT1 transmembrane domain 6 restrains import when intracellular sugar is absent by slowing transport-associated conformational changes.

Highlights

The human equilibrative glucose transporter GLUT1 catalyzes accelerated-exchange transport, but the related isoform GLUT4 does not
Using homology-scanning mutagenesis in which domains of GLUT1 are substituted with equivalent domains from GLUT4 and vice versa, we show that GLUT1 transmembrane domain 6 is both necessary and sufficient for trans-acceleration
We propose that GLUT1 transmembrane domain 6 restrains import when intracellular sugar is absent by slowing transport-associated conformational changes

Summary

Background

The human equilibrative glucose transporter GLUT1 catalyzes accelerated-exchange transport, but the related isoform GLUT4 does not. Using homology-scanning mutagenesis in which domains of GLUT1 are substituted with equivalent domains from GLUT4 and vice versa, we show that GLUT1 transmembrane domain 6 is both necessary and sufficient for trans-acceleration. This region is not directly involved in GLUT1 binding of substrate or inhibitors. We engineered GLUT1 and GLUT4 chimeras in which we substituted progressively smaller domains of one transporter by the corresponding domains of the other transporter These chimeras exhibit sequence-dependent trans-acceleration gain- or loss-of-function. The implications of our findings are discussed in the context of the prevailing models for GLUT-mediated sugar transport

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RESULTS

DISCUSSION