Identification and Analysis of the Domain Required for Trans-Acceleration Kinetics in the Human Glucose Transporter GLUT1: A Dissertation

Abstract

Since the initial characterization of the human glucose transporter GLUT1, it has been observed that the presence of intracellular sugar stimulates the unidirectional rate of sugar uptake by a kinetic phenomenon known as trans-acceleration. Both GLUTs 1 and 3 catalyze transacceleration, while both GLUTs 2 and 4 do not. Although the basis for trans-acceleration is unknown, potential explanations include the requirement of a modulating cofactor, cellular context, or that the behavior is an artifact of imperfect transport measurements. This thesis examines whether trans-acceleration is a sequence-specific property intrinsic to the transporter. A method for detecting trans-acceleration in mammalian cells at physiologic temperature was developed through transport of two different glucose analogs. Homology-scanning mutagenesis was employed to exchange transmembrane domains (TMs) of GLUTs 1 and 4, and thereby test for accelerated-exchange loss- or gain-of-function. This approach was extended to GLUTs 2 and 3. The catalytic rates of these chimeric proteins were determined through transport measurements and expression measured by cell-surface biotinylation. These studies show that the sequence of putative scaffolding domain TM6 is both necessary and sufficient for trans-acceleration in scaffolds of GLUT1, GLUT2, and GLUT4. The substitution of TM6 sequence between these transporters has no effect on the turnover under exchange conditions, yet profoundly modifies turnover in the absence of intracellular sugar. We propose that the sequence-specific interaction of TM6 with other TMs structurally restrains relaxation of the empty carrier in GLUTs which catalyze trans-acceleration, and that binding of intracellular sugar affects these interactions to reduce the overall duration of the transport cycle. In addition, our model suggests that the substrate binding constant and rate of carrier relaxation are inter-dependent. In this model, the dissociation constant determined by substrate binding and dissociation rates at the endofacial sugar binding site must be larger than the equivalent constant at the exofacial site in order for trans-acceleration to occur.