title: Structural requirements for conformational changes and substrate binding of the human facilitative glucose transporter-GLUT3
Creators: name: Porter, Lisa Marie
Publisher: name: University of Glasgow
Year: 1997
Subjects: subject: Molecular biology.

description: The role of proline residues in transport catalysis mediated by GLUT3 has been investigated using site-directed mutagenesis. PCR methodology was used to construct a series of eight mutant GLUTS cDNAs in which individual proline residues were replaced with alanine. Heterologous expression of the GLUTS Pro-Ala mutants in Xenopus laevis oocytes was achieved by microinjection of in vitro transcribed mRNA and has enabled an extensive analysis of the kinetic properties of these mutants. Km values for the zero-trms influx of 2-deoxy-D-glucose into mutant-injected oocytes have been determined, and compared to the values obtained for wild type GLUTS expressed in the same system. The effect of each proline substitution on the integrity of the exofacial and endofacial binding sites was investigated by determining Ki values for the inhibition of 2-deoxy-D-glucose zero-trans influx by maltose and cytochalasin B, respectively. Although no individual proline residue was observed to be absolutely essential for GLUTS catalytic activity, mutation of Pro206, Pro381, Pro385 and Pro451 to alanine resulted in an increase in the Ki for maltose. Replacement of prolines at positions 381, 383 and 385, located within putative transmembrane helix 10 of GLUT3, resulted in a reduction in the Ki for cytochalasin B. The small but significant changes observed in the nature of exofacial substrate binding indicate that proline residues are important for transporter function. In addition, the prolines located within helix 10 are important in the conformational changes of the protein. Measurement of transporter turnover numbers in Xenopus oocytes has not previously been possible due to the lack of an accurate and reproducible method for the isolation of a pure plasma membrane fraction. In order to further characterise the kinetics of the Pro-Ala mutants, a protocol was designed, based on a number of previous attempted methods, for the isolation of a clean plasma membrane fraction from Xenopus oocytes. Results from dot blot and Western blot analysis of plasma membrane fractions isolated from GLUT3-expressing oocytes, indicated that the preparation of a plasma membrane fraction free from other cellular contaminants was extremely problematic, despite various attempts at optimisation. This was attributed to the abundance of glutinous yolk protein and to the extremely fragile nature of the plasma membrane of the oocyte. Consequently, it was concluded that this methodology was not suitable for the accurate quantitation of cell surface transporters and the subsequent determination of turnover numbers in this system. To address the problem of quantitation of cell surface transporters, stable expression of GLUT transporters in a mammalian cell-culture line was investigated as an alternative to expression in oocytes. Simple and reproducible subcellular fractionation procedures are available for this cell line which would enable measurement of transporter turnover numbers. Additionally, this system is more convenient for use in photolabelling studies using ATB- BMPA and cytochalasin B, since large amounts of plasma membranes from cultured mammalian cells can be easily obtained without the need for repeated mRNA injections.