Glucose transport in baker's yeast is regulated by changing the affinity of the glucose carrier. The constitutive carrier undergoes a twenty to fifty fold change in affinity depending on metabolic conditions. Under aerobic conditions it shows low affinity; under anaerobic conditions it shows high affinity. The mechanism of this regulation is not understood. To study the mechanism of regulation the carrier must be isolated and purified and studied in reconstituted liposomes. The carrier will be solubilized by detergents (i.e. octylglucoside) and purified by standard fractionation and chromatographic techniques. The course of purification will be assessed by gel electrophoresis and the specific activity of transport activity after reconstitution in proteoliposomes. To aid in the isolation and purifica-of the carrier, an attempt will be made to clone the carrier gene on a hybrid bacterial-yeast plasmid (i.e. a """"""""shuttle plasmid"""""""") which will allow its selection in E. coli and transfer to yeast. Carrier synthesis should be greatly amplified in yeast cells containing the plasmids. The cloned gene will further allow determination of its primary structure and be useful in in vitro synthesis.
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