Abstract
The maltose transporter of Saccharomyces cerevisiae is subject to rapid, irreversible inactivation in the presence of glucose. Loss of transport function was paralleled by a decrease in amount of transporter protein and most likely involves endocytosis and degradation of the protein in the vacuole. This (catabolite) inactivation of Mal61p was triggered not only by glucose but also by 2-deoxy-D-glucose, which cannot be metabolized beyond 2-deoxy-D-glucose phosphate. The signal that targets membrane proteins specifically for catabolite inactivation is unknown. To investigate whether or not specific modification of Mal61p triggers the inactivation, putative protein kinase A and C phosphorylation sites were removed, and the transport activities and levels of the mutant proteins upon addition of glucose were followed in time. Three Mal61p mutants, i.e. S295A, T363A, and S487A, exhibited significantly reduced rates of inactivation in the presence of glucose. Likewise, in wild-type Mal61p the rate of inactivation and degradation of the protein paralleled each other in the case of T363A. On the contrary, for the S295A and S487A mutants the rates of protein degradation were slowed down more profoundly than was the loss of transport activity. These observations indicate that (i) some form of modification (e.g. phosphorylation) of the protein precedes breakdown, (ii) the modification inactivates Mal61p, and (iii) the inactivation of Mal61p is not necessarily followed by proteolytic degradation.
Highlights
The first step in maltose metabolism in yeast is performed by the maltose transport protein, which catalyzes the uptake of maltose in symport with one proton
In the S295A and the triple mutant, no significant decrease of the protein was observed. These results show that in the T363A mutant both the decrease in maltose uptake activity and the proteolytic degradation of Mal61p are retarded whereas in the S295A and S487A mutants the loss of a putative phosphorylation site has a more profound effect on the degradation than on the inactivation
The maltose transport protein belongs to a family of proteins from prokaryotic as well as eukaryotic origin, of which the members transport different sugars, i.e. MalX1p and Agt1p, maltose; HxtXp and HepG2, glucose; Gal2p, galactose; Lac12p, lactose; XylE, xylose; and AraE, arabinose (Fig. 1B) [20]
Summary
The first step in maltose metabolism in yeast is performed by the maltose transport protein, which catalyzes the uptake of maltose in symport with one proton. MAL gene expression in maltose-fermenting “wild-type” strains is inducible and glucose-repressed. The latter phenomenon is mediated by the main glucose repression/derepression pathway, which constitutes the transcription factor Mig1p, at least one protein kinase (Snf1p), and several other proteins whose functions are unknown [2]. A role for cAMP-dependent protein kinase A activity in catabolite inactivation of the high affinity glucose and galactose transporters was suggested from studies in mutants with varying kinase activities [9] These studies have been disputed more recently [10], it has not been ruled out that phosphorylation of the transport protein forms the trigger for the controlled degradation of the protein. We show that inactivation of the transport system and proteolytic degradation can be uncoupled
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