A PEST-like sequence in the N-terminal cytoplasmic domain of Saccharomyces maltose permease is required for glucose-induced proteolysis and rapid inactivation of transport activity

Abstract

Maltose permease is required for maltose transport into Saccharomyces cells. Glucose addition to maltose-fermenting cells causes selective delivery of this integral plasma membrane protein to the yeast vacuole via endocytosis for degradation by resident proteases. This glucose-induced degradation is independent of the proteasome but requires ubiquitin and certain ubiquitin conjugating enzymes. We used mutation analysis to identify target sequences in Mal61/HA maltose permease involved in its selective glucose-induced degradation. A nonsense mutation was introduced at codon 581, creating a truncated functional maltose permease. Additional missense mutations were introduced into the mal61/HA-581NS allele, altering potential phosphorylation and ubiquitination sites. No significant effect was seen on the rate of glucose-induced degradation of these mutant proteins. Deletion mutations were constructed, removing residues 2-30, 31-60, 61-90, and 49-78 of the N-terminal cytoplasmic domain, as well as a missense mutation of a dileucine motif. Results indicate that the proline-, glutamate-, aspartate-, serine-, and threonine-rich (PEST) sequence found in the N-terminal cytoplasmic domain, particularly residues 49-78, is required for glucose-induced degradation of Mal61/HAp and for the rapid glucose-induced inactivation of maltose transport activity. The decreased rate of glucose-induced degradation correlates with a decrease in the level of glucose-induced ubiquitination of the DeltaPEST mutant permease. In addition, newly synthesized mutant permease proteins lacking residues 49-78 or carrying an alteration in the dileucine motif, residues 69 and 70, are resistant to glucose-induced inactivation of maltose transport activity. This N-terminal PEST-like sequence is the target of both the Rgt2p-dependent and the Glc7p-Reg1p-dependent glucose signaling pathways.