Catabolite inactivation of the yeast maltose transporter occurs in the vacuole after internalization by endocytosis

Abstract

The maltose transporter of Saccharomyces cerevisiae is rapidly degraded during fermentation in the absence of a nitrogen source. The location and mechanism of degradation of the transporter have been investigated. Using mutants defective in endocytosis, we have shown that degradation of this transporter requires internalization by endocytosis. In addition, studies of mutants defective in proteasome or vacuolar proteolysis revealed that degradation occurs in the vacuole and is independent of proteasome function. The results also revealed that degradation of the maltose transporter requires Sec18p and raised the question of whether in the absence of Sec18p activity the internalized maltose transporter is recycled back to the plasma membrane.Sugar transporters are integral plasma membrane proteins that catalyze the first rate-limiting step of glycolysis in Saccharomyces cerevisiae (11). Several strategies are used by this organism to adjust the activities of these proteins to different environmental conditions (22). One of these strategies is the irreversible inactivation of the transporters, which occurs when protein synthesis is impaired upon exhaustion of a nitrogen source in the medium (3-5, 9, 23, 27) and which results in dramatic physiological effects (20,21,23). This inactivation, known as catabolite inactivation (17), affects mainly the V max of the transporters, follows first-order kinetics, and is an energy-dependent process stimulated by fermentable substrates (3,4,9). By using polyclonal antibodies against a recombinant maltose transporter protein, it has been shown that catabolite inactivation is due to proteolysis (26). The experiments reported here attempt to establish the location and mechanism of the maltose transporter degradation. We investigated the inactivation of the maltose transporter by measuring the rate of maltose uptake with radioactive sugar as well as by determining the cellular content of the transporter with polyclonal antibodies. Possible loci investigated are the plasma membrane, which is the locus of transporter action, the cytoplasm, and the vacuole after internalization of the transporter by endocytosis. In this study, we used strains defective in the internalization step of endocytosis as well as strains that show a defect either in the ''chymotrypsin-like'' activity of the proteasome complex or in the two main vacuolar endopeptidases.

Glucose control in Saccharomyces cerevisiae: the role of MIG1 in metabolic functions

“…Carbon catabolite inactivation affects enzymes of peripheral functions, for example maltose permease and galactose permease (Gorts, 1969 ;De Juan & Lagunas, 1986), as well as enzymes of central functions such as the gluconeogenic enzymes fructose-l,6-bisphosphatase and phosphoenolpyruvate carboxykinase (Holzer, 1976). Vacuolar and proteasomal proteolysis have recently been identified as possible mechanisms of degradation, the former particularly for carrier proteins (Schork et al, 1994;Riballo et al, 1995;Horak & Wolf, 1997).…”

Vps34p Is Required for the Decline of Extracellular Fructose-1,6-bisphosphatase in the Vacuole Import and Degradation Pathway

“…Similarly, another vacuole resident protein, carboxypeptidase Y, is transported to the vacuole by the vacuole protein sorting (Vps) 2 pathway (3-7). Extracellular and plasma membrane proteins can be internalized and delivered to the vacuole by endocytosis (11,12). Moreover, organelles such as peroxisomes can also be targeted to the vacuole for degradation by pexophagy (13,14).…”

Gly-46 and His-50 of Yeast Maltose Transporter Mal21p Are Essential for Its Resistance against Glucose-induced Degradation

“…To verify the effect of Gly-46 and His-50, a glucose-induced degradation test was conducted for these mutant proteins (Fig. 5B) MAL61p Is Ubiquitinated to a Higher Degree than MAL21p in the Presence of Glucose-It is known that maltose transporters are not internalized until they are ubiquitinated (14). To examine differences in the efficiency of ubiquitination of Mal61p and Mal21p molecules in the presence of glucose, MAL61 and MAL21 were expressed in end4 and npi1 mutant cells, which are deficient in an early stage endocytosis process (37) and ubiquitin-conjugating activity (32,38), respectively.…”

“…The addition of glucose to maltose-grown yeast cells results in a rapid inactivation of maltose transporters followed by their degradation. This degradation process requires endocytosis of the targeted proteins, their trafficking to the vacuole, and subsequent vacuolar proteolysis (13,14). It has been reported that for various membrane transporters, phosphorylation of Ser, Thr, or Tyr residues and subsequent ubiquitination of the Lys residue are a prerequisite for their internalization on degradation.…”