The maltose transport capacity of fermenting Succharomyces cerevisiue rapidly decreases when protein synthesis is impaired. Using polyclonal antibodies against a recombinant maltose transporter-protein we measured the cellular content of the transporter along this inactivation process. Loss of transport capacity was paralleled by a decrease of cross-reacting material which suggests degradation of the transporter. We also show that in ammonium-starved cells the half-life of the maltose transporter is 1.3 h during catabolism of glucose and > 15 h during catabolism of ethanol.

Endocytosis in Saccharomyces cerevisiae is inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following.

Monoubiquitination of the 12-transmembrane segment (12-TMS) Saccharomyces cerevisiae maltose transporter promoted the maximal internalization rate of this protein. This modification is similar to that of the 7-TMS ␣-factor receptor but different from that of the 12-TMS uracil and general amino acid permeases. This result shows that binding of ubiquitin-Lys63 chains is not required for maximal internalization of all 12-TMScontaining proteins.Binding of ubiquitin (Ub) acts as a signal for at least two different processes in Saccharomyces cerevisiae: for the degradation of cytosolic proteins by the proteasome (3) and for the internalization, for subsequent degradation, of plasma membrane proteins in the vacuole (8). Ub binds through its C terminus to a lysine residue found within target proteins by the action of a cascade of enzymes: Ub-activating enzymes (E1), Ub-conjugating enzymes (E2), and Ub-protein ligase enzymes (E3) (10). Since Ub itself contains seven lysine residues within its sequence, multi-Ub chains bound to proteins can be formed by linking the C terminus of one Ub to a lysine within another Ub. It has been proposed that the differences between the Ub chains bound to cytosolic and plasma membrane proteins could serve for recognition by their respective degradation systems (7,22,23).In yeast cells, Ub chains linked through Lys29, -48, and -63 are present in vivo (1,6,11,21), and it is well established that Ub-Lys48 chains are responsible for the recognition of cytosolic proteins by the proteasome (6). In the case of plasma membrane proteins, it has been shown that Ub-Lys63 chains play a role in the internalization of the uracil (7) and of the general amino acid permeases (22), two 12-transmembrane segment (12-TMS) proteins. However, monoubiquitination is sufficient for internalization of the ␣-factor receptor (23), a 7-TMS protein. It has been postulated that the different ubiquitination requirements for internalization of these two types of plasma membrane proteins might be related to the differences in their size and the TMS number (23).To test this hypothesis, we have examined the type of ubiquitination required for internalization of another 12-TMS protein, the maltose transporter. This transporter is internalized and degraded in the vacuole during nitrogen starvation when a fermentable carbon source is present (12,14,18). This process requires the binding of Ub and the action of both Ub ligase Npi1/Rsp5 and Ub-protein hydrolase Doa4/Npi2 (13, 15). Free Ub is present at low levels in cells lacking Doa4p (16). For this reason, internalization of plasma membrane proteins is substantially reduced in ⌬doa4 mutant cells (7,13,15,22,23). Based on the fact that this phenotype can be complemented with an overproduction of Ub (7, 22, 23), we investigated the effect of overexpressing mutant Ubs carrying Lys3Arg mutations, which prevent the formation of various kinds of Ub chains in ⌬doa4 cells.The following strains and plasmids were used: MHY501 (MAT␣ DOA4 his3-⌬200 leu2-3,112 ura3-52 lys2-801 trp1-1) and it...

The maltose transporter in Saccharomyces cerevisiae is degraded in the vacuole after internalization by endocytosis upon nitrogen starvation in the presence of a fermentable substrate. This degradation, known as catabolite inactivation, is inhibited by the presence of moderate concentrations (2 to 6%, vol/vol) of ethanol. We have investigated the mechanism of this inactivation and have found that it is due to the inhibition of the internalization of the transporter by endocytosis. The results also indicate that this inhibition is due to alterations produced by ethanol in the organization of the plasma membrane which also affects to endocytosis of other plasma membrane proteins. Apparently, endocytosis is particularly sensitive to these alterations compared with other processes occurring at the plasma membrane.