A functional analysis of the yeast ubiquitin ligase Rsp5: the involvement of the ubiquitin-conjugating enzyme Ubc4 and poly-ubiquitination in ethanol-induced down-regulation of targeted proteins.
Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae. We have found that the Ala401Glu rsp5 mutant is hypersensitive to various stresses, suggesting that Rsp5 is a key enzyme for yeast cell growth under stress conditions. The ubiquitination and the subsequent degradation of stress-induced misfolded proteins are indispensable for cell survival under stress conditions. In this study, we analyzed the ubiquitin-conjugating enzyme Ubc4 and the poly-ubiquitination of targeted proteins involved in the function of Rsp5 under ethanol stress conditions. Ubc4 was found to be important in yeast cell growth and poly-ubiquitination of the bulk proteins in the presence of ethanol. The general amino acid permease Gap1 is poly-ubiquitinated via Lys63 and is down-regulated after the addition of ammonium ions through a process requiring Rsp5. We found that Gap1 was removed from the plasma membrane in the presence of ethanol in a Rsp5-dependent manner, and that the disappearance of Gap1 required Ubc4 and involved the lysine residues of ubiquitin. Our results also indicate that Lys6 of ubiquitin might inhibit the disappearance of Gap1. These results suggest that Rsp5 down-regulates the ethanol-induced misfolded forms of Gap1. In addition, it appears that the substrates of Rsp5 are appropriately poly-ubiquitinated via different lysine residues of ubiquitin under various growth conditions.
Hiraishi
Hiroyuki
H
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
Okada
Masahiro
M
Ohtsu
Iwao
I
Takagi
Hiroshi
H
D026905:Amino Acid Transport Systems / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects*; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology*; D008239:Lysine; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological / Q000187:drug effects / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D026905:Amino Acid Transport Systems; D056827:Endosomal Sorting Complexes Required for Transport; C491145:GAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D000431:Ethanol; C501306:Ubc4 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae; D008239:Lysine