Functional analysis of Rpn6p, a lid component of the 26 S proteasome, using temperature-sensitive rpn6 mutants of the yeast Saccharomyces cerevisiae.
Rpn6p is a component of the lid of the 26 S proteasome. We isolated and analyzed two temperature-sensitive rpn6 mutants in the yeast, Saccharomyces cerevisiae. Both mutants showed defects in protein degradation in vivo. However, the affinity-purified 26 S proteasome of the rpn6 mutants grown at the permissive temperature degraded polyubiquitinated Sic1p efficiently, even at a higher temperature. Interestingly, their enzyme activity was even higher at a higher temperature, indicating that once made mutant proteasomes are stable and have little defect in the proteolytic function. These results suggest that the deficiency in protein degradation observed in vivo is rather due to a defect in the assembly of a holoenzyme at the restrictive temperature. Indeed, both rpn6 mutants grown at the restrictive temperature were defective in assembling the 26 S proteasome. A striking feature of the rpn6 mutants at the restrictive temperature was that there appeared a protein complex composed of only four of the nine lid components, Rpn5p, Rpn8p, Rpn9p, and Rpn11p. Altogether, we conclude that Rpn6p is essential for the integrity/assembly of the lid in the sense that it is necessary for the incorporation of Rpn3p, Rpn7p, Rpn12p, and Sem1p (Rpn15p) into the lid, thereby playing an essential role in the proper function of the 26 S proteasome.
Isono
Erika
E
Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
Saito
Naoko
N
Kamata
Naoko
N
Saeki
Yasushi
Y
Toh-E
Akio
A
D018797:Cell Cycle Proteins / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D013696:Temperature; D025801:Ubiquitin / Q000378:metabolism
D018797:Cell Cycle Proteins; C074096:RPN12 protein, S cerevisiae; C403291:RPN3 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D010450:Endopeptidases; C472775:Rpn6 protein, S cerevisiae; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease