P 193 - Redox control of the 20 S proteasome gating: Implications on the chronological life span of yeast cells

Abstract

Two Cys residues located in the α5-subunit of the yeast 20 S proteasome (20SPT) are post-translationally modified by S-glutathionylation. Such modification implies on the opening of the 20SPT gate when compared to the DTT-reduced purified preparations. The site-specific mutation of the highly conserved (yeast to human) α5-Cys76 residue induced either decreased cell viability or life span when compared to the wild type strain. The α5-C76S-mutated strain presented decreased resistance to oxidative stress without any alteration on major redox parameters. The closed gate conformation of the 20SPT pool in that short-lived strain represents 70% of total 20SPT pool against 35% in the wild type counterpart. Mass spectrometry analysis of the α5-subunit in the α5-C76S-mutated strain showed S-glutathionylation of the remaining α5-C221 residue suggesting that redox modification of the conserved α5-C76, but not of the α5-C221, is the one involved in the gate opening. Circular dichroism analyses of recombinant α5-WT and α5-C76S subunits showed similar pattern between them regarding their secondary and tertiary structures. The differences between the wild type and short-lived strains regarding prevalence of 20SPT gate conformation, clearly demonstrate that the 20SPT opening is relevant to maintain cellular homeostasis and life span.