A conditional yeast E1 mutant blocks the ubiquitin-proteasome pathway and reveals a role for ubiquitin conjugates in targeting Rad23 to the proteasome.
E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin-proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo.
Ghaboosi
Nazli
N
Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Deshaies
Raymond J
RJ
D000483:Alleles; D001483:Base Sequence; D001693:Biological Transport, Active; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D025801:Ubiquitin / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics* / Q000378:metabolism*; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C100415:DSK2 protein, S cerevisiae; C490846:RAD23 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D014452:Ubiquitins; D046988:Proteasome Endopeptidase Complex; D044764:Ubiquitin-Activating Enzymes