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Molecular and Cellular Biology, October 1999, p. 6872-6890, Vol. 19, No. 10
Institut Curie-UMR 144, 75248 Paris Cedex 05, France,1 and Scripps Research
Institute, La Jolla, California 920372
Received 6 May 1999/Returned for modification 8 June 1999/Accepted 28 June 1999
By selectively eliminating ubiquitin-conjugated proteins, the 26S
proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been
recently elucidated. In this study, we have investigated the cell cycle
defects resulting from conditional inactivation of one of these RP
components, the essential non-ATPase Rpn3/Sun2 subunit. Using
temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G1/S
transition but cause a metaphase arrest, indicating that the essential
Rpn3 function is limiting for mitosis. rpn3 mutants
appear severely compromised in the ubiquitin-dependent proteolysis of
several physiologically important proteasome substrates. Thus,
RPN3 function is required for the degradation of the
G1-phase cyclin Cln2 targeted by SCF; the S-phase cyclin
Clb5, whose ubiquitination is likely to involve a combination of E3
(ubiquitin protein ligase) enzymes; and anaphase-promoting complex
targets, such as the B-type cyclin Clb2 and the anaphase inhibitor
Pds1. Our results indicate that the Pds1 degradation defect of the
rpn3 mutants most likely accounts for the
metaphase arrest phenotype observed. Surprisingly, but consistent
with the lack of a G1 arrest phenotype in
thermosensitive rpn3 strains, the Cdk inhibitor Sic1
exhibits a short half-life regardless of the RPN3 genotype.
In striking contrast, Sic1 turnover is severely impaired by a
temperature-sensitive mutation in RPN12/NIN1,
encoding another essential RP subunit. While other interpretations
are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle
regulators. The potential implications of these data are
discussed in the context of possible Rpn3 function in
multiubiquitin-protein conjugate recognition by the 19S proteasomal
regulatory particle.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Functional Characterization of Rpn3 Uncovers a Distinct 19S
Proteasomal Subunit Requirement for Ubiquitin-Dependent Proteolysis of
Cell Cycle Regulatory Proteins in Budding Yeast
*
Corresponding author. Present address: Laboratoire
d'Ingenierie des Systèmes Macromoleculaires CNRS, UPR 9027, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Phone: (33) 4 91 16 45 54. Fax: (33) 4 91 71 21 24. E-mail:
ebailly{at}ir2cbm.cnrs-mrs.fr.
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