Functional Characterization of Rpn3 Uncovers a Distinct 19S Proteasomal Subunit Requirement for Ubiquitin-Dependent Proteolysis of Cell Cycle Regulatory Proteins in Budding Yeast

Abstract

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.

ACKNOWLEDGMENTS

We thank Ray Deshaies and Rati Verma for yeast strains; Doris Germain for pGAL-CLB5^HA; Carl Mann for Rpt1/Cim5 antiserum; Etienne Schwob for the D347 construct and the pET15-Sic1 expression vector; Akio Toh-e for the NIN1 and nin1-1 yeast strains; Mike Tyers for Sic1 antibodies; E. Wayner, A. Kahana, and D. Gottschling for the ubiquitin monoclonal antibody; Spencer Brown, Danny Rouillard, and Marie Ange Deugnier for helpful advice on FACScan analysis; Oskar Smrzka for assistance with the sequence alignment programs and Northern experiments; Daniel Meur and Dominique Morineau for the artwork; and Duncan Clarke, Steve Haase, Guillaume Mondésert, Dave Stuart, Mark Watson, Curt Wittenberg, and Meira Wolff for the various DNA constructs and yeast strains used throughout this study. We express special thanks to Marie-Noëlle Simon and Peter Kaiser for stimulating discussions and critical review of the manuscript. Eric Bailly is particularly indebted to Michel Bornens for generosity in sharing laboratory space and for constant interest in this work as well as to all the members of the laboratory for their supporting encouragement and valuable discussions.

E.B. is supported by INSERM and acknowledges an international research fellowship from the Public Health Service Fogarty International Center and financial support from Michel Bornens. This work was supported by the Curie Institute, by CNRS, and by grants from the Association pour la Recherche sur le Cancer to E.B. and from NIH (grant GM38328) to S.I.R.