Dosage Suppressors of pds1 Implicate Ubiquitin-Associated Domains in Checkpoint Control

Abstract

In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated. To identify loci that genetically interact withPDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37°C. Genetic and functional interactions of two suppressors are described. RAD23 andDDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128. rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128. UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them. Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling. Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.

ACKNOWLEDGMENTS

We thank T. Weinert for strains, A. Straight for the TUB1-GFP construct, and L. Prakash for anti-Rad23p antibodies.

M.S. was supported by EMBO and HFSP fellowships, D.J.C. was supported by an EMBO fellowship and a U.S. Army Medical Research Materiel Command Breast Cancer Research Fellowship, B.L.B. was supported by fellowships from the NIH and University of California Office of the President, and S.J. was supported by a fellowship from the Danish Medical Research Council. This work was supported by NIH grant GM38328 (S.I.R.).