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Vol. 19, Issue 9, 3625-3637, September 2008

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Different Domains of the UBL-UBA Ubiquitin Receptor, Ddi1/Vsm1, Are Involved in Its Multiple Cellular Roles

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

Submitted May 18, 2007; Revised June 4, 2008; Accepted June 6, 2008
Monitoring Editor: Akihiko Nakano

Ddi1/Vsm1 is an ubiquitin receptor involved in regulation of the cell cycle and late secretory pathway in Saccharomyces cerevisiae. Ddi1 possesses three domains: an NH₂-terminal ubiquitin-like domain (UBL), a COOH-terminal ubiquitin-associated domain (UBA), and a retroviral aspartyl-protease domain (RVP). Here, we demonstrate the domains involved in homodimerization, checkpoint regulation, localization, and t-SNARE binding. The RVP domain is required for protein homodimerization, whereas the UBL and UBA domains are required for rescue of the pds1-128 checkpoint mutant and enrichment of GFP-Ddi1 in the nucleus. A mutation in aspartate-220, which is necessary for putative aspartyl-protease function, abolished the rescue of pds1-128 cells but not homodimerization. Thus, Ddi1 catalytic activity may be required for checkpoint regulation. The Sso1 t-SNARE-interacting domain maps to residues 344–395 and undergoes phosphorylation on threonines T346 and T348. T348 is necessary for Sso binding, and phosphorylation is important for function, because mutations that lessen phosphorylation (e.g., Ddi1^T346A, Ddi1^T348A) are unable to facilitate growth of the sec9-4 t-SNARE mutant. In contrast, the overproduction of phosphorylatable forms of Ddi1 (e.g., Ddi1, Ddi1^S341A) rescue the growth of sec9-4 cells similar to Sso1 overproduction. Thus, Ddi1 participates in multiple cellular processes via its different domains and phosphorylation may regulate exocytic functions.

Address correspondence to: Jeffrey E. Gerst (jeffrey.gerst{at}weizmann.ac.il)

Abbreviations used: Ddi1, DNA damage inducible 1; GFP, green fluorescent protein; HIV, human immunodeficiency virus; IP, immunoprecipitation; MS, mass spectrometry; Pds1, Precocious Dissociation of Sisters; RVP, retroviral aspartyl protease domain; SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; UBA, ubiquitin-associated domain; UBL, ubiquitin-like domain; WT, wild type; vps, vacuolar protein sorting.