title: Yeast PIAS-type Ull1/Siz1 is composed of SUMO ligase and regulatory domains.
abstract: SUMO (small ubiquitin-like modifier)/Smt3 (suppressor of mif two) is a member of the ubiquitin-related protein family and is known to conjugate with many proteins. In the sumoylation pathway, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme), and E3 (SUMO ligase) functions as an adaptor between E2 and each substrate. Yeast Ull1 (ubiquitin-like protein ligase 1)/Siz1, a PIAS (protein inhibitor of activated STAT)-type SUMO ligase, modifies both cytoplasmic and nuclear proteins. In this paper, we performed a domain analysis of Ull1/Siz1 by constructing various deletion mutants. A novel conserved N-terminal domain, called PINIT, as well as the RING-like domain (SP-RING) were required for the SUMO ligase activity in the in vitro conjugation system and for interaction with Smt3 in an in vitro binding assay. The most distal N-terminal region, which contains a putative DNA-binding SAF-A/B, Acinus, and PIAS (SAP) motif, was not required for the ligase activity but was involved in nuclear localization. A strong SUMO-binding motif was identified, which interacted with Smt3 in the two-hybrid system but was not necessary for the ligase activity. The most distal C-terminal domain was important for stable localization at the bud neck region and thereby for the substrate recognition of septins. Furthermore, the C-terminal half conferred protein instability on Ull1/Siz1. Taken together, we conclude that the SP-RING and PINIT of Ull1/Siz1 are core domains of the SUMO ligase, and the other domains are regulatory for protein stability and subcellular localization.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Kikuchi
mesh_terms: D020816:Amino Acid Motifs; D004926:Escherichia coli / Q000378:metabolism; D005821:Genetic Techniques; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D050828:Protein Inhibitors of Activated STAT / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*
chemical_list: D050828:Protein Inhibitors of Activated STAT; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D049452:Green Fluorescent Proteins; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; C436837:Siz1 protein, S cerevisiae