Yeast Ull1/Siz1 Is a Novel SUMO1/Smt3 Ligase for Septin Components and Functions as an Adaptor between Conjugating Enzyme and Substrates

Abstract

SUMO1/Smt3, a ubiquitin-like protein modifier, is known to conjugate to other proteins and modulate their functions in various important processes. Similar to the ubiquitin conjugation system, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme). In our previous report (Takahashi, Y., Toh-e, A., and Kikuchi, Y. (2001) Gene 275, 223-231), we showed that Siz1/Ull1 (YDR409w) of budding yeast, a member of the human PIAS family containing a RINGlike domain, is a strong candidate for SUMO1/Smt3 ligase because the SUMO1/Smt3 modification of septin components was abolished in the ull1 mutant and Ull1 associated with E2 (Ubc9) and the substrates (septin components) in immunoprecipitation experiments. Here we have developed an in vitro Smt3 conjugation system for a septin component (Cdc3) using purified recombinant proteins. In this system, Ull1 is additionally required as well as E1 (Sua1⅐Uba2 complex), E2 (Ubc9), and ATP. A cysteine residue of the RING-like domain was essential for the conjugation both in vivo and in vitro. Furthermore, a region containing the RING-like domain directly interacted with Ubc9 and Cdc3. Thus, this SUMO/Smt3 ligase functions as an adaptor between E2 and the target proteins.SUMO 1 (small ubiquitin-like modifier)/Smt3 is a member of a growing family of ubiquitin-related proteins and is known to be conjugated to RanGAP1, PML (promyelocytic leukemia), I B␣, p53, septins, etc. (1-4). Not only are the amino acid sequences and the three-dimensional structures similar between SUMO/ Smt3 and ubiquitin, but their conjugation systems and the enzymes involved are highly related (5-9). In the ubiquitin pathway a third enzyme, ubiquitin ligase (E3), is often required for the final transfer of this modifier and plays a crucial role by recognizing target proteins and by promoting their conjugation (10). It remains unknown, however, whether any SUMO1/Smt3 ligases (E3s) are involved in this conjugation pathway.In the ubiquitin pathway, some E3 components such as Apc11 of the anaphase-promoting complex and Rbx1 of the SCF (Skp1, cullin, F-box protein)⅐ubiquitin ligase complex contain a zinc-binding RING domain with an octet of ordered cysteine and histidine residues forming a cross-brace around two zinc atoms (11,12). This type of ubiquitin ligases (E3s) has to interact with E2 and the substrate at the same time because apparently they do not form the thioester bond with ubiquitin. In the case of c-Cbl proto-oncoprotein, its RING domain interacts with UbcH7 (E2), and the tyrosine kinase binding domain, a region close to the RING domain, recognizes its substrate. Thus, c-Cbl proto-oncoprotein functions as a bridging molecule between E2 and its substrate (13).In budding yeast, Smt3 is the only member of the SUMO family, and the Smt3 conjugation system is essential for mitotic growth. The lethality of the smt3 deletion mutant can be suppressed by expressing human SUMO1, suggesting that SUMO1 is a functional homologue of yeast Smt...

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

“…Most SUMO E3 ligases are characterized by an SP-RING domain that is essential for SUMO ligase activity [10,14,42,44,45]. To test the role of the RING finger in sumoylation activity, we expressed and purified some mutant proteins lacking this domain for in vitro assays.…”

“…These proteins share a RINGfinger related sequence motif, the SP-RING domain, with the PIAS1 (Protein Inhibitor of Activated Signal transducer and activator of transcription) protein of human cells [11,12]. The Siz1 and Siz2 ligases are active on septins and may have partially overlapping specificities [10,13,14]. Together, Siz1 and Siz2 account for 90% of the total sumoylation in yeast, and cells lacking both SIZ1 and SIZ2 are viable but slow growing [9,10].…”

PIASy-Deficient Mice Display Modest Defects in IFN and Wnt Signaling

“…An additional role of PIAS in the regulation of chromosome structure and function was inferred from the identification and characterization of dPIAS as a suppressor of positioneffect-variegation, Su(var)2-10 (46). Likewise, a yeast orthologue of PIAS proteins, termed Siz1 was identified through its genetic interaction with the condensing complex (47) and shown to act as a SUMO E3 ligase for septin proteins (14,16). However, the function of PIAS proteins in the mouse has not yet been elucidated.…”

“…These include nuclear hormone receptors, such as the androgen receptor (AR), p53, Smad4, Sp3, HMGI-C, Gfi-1, IRF-1, TFII-I and yeast septins (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). In mouse and man, four Pias genes (Pias1, Pias3, Piasx, and Piasy) have been identified, which encode proteins that share a similar domain structure but differ in their specificity of interaction with other proteins.…”

“…Total RNA was isolated with TRIzol (Invitrogen Life Technologies) and 2 g of RNA were transcribed to cDNA using 200 U SuperscriptII (Invitrogen Life Technologies) and 0.1 nM oligo(dT) [12][13][14][15][16][17][18] in a total volume of 20 l. For one real-time reaction, 2ϫ SYBR Green Mix (Applied Biosystems) was supplemented with 1/40 of synthesized cDNA in addition to the appropriate oligonucleotide primer pair and run on the AbiPrism 7000 sequence detection system (Applied Biosystems, Foster City, CA). Reverse transcriptase controls were done in parallel without adding enzyme.…”