A novel factor required for the SUMO1/Smt3 conjugation of yeast septins

Takahashi, Yoshimitsu; Toh‐e, Akio; Kikuchi, Yo

doi:10.1016/s0378-1119(01)00662-x

Cited by 116 publications

(131 citation statements)

References 24 publications

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“…Strains of Saccharomyces cerevisiae, T-13 (ull1::cgHIS3) and T-20 (ull1::cgHIS3 CDC3HA-TRP1), isogenic to W303-1A (MATa ade2 ura3 trp1 leu2 his3 can1 ssd-d2), were described previously (19). PJ69-4A (MATa ura3 trp1 leu2 his3 gal4 gal80 LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) was used for the two-hybrid system (20).…”

Section: Methodsmentioning

confidence: 99%

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

Takahashi¹,

Kahyo²,

Toh‐e³

et al. 2001

Journal of Biological Chemistry

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176

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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...

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Section: Methodsmentioning

confidence: 99%

“…In our previous work, we showed that Siz1 (YDR409w), a member of a new family including human PIAS3 (18), containing a RING-like domain, is required for the Smt3 conjugation to septins in vivo and associates with Ubc9 and septins as assayed by immunoprecipitation experiments (19). Thereby, Siz1 could be a novel Smt3/SUMO1 ligase.…”

mentioning

confidence: 99%

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

Takahashi¹,

Kahyo²,

Toh‐e³

et al. 2001

Journal of Biological Chemistry

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156

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“…This defect was further exacerbated in siz1 siz2 slx5 or siz1 siz2 slx8 triple mutants (Table 2). On their own, siz1 siz2 double mutants displayed a synthetic growth defect [10,42] with a doubling time (DT) of 123 min, and loss of SLX5 or SLX8Δ in this background exacerbated this defect (DT = 200 min). The fact that all of these proteins contain RING-or SP-RING motifs suggested that Siz1 and/or Siz2 may functionally overlap with, or regulate the levels of Slx5 and Slx8.…”

Section: Genetic Interactions Between Slx5-8 and Components Of The Sumentioning

confidence: 99%

“…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.…”

Section: Role Of the Ring Finger In In-vitro Sumoylationmentioning

confidence: 99%

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

et al. 2007

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The yeast genes SLX5 and SLX8 were identified based on their requirement for viability in the absence of the Sgs1 DNA helicase. Loss of these genes results in genome instability, nibbled colonies, and other phenotypes associated with defects in sumoylation. The Slx5 and Slx8 proteins form a stable complex and each subunit contains a single RING-finger domain at its C-terminus. To determine the physiological function of the Slx5-8 complex, we explored its interaction with the SUMO pathway. Curing 2μ circle from the mutants suppressed their nibbled colony phenotype and partially improved their growth rate, but did not affect their sensitivity to hydroxyurea. The increase in sumoylation observed in slx5Δ and slx8Δ mutants was found to be dependent on the Siz1 SUMO ligase. Physical interactions between the Slx5-8 complex and both Ubc9 and Smt3 were identified and characterized. Using in vitro reactions, we show that Slx5, Slx8, or the Slx5-8 complex stimulates the formation of SUMO chains and the sumoylation of a test substrate. Interestingly, a functional RING-finger domain is not required for this stimulation in vitro. These biochemical data demonstrate for the first time that the Slx5-8 complex is capable of interacting directly with the SUMO pathway.

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“…Interestingly, the Smc5ϩ6 non-SMC subunits Nse1 and Nse2 contain zinc finger domains related to the RING and Miztype domains, respectively (Fujioka et al, 2002;. This suggests that they are not only structural components of the complex, but likely act as E3-ligases to modify target proteins with ubiquitin or SUMO, thereby modulating their functions (Wu et al, 1997;Freemont, 2000;Joazeiro and Weissman, 2000;Hari et al, 2001;Takahashi et al, 2001).…”

Section: Smc6 Of the Smc5ϩ6 Heterodimer Was Initially Identified By Amentioning

confidence: 99%

Nse1, Nse2, and a Novel Subunit of the Smc5-Smc6 Complex, Nse3, Play a Crucial Role in Meiosis

Pébernard

McDonald

Pavlova

et al. 2004

MBoC

113

150

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The structural maintenance of chromosomes (SMC) family of proteins play key roles in the organization, packaging, and repair of chromosomes. Cohesin (Smc1؉3) holds replicated sister chromatids together until mitosis, condensin (Smc2؉4) acts in chromosome condensation, and Smc5؉6 performs currently enigmatic roles in DNA repair and chromatin structure. The SMC heterodimers must associate with non-SMC subunits to perform their functions. Using both biochemical and genetic methods, we have isolated a novel subunit of the Smc5؉6 complex, Nse3. Nse3 is an essential nuclear protein that is required for normal mitotic chromosome segregation and cellular resistance to a number of genotoxic agents. Epistasis with Rhp51 (Rad51) suggests that like Smc5؉6, Nse3 functions in the homologous recombination based repair of DNA damage. We previously identified two non-SMC subunits of Smc5؉6 called Nse1 and Nse2. Analysis of nse1-1, nse2-1, and nse3-1 mutants demonstrates that they are crucial for meiosis. The Nse1 mutant displays meiotic DNA segregation and homologous recombination defects. Spore viability is reduced by nse2-1 and nse3-1, without affecting interhomolog recombination. Finally, genetic interactions shared by the nse mutants suggest that the Smc5؉6 complex is important for replication fork stability. INTRODUCTIONBoth endogenous and exogenous agents constantly threaten genomic integrity. The DNA double-strand break (DSB) is a potentially life-threatening lesion for a cell and can occur spontaneously during growth, as part of a programmed event such as meiosis or as the result of exposure to environmental genotoxic agents. Multiple mechanisms exist to repair DSBs, including nonhomologous end joining and homologous recombination (HR) (Paques and Haber, 1999;Symington, 2002). The HR pathway serves to maintain all original genetic information during DSB repair. Many components of that pathway have been identified and characterized (Paques and Haber, 1999;Symington, 2002). HR involves multiple steps. The DSB is first resected to generate a recombinogenic 3Ј overhang that invades a homologous sequence (e.g., sister chromatid), forming a displacement or D-loop. The invasion step is catalyzed by a number of proteins including the Escherichia coli RecA homologue, Rad51. D-loop formation primes repair synthesis, which is followed by resolution of the recombined duplexes and ligation, producing intact duplex DNA molecules (Paques and Haber, 1999;Symington, 2002). The resolution of recombined DNA duplexes can yield products in which there has been reciprocal exchange of flanking markers (crossover) or not. During mitotic growth gene conversion is infrequently accompanied by crossover, whereas during meiosis, crossover recombination is much more prevalent (Paques and Haber, 1999).Efficient DNA repair requires modulation of both local and higher order chromatin structure. Interestingly, the structural maintenance of chromosomes (SMC) proteins have recently been recognized as important players in DNA repair (Hirano, 2002;Jessberger...

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A novel factor required for the SUMO1/Smt3 conjugation of yeast septins

Cited by 116 publications

References 24 publications

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

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

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

Nse1, Nse2, and a Novel Subunit of the Smc5-Smc6 Complex, Nse3, Play a Crucial Role in Meiosis

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