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Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins.

Johnson ES, Blobel G - J. Cell Biol. (1999)

Bottom Line: We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis.Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G(2)/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle.This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021, USA. Erica.Johnson@mail.tju.edu

ABSTRACT
SUMO is a ubiquitin-related protein that functions as a posttranslational modification on other proteins. SUMO conjugation is essential for viability in Saccharomyces cerevisiae and is required for entry into mitosis. We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis. Septins are components of a belt of 10-nm filaments encircling the yeast bud neck. Intriguingly, only septins on the mother cell side of the bud neck are sumoylated. We have identified four major SUMO attachment-site lysine residues in Cdc3, one in Cdc11, and two in Shs1, all within the consensus sequence (IVL)KX(ED). Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G(2)/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle. This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites. Thus, SUMO conjugation plays a role in regulating septin ring dynamics during the cell cycle.

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Single SUMO moieties are attached to multiple Lys residues in the NH2-terminal domain of Cdc3. a, Diagram of TEV protease cleavage site-containing constructs, showing the six Lys residues in the NH2-terminal domain of Cdc3 and the positions of introduced TEV cleavage sites. b–e, Exponential cultures of EJY300 (cdc3Δ) bearing plasmids expressing Cdc3-HA or one of the TEV site-containing Cdc3-HA constructs were arrested with nocodazole, lysed, and the lysates immunoprecipitated with an mAb against the HA epitope. Immunoprecipitated protein was treated with TEV protease while bound to the beads, and the resulting cleavage products were separated into an unbound fraction (e) and a bound fraction (b and d). The uncleaved proteins are shown in c. Fractions were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (b) or a polyclonal antibody against SUMO (c–e). Lanes are numbered according to construct numbers in a. Asterisks indicate bands containing IgG. Arrow indicates uncleaved, unmodified Cdc3-HA. Open circles in d indicate the TEV cleavage products.
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Figure 4: Single SUMO moieties are attached to multiple Lys residues in the NH2-terminal domain of Cdc3. a, Diagram of TEV protease cleavage site-containing constructs, showing the six Lys residues in the NH2-terminal domain of Cdc3 and the positions of introduced TEV cleavage sites. b–e, Exponential cultures of EJY300 (cdc3Δ) bearing plasmids expressing Cdc3-HA or one of the TEV site-containing Cdc3-HA constructs were arrested with nocodazole, lysed, and the lysates immunoprecipitated with an mAb against the HA epitope. Immunoprecipitated protein was treated with TEV protease while bound to the beads, and the resulting cleavage products were separated into an unbound fraction (e) and a bound fraction (b and d). The uncleaved proteins are shown in c. Fractions were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (b) or a polyclonal antibody against SUMO (c–e). Lanes are numbered according to construct numbers in a. Asterisks indicate bands containing IgG. Arrow indicates uncleaved, unmodified Cdc3-HA. Open circles in d indicate the TEV cleavage products.

Mentions: Examination of the sequence surrounding Lys11 revealed that three other Lys residues in the NH2-terminal domain of Cdc3 were embedded in similar sequence motifs having the consensus (IVL)KXE, which appeared to be a potential sumoylation site consensus sequence. Since there are multiple SUMO-Cdc3 conjugate bands (Fig. 1), such a consensus sequence might be used in either of two different ways. SUMO might be attached at one or the other of these Lys residues as a chain containing SUMO–SUMO linkages, analogous to the multi-Ub chain. Alternatively, multiple single copies of SUMO might be attached with one SUMO moiety per Lys residue. To distinguish between these possibilities, we designed a series of constructs containing cleavage sites for the TEV protease at different sites along the NH2-terminal domain of Cdc3, either after the first two Lys residues (TEV-K2), after the third (TEV-K3), after the fourth (TEV-K4), or after the sixth (TEV-K6; see Fig. 4 a). These constructs also contained a COOH-terminal HA tag, which was used to immunoprecipitate the Cdc3 variants from lysates of nocodazole-arrested yeast. Immunoprecipitated proteins were cleaved with TEV protease while still bound to the beads and separated into an unbound fraction containing the fragment NH2-terminal to the cleavage site, and a bound fraction containing the COOH-terminal cleavage product. This experiment showed that for each additional Lys residue included NH2-terminal to the TEV site, an additional sumoylated species appeared on the NH2-terminal product and disappeared from the COOH-terminal product. There was one major NH2-terminal SUMO-containing species from TEV-K2, two from TEV-K3, and three from TEV-K4 and TEV-K6 (Fig. 4 e), and there were two major SUMO-containing COOH-terminal cleavage products from TEV-K2, one from TEV-K3, and none from TEV-K4 or TEV-K6 (Fig. 4 d, cleavage products are indicated; lanes 2, 3, and 5 contained uncleaved species, see Fig. 4 b). These results were generally consistent with a model where single copies of SUMO are attached to Cdc3 at three to four major sites, at Lys-4 and/or Lys-11, at Lys-30, and at Lys-63. A separate experiment in which these Lys residues were altered by site-directed mutagenesis showed that both Lys-4 and Lys-11 can serve as sumoylation sites (data not shown).


Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins.

Johnson ES, Blobel G - J. Cell Biol. (1999)

Single SUMO moieties are attached to multiple Lys residues in the NH2-terminal domain of Cdc3. a, Diagram of TEV protease cleavage site-containing constructs, showing the six Lys residues in the NH2-terminal domain of Cdc3 and the positions of introduced TEV cleavage sites. b–e, Exponential cultures of EJY300 (cdc3Δ) bearing plasmids expressing Cdc3-HA or one of the TEV site-containing Cdc3-HA constructs were arrested with nocodazole, lysed, and the lysates immunoprecipitated with an mAb against the HA epitope. Immunoprecipitated protein was treated with TEV protease while bound to the beads, and the resulting cleavage products were separated into an unbound fraction (e) and a bound fraction (b and d). The uncleaved proteins are shown in c. Fractions were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (b) or a polyclonal antibody against SUMO (c–e). Lanes are numbered according to construct numbers in a. Asterisks indicate bands containing IgG. Arrow indicates uncleaved, unmodified Cdc3-HA. Open circles in d indicate the TEV cleavage products.
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Related In: Results  -  Collection

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Figure 4: Single SUMO moieties are attached to multiple Lys residues in the NH2-terminal domain of Cdc3. a, Diagram of TEV protease cleavage site-containing constructs, showing the six Lys residues in the NH2-terminal domain of Cdc3 and the positions of introduced TEV cleavage sites. b–e, Exponential cultures of EJY300 (cdc3Δ) bearing plasmids expressing Cdc3-HA or one of the TEV site-containing Cdc3-HA constructs were arrested with nocodazole, lysed, and the lysates immunoprecipitated with an mAb against the HA epitope. Immunoprecipitated protein was treated with TEV protease while bound to the beads, and the resulting cleavage products were separated into an unbound fraction (e) and a bound fraction (b and d). The uncleaved proteins are shown in c. Fractions were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (b) or a polyclonal antibody against SUMO (c–e). Lanes are numbered according to construct numbers in a. Asterisks indicate bands containing IgG. Arrow indicates uncleaved, unmodified Cdc3-HA. Open circles in d indicate the TEV cleavage products.
Mentions: Examination of the sequence surrounding Lys11 revealed that three other Lys residues in the NH2-terminal domain of Cdc3 were embedded in similar sequence motifs having the consensus (IVL)KXE, which appeared to be a potential sumoylation site consensus sequence. Since there are multiple SUMO-Cdc3 conjugate bands (Fig. 1), such a consensus sequence might be used in either of two different ways. SUMO might be attached at one or the other of these Lys residues as a chain containing SUMO–SUMO linkages, analogous to the multi-Ub chain. Alternatively, multiple single copies of SUMO might be attached with one SUMO moiety per Lys residue. To distinguish between these possibilities, we designed a series of constructs containing cleavage sites for the TEV protease at different sites along the NH2-terminal domain of Cdc3, either after the first two Lys residues (TEV-K2), after the third (TEV-K3), after the fourth (TEV-K4), or after the sixth (TEV-K6; see Fig. 4 a). These constructs also contained a COOH-terminal HA tag, which was used to immunoprecipitate the Cdc3 variants from lysates of nocodazole-arrested yeast. Immunoprecipitated proteins were cleaved with TEV protease while still bound to the beads and separated into an unbound fraction containing the fragment NH2-terminal to the cleavage site, and a bound fraction containing the COOH-terminal cleavage product. This experiment showed that for each additional Lys residue included NH2-terminal to the TEV site, an additional sumoylated species appeared on the NH2-terminal product and disappeared from the COOH-terminal product. There was one major NH2-terminal SUMO-containing species from TEV-K2, two from TEV-K3, and three from TEV-K4 and TEV-K6 (Fig. 4 e), and there were two major SUMO-containing COOH-terminal cleavage products from TEV-K2, one from TEV-K3, and none from TEV-K4 or TEV-K6 (Fig. 4 d, cleavage products are indicated; lanes 2, 3, and 5 contained uncleaved species, see Fig. 4 b). These results were generally consistent with a model where single copies of SUMO are attached to Cdc3 at three to four major sites, at Lys-4 and/or Lys-11, at Lys-30, and at Lys-63. A separate experiment in which these Lys residues were altered by site-directed mutagenesis showed that both Lys-4 and Lys-11 can serve as sumoylation sites (data not shown).

Bottom Line: We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis.Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G(2)/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle.This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021, USA. Erica.Johnson@mail.tju.edu

ABSTRACT
SUMO is a ubiquitin-related protein that functions as a posttranslational modification on other proteins. SUMO conjugation is essential for viability in Saccharomyces cerevisiae and is required for entry into mitosis. We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis. Septins are components of a belt of 10-nm filaments encircling the yeast bud neck. Intriguingly, only septins on the mother cell side of the bud neck are sumoylated. We have identified four major SUMO attachment-site lysine residues in Cdc3, one in Cdc11, and two in Shs1, all within the consensus sequence (IVL)KX(ED). Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G(2)/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle. This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites. Thus, SUMO conjugation plays a role in regulating septin ring dynamics during the cell cycle.

Show MeSH