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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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Septins are stable in a ubc9 mutant. Strain YWO102 (ubc9) was grown to log phase in rich medium (YPD) at 25°C and shifted to 37°C for the indicated period of time. a, Cdc11 was visualized by immunofluorescence microscopy using a polyclonal antibody against Cdc11 and DNA by DAPI staining. Bar, 5 μm. b, Whole cell lysates were analyzed by SDS-PAGE and immunoblotting with a polyclonal antibody against Cdc3 (top) or against Cdc11 (bottom).
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Figure 7: Septins are stable in a ubc9 mutant. Strain YWO102 (ubc9) was grown to log phase in rich medium (YPD) at 25°C and shifted to 37°C for the indicated period of time. a, Cdc11 was visualized by immunofluorescence microscopy using a polyclonal antibody against Cdc11 and DNA by DAPI staining. Bar, 5 μm. b, Whole cell lysates were analyzed by SDS-PAGE and immunoblotting with a polyclonal antibody against Cdc3 (top) or against Cdc11 (bottom).

Mentions: There are two simple reasons why the triple sumoylation site mutant might be defective in septin ring disassembly. One is that attachment of SUMO to the septin ring promotes disassembly. The other is that the sumoylation-site Lys residues also have some other function, and that this other function promotes septin ring disassembly. For example, the purpose of these Lys residues could be to serve as ubiquitination sites, or perhaps acetylation or methylation sites. One way to distinguish between these possibilities might be to determine whether mutants in the SUMO conjugation pathway also have septin ring disassembly defects. Examination of Cdc11 localization in ubc9 (Fig. 7 a) and uba2 (data not shown) ts mutants showed that no extra septin rings were visible in cells from either strain, either when grown at the permissive temperature or after transfer to the restrictive temperature. This result may suggest that SUMO conjugation, per se, does not promote septin ring disassembly. Alternatively, it may indicate that, although SUMO conjugation may promote septin ring disassembly, as long as these ts mutants retain enough SUMO conjugating activity to divide, they also retain sufficient SUMO conjugation to disassemble their septin rings.


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

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

Septins are stable in a ubc9 mutant. Strain YWO102 (ubc9) was grown to log phase in rich medium (YPD) at 25°C and shifted to 37°C for the indicated period of time. a, Cdc11 was visualized by immunofluorescence microscopy using a polyclonal antibody against Cdc11 and DNA by DAPI staining. Bar, 5 μm. b, Whole cell lysates were analyzed by SDS-PAGE and immunoblotting with a polyclonal antibody against Cdc3 (top) or against Cdc11 (bottom).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169351&req=5

Figure 7: Septins are stable in a ubc9 mutant. Strain YWO102 (ubc9) was grown to log phase in rich medium (YPD) at 25°C and shifted to 37°C for the indicated period of time. a, Cdc11 was visualized by immunofluorescence microscopy using a polyclonal antibody against Cdc11 and DNA by DAPI staining. Bar, 5 μm. b, Whole cell lysates were analyzed by SDS-PAGE and immunoblotting with a polyclonal antibody against Cdc3 (top) or against Cdc11 (bottom).
Mentions: There are two simple reasons why the triple sumoylation site mutant might be defective in septin ring disassembly. One is that attachment of SUMO to the septin ring promotes disassembly. The other is that the sumoylation-site Lys residues also have some other function, and that this other function promotes septin ring disassembly. For example, the purpose of these Lys residues could be to serve as ubiquitination sites, or perhaps acetylation or methylation sites. One way to distinguish between these possibilities might be to determine whether mutants in the SUMO conjugation pathway also have septin ring disassembly defects. Examination of Cdc11 localization in ubc9 (Fig. 7 a) and uba2 (data not shown) ts mutants showed that no extra septin rings were visible in cells from either strain, either when grown at the permissive temperature or after transfer to the restrictive temperature. This result may suggest that SUMO conjugation, per se, does not promote septin ring disassembly. Alternatively, it may indicate that, although SUMO conjugation may promote septin ring disassembly, as long as these ts mutants retain enough SUMO conjugating activity to divide, they also retain sufficient SUMO conjugation to disassemble their septin rings.

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
Related in: MedlinePlus