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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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The steady-state levels of Cdc3 and Cdc11 do not fluctuate with the cell cycle. EJY321 (cdc15-2) was grown to exponential phase at 25°C in synthetic (SD–URA) medium, incubated at 37°C for 3 h, cooled briefly on ice, and returned to growth at 25°C. Samples taken at the indicated times after the culture was cooled were used to make whole cell lysates, which were analyzed by SDS-PAGE and immunoblotting with a polyclonal antibody against Cdc3 (a and b) or against Cdc11 (c and d). b and d are shorter exposures of a and c, respectively. Bands corresponding to unmodified Cdc3 and Cdc11 are indicated. A half-open square bracket designates high molecular weight SUMO-Cdc3 conjugates. A circle indicates a band that cross-reacts with the anti-Cdc3 antibody. A SUMO-Cdc11 conjugate is indicated with an arrow, and a possible proteolytic breakdown product of Cdc11 with an arrowhead. The percentage of cells in which the actin patches are polarized toward the bud neck, an indication of cells undergoing cytokinesis, is listed over the lanes. A dash indicates <1%.
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Figure 8: The steady-state levels of Cdc3 and Cdc11 do not fluctuate with the cell cycle. EJY321 (cdc15-2) was grown to exponential phase at 25°C in synthetic (SD–URA) medium, incubated at 37°C for 3 h, cooled briefly on ice, and returned to growth at 25°C. Samples taken at the indicated times after the culture was cooled were used to make whole cell lysates, which were analyzed by SDS-PAGE and immunoblotting with a polyclonal antibody against Cdc3 (a and b) or against Cdc11 (c and d). b and d are shorter exposures of a and c, respectively. Bands corresponding to unmodified Cdc3 and Cdc11 are indicated. A half-open square bracket designates high molecular weight SUMO-Cdc3 conjugates. A circle indicates a band that cross-reacts with the anti-Cdc3 antibody. A SUMO-Cdc11 conjugate is indicated with an arrow, and a possible proteolytic breakdown product of Cdc11 with an arrowhead. The percentage of cells in which the actin patches are polarized toward the bud neck, an indication of cells undergoing cytokinesis, is listed over the lanes. A dash indicates <1%.

Mentions: To test whether septins are ubiquitinated or degraded during cytokinesis, we synchronized a culture of cdc15-2 cells by incubating them at 37°C, which arrests them in late anaphase, and releasing them at 25°C to allow them to complete cytokinesis (Fig. 8). Between 30 and 50 min after release, all the SUMO-conjugated forms of Cdc3 and Cdc11 disappeared from these cells. However, there was no significant reduction in the steady-state level of either Cdc3 or Cdc11 at this point, which is consistent with the observation by immunofluorescence microscopy that septin rings do not disappear suddenly at cytokinesis. Also, in several different experiments, we never observed high molecular weight ubiquitinated Cdc3 species. However, we did sometimes observe minor bands that might be degradation products of Cdc11 (Fig. 8 c) and of Cdc3-HA (data not shown). We conclude that, at most, a small fraction of Cdc3 and Cdc11 is degraded immediately at cytokinesis.


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

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

The steady-state levels of Cdc3 and Cdc11 do not fluctuate with the cell cycle. EJY321 (cdc15-2) was grown to exponential phase at 25°C in synthetic (SD–URA) medium, incubated at 37°C for 3 h, cooled briefly on ice, and returned to growth at 25°C. Samples taken at the indicated times after the culture was cooled were used to make whole cell lysates, which were analyzed by SDS-PAGE and immunoblotting with a polyclonal antibody against Cdc3 (a and b) or against Cdc11 (c and d). b and d are shorter exposures of a and c, respectively. Bands corresponding to unmodified Cdc3 and Cdc11 are indicated. A half-open square bracket designates high molecular weight SUMO-Cdc3 conjugates. A circle indicates a band that cross-reacts with the anti-Cdc3 antibody. A SUMO-Cdc11 conjugate is indicated with an arrow, and a possible proteolytic breakdown product of Cdc11 with an arrowhead. The percentage of cells in which the actin patches are polarized toward the bud neck, an indication of cells undergoing cytokinesis, is listed over the lanes. A dash indicates <1%.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169351&req=5

Figure 8: The steady-state levels of Cdc3 and Cdc11 do not fluctuate with the cell cycle. EJY321 (cdc15-2) was grown to exponential phase at 25°C in synthetic (SD–URA) medium, incubated at 37°C for 3 h, cooled briefly on ice, and returned to growth at 25°C. Samples taken at the indicated times after the culture was cooled were used to make whole cell lysates, which were analyzed by SDS-PAGE and immunoblotting with a polyclonal antibody against Cdc3 (a and b) or against Cdc11 (c and d). b and d are shorter exposures of a and c, respectively. Bands corresponding to unmodified Cdc3 and Cdc11 are indicated. A half-open square bracket designates high molecular weight SUMO-Cdc3 conjugates. A circle indicates a band that cross-reacts with the anti-Cdc3 antibody. A SUMO-Cdc11 conjugate is indicated with an arrow, and a possible proteolytic breakdown product of Cdc11 with an arrowhead. The percentage of cells in which the actin patches are polarized toward the bud neck, an indication of cells undergoing cytokinesis, is listed over the lanes. A dash indicates <1%.
Mentions: To test whether septins are ubiquitinated or degraded during cytokinesis, we synchronized a culture of cdc15-2 cells by incubating them at 37°C, which arrests them in late anaphase, and releasing them at 25°C to allow them to complete cytokinesis (Fig. 8). Between 30 and 50 min after release, all the SUMO-conjugated forms of Cdc3 and Cdc11 disappeared from these cells. However, there was no significant reduction in the steady-state level of either Cdc3 or Cdc11 at this point, which is consistent with the observation by immunofluorescence microscopy that septin rings do not disappear suddenly at cytokinesis. Also, in several different experiments, we never observed high molecular weight ubiquitinated Cdc3 species. However, we did sometimes observe minor bands that might be degradation products of Cdc11 (Fig. 8 c) and of Cdc3-HA (data not shown). We conclude that, at most, a small fraction of Cdc3 and Cdc11 is degraded immediately at cytokinesis.

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