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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 cdc3 attachment site mutant is synthetically lethal with cdc12-1. EJY323 (cdc3-R4,11,30,63 cdc12-1) carrying p315-PGAL-CDC3-HA was grown to exponential phase at 25°C in YPG (a); or YPG-grown cells were isolated, resuspended in YPD, and then incubated at 25°C for 24 h (b). Bar, 10 μm.
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Figure 9: The cdc3 attachment site mutant is synthetically lethal with cdc12-1. EJY323 (cdc3-R4,11,30,63 cdc12-1) carrying p315-PGAL-CDC3-HA was grown to exponential phase at 25°C in YPG (a); or YPG-grown cells were isolated, resuspended in YPD, and then incubated at 25°C for 24 h (b). Bar, 10 μm.

Mentions: We also tested whether the sumoylation site triple mutant interacted genetically with either SUMO conjugation pathway mutants or septin mutants. A quadruple mutant also containing a chromosomally integrated version of the uba2 ts10 allele grew at a range of rates similar to those seen with the uba2 ts10 strain alone (data not shown), which grew very poorly and was heterogeneous. Thus, the attachment site mutations did not strongly exacerbate or suppress the phenotypes of the uba2 ts10 mutant. However, when we crossed the sumoylation site triple mutant to a cdc12-1 mutant, we obtained the surprising result that the cdc3-R4,11,30,63 mutation alone was synthetically lethal with the cdc12-1 mutation. cdc12-1 single mutants grow well with near normal morphology at 25°C, but lose septin rings and develop the distinctive phenotypes of septin mutants at 37°C (see introduction). At 25°C, cdc3-R4,11,30,63 cdc12-1 cells overexpressing wild-type CDC3-HA exhibited near normal morphology (Fig. 9 a). When CDC3-HA expression was repressed, the double mutant developed the phenotypes of septin mutants, even at 25°C, producing branched, elongated cells and failing to undergo cytokinesis (Fig. 9 b). This effect was not general for all septin mutants, as the sumoylation site mutations did not exacerbate the phenotypes of the cdc10-1 mutant (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)

The cdc3 attachment site mutant is synthetically lethal with cdc12-1. EJY323 (cdc3-R4,11,30,63 cdc12-1) carrying p315-PGAL-CDC3-HA was grown to exponential phase at 25°C in YPG (a); or YPG-grown cells were isolated, resuspended in YPD, and then incubated at 25°C for 24 h (b). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169351&req=5

Figure 9: The cdc3 attachment site mutant is synthetically lethal with cdc12-1. EJY323 (cdc3-R4,11,30,63 cdc12-1) carrying p315-PGAL-CDC3-HA was grown to exponential phase at 25°C in YPG (a); or YPG-grown cells were isolated, resuspended in YPD, and then incubated at 25°C for 24 h (b). Bar, 10 μm.
Mentions: We also tested whether the sumoylation site triple mutant interacted genetically with either SUMO conjugation pathway mutants or septin mutants. A quadruple mutant also containing a chromosomally integrated version of the uba2 ts10 allele grew at a range of rates similar to those seen with the uba2 ts10 strain alone (data not shown), which grew very poorly and was heterogeneous. Thus, the attachment site mutations did not strongly exacerbate or suppress the phenotypes of the uba2 ts10 mutant. However, when we crossed the sumoylation site triple mutant to a cdc12-1 mutant, we obtained the surprising result that the cdc3-R4,11,30,63 mutation alone was synthetically lethal with the cdc12-1 mutation. cdc12-1 single mutants grow well with near normal morphology at 25°C, but lose septin rings and develop the distinctive phenotypes of septin mutants at 37°C (see introduction). At 25°C, cdc3-R4,11,30,63 cdc12-1 cells overexpressing wild-type CDC3-HA exhibited near normal morphology (Fig. 9 a). When CDC3-HA expression was repressed, the double mutant developed the phenotypes of septin mutants, even at 25°C, producing branched, elongated cells and failing to undergo cytokinesis (Fig. 9 b). This effect was not general for all septin mutants, as the sumoylation site mutations did not exacerbate the phenotypes of the cdc10-1 mutant (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