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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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SUMO localizes to the mother cell side of the septin ring during mitosis. Double-label immunofluorescence microscopy of EJY306 (CDC3-HA/CDC3-HA) using a polyclonal antibody against SUMO (red; a, e, j, and o) and an mAb against the HA epitope (green; b, f, k, and p). Cells were visualized by differential interference contrast (DIC; d, h, m, and r) and DNA by DAPI staining (c, g, l, and q). Panels i and n are overlays of the anti-SUMO and anti-HA signals in the fields shown in e–h and j–m, respectively. Colocalization is represented by yellow in the overlays. Bars, 5 μm.
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Figure 3: SUMO localizes to the mother cell side of the septin ring during mitosis. Double-label immunofluorescence microscopy of EJY306 (CDC3-HA/CDC3-HA) using a polyclonal antibody against SUMO (red; a, e, j, and o) and an mAb against the HA epitope (green; b, f, k, and p). Cells were visualized by differential interference contrast (DIC; d, h, m, and r) and DNA by DAPI staining (c, g, l, and q). Panels i and n are overlays of the anti-SUMO and anti-HA signals in the fields shown in e–h and j–m, respectively. Colocalization is represented by yellow in the overlays. Bars, 5 μm.

Mentions: To define the stages when septins are sumoylated in more detail, SUMO was visualized by double label immunofluorescence microscopy. The protein detected in addition to SUMO was either Cdc3-HA (Fig. 3) or tubulin (Fig. 2 b), whose localization was used to determine the stage of the cell cycle occupied by individual cells. SUMO localized to the nucleus at all points in the cell cycle (Fig. 3, a, e, j, and o; data not shown), but more strikingly it localized to a ring at the bud neck only during mitosis. Specifically, a ring of SUMO appeared at the bud neck of large budded cells just before initiation of anaphase spindle elongation and nuclear division (Fig. 2 b and 3 e). This SUMO ring persisted through anaphase (Fig. 2 b and 3 j) and it disappeared abruptly at cytokinesis, virtually simultaneously with septin ring separation (Fig. 3o and Fig. p) and the beginning of spindle breakdown (Fig. 2 b). Surprisingly, the SUMO ring did not completely colocalize with the septins. SUMO coincided only with the mother cell side of the septin “double ring,” appearing on the side next to the undivided nucleus or on the side of the larger cell in mitotic cells (Fig. 3e, Fig. i, Fig. j, and Fig. n). Thus, SUMO conjugation to the septins was asymmetrical with respect to the mother–bud axis.


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

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

SUMO localizes to the mother cell side of the septin ring during mitosis. Double-label immunofluorescence microscopy of EJY306 (CDC3-HA/CDC3-HA) using a polyclonal antibody against SUMO (red; a, e, j, and o) and an mAb against the HA epitope (green; b, f, k, and p). Cells were visualized by differential interference contrast (DIC; d, h, m, and r) and DNA by DAPI staining (c, g, l, and q). Panels i and n are overlays of the anti-SUMO and anti-HA signals in the fields shown in e–h and j–m, respectively. Colocalization is represented by yellow in the overlays. Bars, 5 μm.
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Related In: Results  -  Collection

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Figure 3: SUMO localizes to the mother cell side of the septin ring during mitosis. Double-label immunofluorescence microscopy of EJY306 (CDC3-HA/CDC3-HA) using a polyclonal antibody against SUMO (red; a, e, j, and o) and an mAb against the HA epitope (green; b, f, k, and p). Cells were visualized by differential interference contrast (DIC; d, h, m, and r) and DNA by DAPI staining (c, g, l, and q). Panels i and n are overlays of the anti-SUMO and anti-HA signals in the fields shown in e–h and j–m, respectively. Colocalization is represented by yellow in the overlays. Bars, 5 μm.
Mentions: To define the stages when septins are sumoylated in more detail, SUMO was visualized by double label immunofluorescence microscopy. The protein detected in addition to SUMO was either Cdc3-HA (Fig. 3) or tubulin (Fig. 2 b), whose localization was used to determine the stage of the cell cycle occupied by individual cells. SUMO localized to the nucleus at all points in the cell cycle (Fig. 3, a, e, j, and o; data not shown), but more strikingly it localized to a ring at the bud neck only during mitosis. Specifically, a ring of SUMO appeared at the bud neck of large budded cells just before initiation of anaphase spindle elongation and nuclear division (Fig. 2 b and 3 e). This SUMO ring persisted through anaphase (Fig. 2 b and 3 j) and it disappeared abruptly at cytokinesis, virtually simultaneously with septin ring separation (Fig. 3o and Fig. p) and the beginning of spindle breakdown (Fig. 2 b). Surprisingly, the SUMO ring did not completely colocalize with the septins. SUMO coincided only with the mother cell side of the septin “double ring,” appearing on the side next to the undivided nucleus or on the side of the larger cell in mitotic cells (Fig. 3e, Fig. i, Fig. j, and Fig. n). Thus, SUMO conjugation to the septins was asymmetrical with respect to the mother–bud axis.

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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