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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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Cell-cycle dependent attachment of SUMO to the septins. a, Whole cell lysates from EJY301 (CDC3-HA), growing exponentially (log) or arrested with α-factor, hydroxyurea (HU), or nocodazole (NOC), or from EJY322 (CDC3-HA cdc15-2) that had been arrested at 37°C for 3 h (cdc15), were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (top) or a polyclonal antibody against Cdc11 (bottom). Bands corresponding to unmodified Cdc3-HA and to Cdc11 are indicated. Arrows indicate the positions of SUMO-modified species. Circles indicate bands that cross-react with the anti-HA antibody. b, Exponentially growing wild-type (DF5) cells were analyzed by double-label immunofluorescence microscopy with a polyclonal antibody against SUMO and an mAb against tubulin. Height of bars represents the percentage of total cells in each of the five categories illustrated below the histogram: unbudded cells, small-budded cells (buds less than one third the size of the mother cell), large-budded cells with a short spindle, cells undergoing nuclear division with an intact elongated spindle, and cells with a divided nucleus and broken spindle. Black bars represent the portion containing a SUMO ring at the bud neck. 424 total cells were counted.
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Figure 2: Cell-cycle dependent attachment of SUMO to the septins. a, Whole cell lysates from EJY301 (CDC3-HA), growing exponentially (log) or arrested with α-factor, hydroxyurea (HU), or nocodazole (NOC), or from EJY322 (CDC3-HA cdc15-2) that had been arrested at 37°C for 3 h (cdc15), were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (top) or a polyclonal antibody against Cdc11 (bottom). Bands corresponding to unmodified Cdc3-HA and to Cdc11 are indicated. Arrows indicate the positions of SUMO-modified species. Circles indicate bands that cross-react with the anti-HA antibody. b, Exponentially growing wild-type (DF5) cells were analyzed by double-label immunofluorescence microscopy with a polyclonal antibody against SUMO and an mAb against tubulin. Height of bars represents the percentage of total cells in each of the five categories illustrated below the histogram: unbudded cells, small-budded cells (buds less than one third the size of the mother cell), large-budded cells with a short spindle, cells undergoing nuclear division with an intact elongated spindle, and cells with a divided nucleus and broken spindle. Black bars represent the portion containing a SUMO ring at the bud neck. 424 total cells were counted.

Mentions: A rabbit polyclonal antibody was raised against NH2 terminally His6-tagged SUMO(G98) (Cocalico Biologicals) and was affinity purified on a His6-FLAG-SUMO(G98) affinity column (Johnson et al. 1997) as described (Harlow and Lane 1988). In a whole cell lysate from an arrested ubc9 mutant, the affinity-purified anti-SUMO antibody recognized only free unconjugated SUMO (data not shown), demonstrating its specificity. A rabbit polyclonal antibody against Cdc3, a generous gift of J. Pringle (University of North Carolina, Chapel Hill, NC), was affinity purified on a nitrocellulose filter containing Cdc3-lacZ (expressed from pUR-PH1 [Kim et al. 1991] a gift of J. Pringle) as described (Pringle et al. 1991). The unpurified anti-Cdc3 serum recognized primarily an ∼100-kD band unrelated to Cdc3. The affinity-purified antibody also contained a small amount of antibody against this other protein. Other antibodies used were the 16B12 mAb against the HA epitope (in Fig. 1 b, 3, 4 b, 5 b, and 6; Berkeley Antibody Co.), the 3F10 mAb against the HA epitope (in Fig. 2 a; Boehringer Mannheim Corp.), the B5-1-2 mAb against tubulin (a generous gift of Mike Rout, Rockefeller University, NY, NY), and a rabbit polyclonal antibody against Cdc11 (Santa Cruz Biotechnology).


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

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

Cell-cycle dependent attachment of SUMO to the septins. a, Whole cell lysates from EJY301 (CDC3-HA), growing exponentially (log) or arrested with α-factor, hydroxyurea (HU), or nocodazole (NOC), or from EJY322 (CDC3-HA cdc15-2) that had been arrested at 37°C for 3 h (cdc15), were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (top) or a polyclonal antibody against Cdc11 (bottom). Bands corresponding to unmodified Cdc3-HA and to Cdc11 are indicated. Arrows indicate the positions of SUMO-modified species. Circles indicate bands that cross-react with the anti-HA antibody. b, Exponentially growing wild-type (DF5) cells were analyzed by double-label immunofluorescence microscopy with a polyclonal antibody against SUMO and an mAb against tubulin. Height of bars represents the percentage of total cells in each of the five categories illustrated below the histogram: unbudded cells, small-budded cells (buds less than one third the size of the mother cell), large-budded cells with a short spindle, cells undergoing nuclear division with an intact elongated spindle, and cells with a divided nucleus and broken spindle. Black bars represent the portion containing a SUMO ring at the bud neck. 424 total cells were counted.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169351&req=5

Figure 2: Cell-cycle dependent attachment of SUMO to the septins. a, Whole cell lysates from EJY301 (CDC3-HA), growing exponentially (log) or arrested with α-factor, hydroxyurea (HU), or nocodazole (NOC), or from EJY322 (CDC3-HA cdc15-2) that had been arrested at 37°C for 3 h (cdc15), were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (top) or a polyclonal antibody against Cdc11 (bottom). Bands corresponding to unmodified Cdc3-HA and to Cdc11 are indicated. Arrows indicate the positions of SUMO-modified species. Circles indicate bands that cross-react with the anti-HA antibody. b, Exponentially growing wild-type (DF5) cells were analyzed by double-label immunofluorescence microscopy with a polyclonal antibody against SUMO and an mAb against tubulin. Height of bars represents the percentage of total cells in each of the five categories illustrated below the histogram: unbudded cells, small-budded cells (buds less than one third the size of the mother cell), large-budded cells with a short spindle, cells undergoing nuclear division with an intact elongated spindle, and cells with a divided nucleus and broken spindle. Black bars represent the portion containing a SUMO ring at the bud neck. 424 total cells were counted.
Mentions: A rabbit polyclonal antibody was raised against NH2 terminally His6-tagged SUMO(G98) (Cocalico Biologicals) and was affinity purified on a His6-FLAG-SUMO(G98) affinity column (Johnson et al. 1997) as described (Harlow and Lane 1988). In a whole cell lysate from an arrested ubc9 mutant, the affinity-purified anti-SUMO antibody recognized only free unconjugated SUMO (data not shown), demonstrating its specificity. A rabbit polyclonal antibody against Cdc3, a generous gift of J. Pringle (University of North Carolina, Chapel Hill, NC), was affinity purified on a nitrocellulose filter containing Cdc3-lacZ (expressed from pUR-PH1 [Kim et al. 1991] a gift of J. Pringle) as described (Pringle et al. 1991). The unpurified anti-Cdc3 serum recognized primarily an ∼100-kD band unrelated to Cdc3. The affinity-purified antibody also contained a small amount of antibody against this other protein. Other antibodies used were the 16B12 mAb against the HA epitope (in Fig. 1 b, 3, 4 b, 5 b, and 6; Berkeley Antibody Co.), the 3F10 mAb against the HA epitope (in Fig. 2 a; Boehringer Mannheim Corp.), the B5-1-2 mAb against tubulin (a generous gift of Mike Rout, Rockefeller University, NY, NY), and a rabbit polyclonal antibody against Cdc11 (Santa Cruz Biotechnology).

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