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. 1999 Nov 29;147(5):981-94.
doi: 10.1083/jcb.147.5.981.

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

E S Johnson  1 , G Blobel
Affiliations

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

E S Johnson et al. J Cell Biol. .

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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Figures

Figure 1
Figure 1
SUMO is attached to the septins Cdc3, Cdc11, and Shs1. a, Lysate from cells expressing His6- and FLAG epitope-tagged SUMO was subjected to affinity chromatography on Ni-NTA agarose and anti-FLAG agarose. The resulting fraction was analyzed by SDS-PAGE and Coomassie brilliant blue staining. Bands determined to contain Cdc3 by MALDI-TOF mass spectrometry are indicated by arrows. b, Whole cell lysates (lanes 1–5) or anti-HA immunoprecipitates (lanes 6–10) from nocodazole-arrested cells expressing HA-tagged septins were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (lanes 1–5) or with a polyclonal antibody against SUMO (lanes 6–10). Lanes 1 and 6, EJY301; lanes 2 and 7, EJY303; lanes 3 and 8, EJY305; lanes 4 and 9, EJY302; lanes 5 and 10, EJY304. The HA-tagged septin in each lane is indicated. Bands corresponding to unmodified HA-tagged septins are indicated.
Figure 2
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.
Figure 3
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.
Figure 4
Figure 4
Single SUMO moieties are attached to multiple Lys residues in the NH2-terminal domain of Cdc3. a, Diagram of TEV protease cleavage site-containing constructs, showing the six Lys residues in the NH2-terminal domain of Cdc3 and the positions of introduced TEV cleavage sites. b–e, Exponential cultures of EJY300 (cdc3Δ) bearing plasmids expressing Cdc3-HA or one of the TEV site-containing Cdc3-HA constructs were arrested with nocodazole, lysed, and the lysates immunoprecipitated with an mAb against the HA epitope. Immunoprecipitated protein was treated with TEV protease while bound to the beads, and the resulting cleavage products were separated into an unbound fraction (e) and a bound fraction (b and d). The uncleaved proteins are shown in c. Fractions were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (b) or a polyclonal antibody against SUMO (c–e). Lanes are numbered according to construct numbers in a. Asterisks indicate bands containing IgG. Arrow indicates uncleaved, unmodified Cdc3-HA. Open circles in d indicate the TEV cleavage products.
Figure 5
Figure 5
Mutational analysis of SUMO attachment site Lys residues in the septins. a, Sequences surrounding SUMO attachment sites in the septins. b and c, Whole cell lysates from nocodazole-arrested cultures were analyzed by SDS-PAGE and immunoblotting with an mAb against the HA epitope (b), or a polyclonal antibody against SUMO (c). b, Lane 1, EJY301; lane 2, EJY307; lane 3, EJY303; lane 4, EJY308; lane 5, EJY305; lane 6, EJY309. The HA-tagged septin in each strain is indicated over the lane. c, Lane 1, JD52 (wild-type); lane 2, EJY307 (cdc3-Δ94-HA); lane 3, EJY310 (cdc3-Δ94-HA cdc11-R412-HA), lane 4, EJY311 (cdc3-Δ94-HA cdc11-R412-HA shs1-R426,437-HA).
Figure 6
Figure 6
The triple sumoylation site septin mutant accumulates extra septin rings. a–h, EJY306 (CDC3-HA; a and b) or EJY318 (cdc3-R4,11,30,63 cdc11-R412-HA shs1-R426,437-HA; c–h) were analyzed by double-label immunofluorescence microscopy with a polyclonal antibody against SUMO (a, c, e, and f) and an mAb against the HA epitope (b, d, g, and h). i–n, Fluorescence microscopy of living log phase cells stained with Calcofluor, visualizing Calcofluor staining (i, k, and m) or Cdc12-GFP (j, l, and n). Strains used were EJY319 (CDC12-GFP; i and j) and EJY320 (cdc3-R4,11,30,63 cdc11-R412-HA shs1-R426,437-HA CDC12-GFP; k–n). Arrowheads indicate septin rings remaining from previous cell cycles. Bars, 5 μm.
Figure 7
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).
Figure 8
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%.
Figure 9
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.
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