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. 1998 Nov 2;143(3):709-17.
doi: 10.1083/jcb.143.3.709.

The septins are required for the mitosis-specific activation of the Gin4 kinase

C W Carroll  1 , R Altman, D Schieltz, J R Yates, D Kellogg
Affiliations

The septins are required for the mitosis-specific activation of the Gin4 kinase

C W Carroll et al. J Cell Biol. .

Abstract

In budding yeast, a protein kinase called Gin4 is specifically activated during mitosis and functions in a pathway initiated by the Clb2 cyclin to control bud growth. We have used genetics and biochemistry to identify additional proteins that function with Gin4 in this pathway, and both of these approaches have identified members of the septin family. Loss of septin function produces a phenotype that is very similar to the phenotype caused by loss of Gin4 function, and the septins are required early in mitosis to activate Gin4 kinase activity. Furthermore, septin mutants display a prolonged mitotic delay at the short spindle stage, consistent with a role for the septins in the control of mitotic events. Members of the septin family bind directly to Gin4, demonstrating that the functions of Gin4 and the septins must be closely linked within the cell. These results demonstrate that the septins in budding yeast play an integral role in the mitosis-specific regulation of the Gin4 kinase and that they carry out functions early in mitosis.

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Figures

Figure 2
Figure 2
(A) Affinity purification of Gin4 binding proteins. The lane marked GST-Gin4-6His shows the purified fusion protein used to construct a Gin4 affinity column. The major band migrating above the 97-kD marker is the full-length fusion protein, while the other bands represent breakdown products. A Gin4 affinity column was loaded with a crude extract made from log-phase yeast cells, washed with buffer, and then eluted with a salt gradient. Eluted fractions were precipitated with TCA, resuspended in gel sample buffer, and then resolved on a 12.5% SDS-polyacrylamide gel as previously described (Kellogg et al., 1995). The gel is stained with Coommassie blue. LSS, the low speed supernatant (12,000 g, 5 min); HSS, the high speed supernatant that was loaded onto the column (100,000 g, 90 min); Flow Through, shows the extract after it passed through the column. (B) A Western blot confirming that the Cdc11 septin binds to the Gin4 affinity column. The same elution fractions shown in A were subjected to Western blotting using an anti-Cdc11 antibody. For the Western blot, we loaded only 1/10 the amount of sample that was loaded onto the gel shown in A so that the signal would not be too strong.
Figure 4
Figure 4
(A) Loss of septin function results in a mitotic delay. Log phase cultures of cdc12-6 and wild-type cells were grown at 23°C and were then shifted to 33°C and α-factor was added. Samples were taken at the indicated time points after the addition of α-factor, and Western blots were performed using anti-Clb2 antibodies. (B) Septin mutants arrest at the short spindle stage of mitosis. A time course was carried out as described for A, except that samples were taken at each time point and cells were stained with an anti-tubulin antibody. The percentage of cells with a short spindle at each time point was quantitated, counting at least 200 cells for each time point.
Figure 1
Figure 1
The temperature-sensitive elongated bud phenotype of the mutant strain identified in a screen for mutations that cause the formation of elongated buds. The mutant strain was grown overnight to log phase at room temperature and then either left at room temperature or shifted to 30°C for 6 h and photographed by Nomarski optics.
Figure 3
Figure 3
(A) Septin function is required for the mitosis-specific activation of the Gin4 kinase. Wild-type and cdc11 cells were arrested in G1 by the addition of α-factor, and were then released from the arrest to begin the cell cycle at the restrictive temperature for cdc11. At each of the indicated time points, samples were taken and the mitosis-specific activation of Gin4 was assayed using Western blotting to detect the hyperphosphorylation of Gin4 that leads to activation. (B) The mitotic cyclin Clb2 appears with normal kinetics in cells lacking septin function. Wild-type and cdc11 cells were synchronized and released at the restrictive temperature as in Fig. 3 A, and the appearance of the Clb2 cyclin was assayed by Western blotting. This experiment was carried out independently of the experiment shown in Fig. 3 A, and the cells entered mitosis slightly later than the cells shown in Fig. 3 A. (C) Septin function is required for the activation of Gin4 kinase activity. Log phase cultures of wild-type and cdc12-6 were arrested in mitosis at 37°C with 20 μg/ml nocodazole and 30 μg/ml benomyl for 2.5 h. Gin4 was then immunoprecipitated and assayed for its ability to phosphorylate histone H1 in vitro (left). In addition, Western blotting was used to assay Gin4 hyperphosphorylation in each sample (right). Similar results were obtained using the allele of cdc11 that we isolated in our screen.
Figure 5
Figure 5
An alignment of the septins expressed in vegetative budding yeast cells. Conserved residues found in the GTP-binding domains G1, G3, and G4 are marked with asterisks (for a discussion of GTP-binding consensus sequences see Bourne et al., 1991).
Figure 6
Figure 6
(A) Sep7 colocalizes with Cdc11. Cells carrying a 3×HA-tagged version of Sep7 were grown to log phase and then fixed with formaldehyde. The localization of the Cdc11 protein was determined using an affinity-purified polyclonal anti-Cdc11 antibody, while the localization of the Sep7 protein was determined using the 12CA5 anti-HA mAb. (B) Deletion of the SEP7 gene in Clb2-dependent cells causes the formation of elongated buds. The indicated strains were grown overnight to log phase at 30°C and were then photographed with Nomarski optics. (C) Sep7 is required for the mitosis-specific activation of the Gin4 kinase. A wild-type strain and a ΔSep7 strain were arrested in G1 with α-factor for 2.5 h, and then released from the arrest to begin the cell cycle at 30°C. At each of the indicated time points, samples were taken and the mitosis-specific activation of Gin4 was assayed by Western blotting. Both strains used in this experiment are in the Clb2-dependent background, and identical results are obtained in a wild-type background.
Figure 6
Figure 6
(A) Sep7 colocalizes with Cdc11. Cells carrying a 3×HA-tagged version of Sep7 were grown to log phase and then fixed with formaldehyde. The localization of the Cdc11 protein was determined using an affinity-purified polyclonal anti-Cdc11 antibody, while the localization of the Sep7 protein was determined using the 12CA5 anti-HA mAb. (B) Deletion of the SEP7 gene in Clb2-dependent cells causes the formation of elongated buds. The indicated strains were grown overnight to log phase at 30°C and were then photographed with Nomarski optics. (C) Sep7 is required for the mitosis-specific activation of the Gin4 kinase. A wild-type strain and a ΔSep7 strain were arrested in G1 with α-factor for 2.5 h, and then released from the arrest to begin the cell cycle at 30°C. At each of the indicated time points, samples were taken and the mitosis-specific activation of Gin4 was assayed by Western blotting. Both strains used in this experiment are in the Clb2-dependent background, and identical results are obtained in a wild-type background.
Figure 6
Figure 6
(A) Sep7 colocalizes with Cdc11. Cells carrying a 3×HA-tagged version of Sep7 were grown to log phase and then fixed with formaldehyde. The localization of the Cdc11 protein was determined using an affinity-purified polyclonal anti-Cdc11 antibody, while the localization of the Sep7 protein was determined using the 12CA5 anti-HA mAb. (B) Deletion of the SEP7 gene in Clb2-dependent cells causes the formation of elongated buds. The indicated strains were grown overnight to log phase at 30°C and were then photographed with Nomarski optics. (C) Sep7 is required for the mitosis-specific activation of the Gin4 kinase. A wild-type strain and a ΔSep7 strain were arrested in G1 with α-factor for 2.5 h, and then released from the arrest to begin the cell cycle at 30°C. At each of the indicated time points, samples were taken and the mitosis-specific activation of Gin4 was assayed by Western blotting. Both strains used in this experiment are in the Clb2-dependent background, and identical results are obtained in a wild-type background.
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