The elm1 kinase functions in a mitotic signaling network in budding yeast

Abstract

In budding yeast, the Clb2 mitotic cyclin initiates a signaling network that negatively regulates polar bud growth during mitosis. This signaling network appears to require the function of a Clb2-binding protein called Nap1, the Cdc42 GTPase, and two protein kinases called Gin4 and Cla4. In this study, we demonstrate that the Elm1 kinase also plays a role in the control of bud growth during mitosis. Cells carrying a deletion of the ELM1 gene undergo a prolonged mitotic delay, fail to negatively regulate polar bud growth during mitosis, and show defects in septin organization. In addition, Elm1 is required in vivo for the proper regulation of both the Cla4 and Gin4 kinases and interacts genetically with Cla4, Gin4, and the mitotic cyclins. Previous studies have suggested that Elm1 may function to negatively regulate the Swe1 kinase. To further understand the functional relationship between Elm1 and Swe1, we have characterized the phenotype of Deltaelm1 Deltaswe1 cells. We found that Deltaelm1 Deltaswe1 cells are inviable at 37 degrees C and that a large proportion of Deltaelm1 Deltaswe1 cells grown at 30 degrees C contain multiple nuclei, suggesting severe defects in cytokinesis. In addition, we found that Elm1 is required for the normal hyperphosphorylation of Swe1 during mitosis. We propose a model in which the Elm1 kinase functions in a mitotic signaling network that controls events required for normal bud growth and cytokinesis, while the Swe1 kinase functions in a checkpoint pathway that delays nuclear division in response to defects in these events.

FIG. 1

The phenotype of cells lacking the function of the Elm1 kinase. The cells shown in panels A, B, and C were grown to log phase in liquid YPD medium at 30°C and photographed with Nomarski optics. (A) The phenotype of the ecm41 mutation identified in a screen for mutations that cause an elongated cell morphology. (B) Deletion of the ELM1 gene in a wild-type background results in an elongated bud phenotype. (C) Deletion of the ELM1 gene results in a severe elongated bud phenotype in a Clb2-dependent background. (D) Cells carrying a deletion of the ELM1 gene in a Clb2-dependent background are barely viable. The indicated strains were grown on a YPD plate at 37°C.

FIG. 2

ELM1 interacts genetically with CLA4. (A) Strains carrying deletions of the genes for ELM1 and CLA4 either alone or in combination were grown on a YPD plate at 37°C. (B) Cells carrying deletions of both the ELM1 and CLA4 genes have a severe elongated bud phenotype. Cells were grown to log phase in liquid YPD medium at 30°C and photographed with Nomarski optics.

FIG. 3

Elm1 is required for normal progression through mitosis. (A) Wild-type and Δelm1 cells were released from an α-factor arrest, and the percentage of cells with a short mitotic spindle was determined as a function of time during the cell cycle. (B) Western blots show the amount of the Clb2 protein present in wild-type and Δelm1 cells as a function of time after release from an α-factor arrest. (C) Western blots showing the amount of the Clb2 protein present as a function of time after addition of α-factor to Δclb1,3,4 and Δelm1 Δclb1,3,4.

FIG. 4

Elm1 is not required for the formation of active Clb2-Cdc28 kinase complexes. A time course shows the appearance of Clb2-associated kinase activity during mitosis in wild-type and Δelm1 cells. Cells were released from an α-factor arrest and then assayed for Clb2-associated kinase activity during the cell cycle as previously described (21).

FIG. 5

Elm1 is required for the mitosis-specific hyperphosphorylation of the Gin4 and Cla4 kinases. (A) A time course showing the appearance Gin4 hyperphosphorylation during mitosis. Wild-type and Δelm1 cells were released from α-factor arrest and then assayed for Gin4 hyperphosphorylation during the cell cycle as previously described (1). (B) Cells were arrested in interphase with α-factor and induced to express Cdc42^V12 and Clb2^Δ176 in a control strain and in a Δelm1 strain. Samples were taken every hour for 3 hours and were then immunoblotted with affinity-purified anti-Cla4 antibodies to observe Cla4 hyperphosphorylation as previously described (44).

FIG. 6

Deletion of the SWE1 gene does not restore the hyperphosphorylation and activation of the Gin4 kinase in Δelm1 cells. A wild-type control strain and strains carrying deletions of the SWE1 or ELM1 genes either alone or in combination were grown to log phase in liquid YPD medium and arrested with either α-factor or benomyl. Gin4 hyperphosphorylation was assayed by Western blotting, and Gin4 kinase activity was assayed by an immunoprecipitation kinase assay as previously described (1). The same samples were also probed with an anti-Clb2 antibody to demonstrate that the cells were arrested in mitosis. We always observed that there is a small amount of the Clb2 protein present after treatment with α-factor in Δelm1 and in Δelm1 Δswe1 cells, a finding consistent with the idea that some of the cells in these strains have difficulty exiting mitosis. The nature of the band that migrates slightly more slowly than Clb2 is unknown. This band also appears in the Δelm1 Δswe1 strain upon longer exposure of the blot.

FIG. 7

Δelm1 Δswe1 cells are multinucleate. Δelm1 Δswe1 cells were grown to log phase at 30°C and stained with antitubulin antibodies and the DNA stain DAPI (4′,6′-diamidino-2-phenylindole). A high-magnification image (A) and a lower-magnification view that includes more cells (B) are shown. In panel C, the indicated strains were grown to log phase at 30°C, stained with propidium iodide, and analyzed for DNA content by flow cytometry.

FIG. 8

Δelm1 Δswe1 cells are inviable at 37°C and are morphologically abnormal. (A) Strains carrying deletions of SWE1 and ELM1 either alone or in combination were grown on YPD plates at 30 and 37°C. (B) Morphology of Δelm1 Δswe1 cells. Cells were grown to log phase at 30°C in YPD liquid medium and photographed with Nomarski optics.

FIG. 9

The septins are mislocalized in Δelm1 and Δelm1 Δswe1 cells. Wild-type, Δelm1, and Δelm1 Δswe1 cells were grown to log phase at 30°C in YPD liquid medium and stained with a DNA stain and a polyclonal antibody that recognizes Cdc11.

FIG. 10

Elm1 is required for the mitosis-specific hyperphosphorylation of the Swe1 kinase. (A) A time course shows the appearance of Swe1 hyperphosphorylation during mitosis. Wild-type and Δelm1 cells were released from α-factor arrest and then assayed for Swe1 hyperphosphorylation during the cell cycle by using affinity-purified anti-Swe1 antibodies. In this experiment, less total protein was loaded for the Δelm1 samples because the starting culture had fewer cells (due to the aberrant morphology of Δelm1 cells, it is difficult to quantitate the total number of cells in culture). We have not found evidence for decreased levels of the Swe1 protein in Δelm1 cells. (B) Western blot of phosphatase-treated and untreated samples of Swe1 immunoprecipitates probed with Swe1 polyclonal antibody.