Specific inhibition of Elm1 kinase activity reveals functions required for early G1 events

Abstract

In budding yeast, the Elm1 kinase is required for coordination of cell growth and cell division at G(2)/M. Elm1 is also required for efficient cytokinesis and for regulation of Swe1, the budding yeast homolog of the Wee1 kinase. To further characterize Elm1 function, we engineered an ELM1 allele that can be rapidly and selectively inhibited in vivo. We found that inhibition of Elm1 kinase activity during G(2) results in a phenotype similar to the phenotype caused by deletion of the ELM1 gene, as expected. However, inhibition of Elm1 kinase activity earlier in the cell cycle results in a prolonged G(1) delay. The G(1) requirement for Elm1 kinase activity occurs before bud emergence, polarization of the septins, and synthesis of G(1) cyclins. Inhibition of Elm1 kinase activity during early G(1) also causes defects in the organization of septins, and inhibition of Elm1 kinase activity in a strain lacking the redundant G(1) cyclins CLN1 and CLN2 is lethal. These results demonstrate that the Elm1 kinase plays an important role in G(1) events required for bud emergence and septin organization.

FIG. 1.

The phenotype caused by inhibition of elm1-as activity. (A) Threonine 200 in subdomain V was changed to a glycine to generate the elm1-as mutant. (B) Log-phase elm1-as cells (strain AS79) growing at 30°C in YEPD medium were mock treated with DMSO (Control) or treated with 25 μM 1NM-PP1 for 8 h. elm1Δ cells (strain AS1) are shown for comparison.

FIG. 2.

Inhibition of elm1-as kinase activity causes a prolonged delay in bud emergence. (A) elm1-as cells (strain AS79) were released from an α-factor arrest and treated with 25 μM 1NM-PP1 or mock treated at 10 min after release from the arrest. Samples were taken every 10 min for 140 min, and the percentages of budded cells were calculated. (B) elm1-as cells (strain AS79) were released from an α-factor arrest and treated with 25 μM 1NM-PP1 at 10, 20, 30, 40, and 50 min after release from α-factor arrest. Samples were taken every 10 min for 130 min, and the percentages of budded cells were determined.

FIG. 3.

Inhibition of Elm1 kinase activity causes a delay in septin localization and aberrant septin organization. (A) elm1-as cells were released from an α-factor arrest and treated with 25 μM 1NM-PP1 or mock treated at 10 min after release from the arrest. Samples were taken every 15 min, and the percentages of cells with polarized septin staining were determined after staining with an anti-Cdc11 polyclonal antibody. (B) elm1-as cells were released from an α-factor arrest and treated with 1NM-PP1 as described for Fig. 3A. Examples of the septin staining observed at the indicated times are shown.

FIG. 4.

The requirement for Elm1 kinase activity occurs before transcription of the G₁ cyclin CLN2. (A) Western blots showing the behavior of a Cln2-3XHA fusion protein in 1NM-PP1-treated and mock-treated elm1-as cells (strain AS88) as a function of time after release from an α-factor arrest. 1NM-PP1 was added at 10 min after release from the arrest. (B) Cln2 Northern blots from an experiment identical to the one whose results are shown in panel A.

FIG. 5.

Expression of CLN2 from the GAL1 promoter does not rescue the defect in bud emergence caused by inhibition of elm1-as kinase activity. (A) elm1-as cells (strain SA49) that express CLN2 from the GAL1 promoter were released from an α-factor arrest and treated with 25 μM 1NM-PP1 or mock treated with DMSO 15 min after release from the arrest. The percentages of cells undergoing bud emergence were then determined at 15-min intervals and plotted as a function of time. (B) Examples of cells at the 105-min time point in panel A.

FIG. 6.

Inhibition of Elm1 kinase activity late in the cell cycle causes a mitotic arrest with high Clb2 levels and a short spindle. (A) elm1-as cells (strain AS79) were released from α-factor arrest and treated with 1NM-PP1 70 min after release. Samples were taken every 15 min, and the percentage of budded cells was determined at each time point. (B) elm1-as cells (strain AS79) were released from α-factor arrest and treated with 1NM-PP1 at 10 or 70 min after release. Samples were taken every 20 min, and Western blotting was used to assay levels of the Clb2 mitotic cyclin. (C) elm1-as cells (strain AS79) were released from α-factor arrest and treated with 1NM-PP1 70 min after release. Samples were taken every 15 min, and the percentage of cells with a short mitotic spindle was determined at each time point.

FIG. 7.

Inhibition of Elm1 kinase activity in elm1-asΔ cln1 cln2 cells is lethal. (A) The indicated strains were grown to saturation, plated onto YEPD, and analyzed for sensitivity to 1NM-PP1 by a halo assay (strains DK186, AS79, AS96, and AS105). WT, wild type. (B) The elm1-as cln1Δ cln2Δ strain (AS105) was nutrient arrested and released onto a YEPD plate containing 40 μM 1NM-PP1 for 24 h. Cells were removed from the plate with a toothpick and photographed with Nomarski optics. (C) Western blots showing the behavior of the Clb2 protein in elm1-as cln1Δ cln2Δ cells (strain AS105) as a function of time after release from nutrient arrest in the presence of 25 μM 1NM-PP1.