The puc1 cyclin regulates the G1 phase of the fission yeast cell cycle in response to cell size

Abstract

Eukaryotic cells coordinate cell size with cell division by regulating the length of the G1 and G2 phases of the cell cycle. In fission yeast, the length of the G1 phase depends on a precise balance between levels of positive (cig1, cig2, puc1, and cdc13 cyclins) and negative (rum1 and ste9-APC) regulators of cdc2. Early in G1, cyclin proteolysis and rum1 inhibition keep the cdc2/cyclin complexes inactive. At the end of G1, the balance is reversed and cdc2/cyclin activity down-regulates both rum1 and the cyclin-degrading activity of the APC. Here we present data showing that the puc1 cyclin, a close relative of the Cln cyclins in budding yeast, plays an important role in regulating the length of G1. Fission yeast cells lacking cig1 and cig2 have a cell cycle distribution similar to that of wild-type cells, with a short G1 and a long G2. However, when the puc1(+) gene is deleted in this genetic background, the length of G1 is extended and these cells undergo S phase with a greater cell size than wild-type cells. This G1 delay is completely abolished in cells lacking rum1. Cdc2/puc1 function may be important to down-regulate the rum1 Cdk inhibitor at the end of G1.

Figure 1

Deletion of cig1⁺, cig2⁺, and puc1⁺ causes a cell cycle delay in G1. (A) Flow cytometry analysis of the wild type and cig1, cig2, and puc1 single, double, and triple mutants in wild-type and wee1-50 backgrounds at 25°C. (B) Flow cytometry analysis of wild type, cig1Δ cig2Δ puc1Δ, wee1-50, and wee1-50 cig1Δ cig2Δ puc1Δ at 35°C. For each strain, there is a histogram (left) and a dot-plot (right) representation of the data. The vertical lines in the dot plots correspond to the biggest cells in the wild-type culture. (C) Quantification of the data shown in A and B to indicate the percentage of cells in G1 in the different cyclin mutants. (D) Cell size of the different cyclin mutants in arbitrary units considering 100 as the size of the wild type, wee1-50 at 25°C, or wee1-50 at 36°C. Cells were grown in minimal medium to midexponential phase.

Figure 2

The cig1Δ cig2Δ puc1Δ mutant arrest in G1 after a single round of cell division in nitrogen-free medium. Wild-type and cig1Δ cig2Δ puc1Δ mutant cells grown at 25°C in minimal medium were centrifuged, washed four times in minimal medium without nitrogen, and resuspended in minimal medium without nitrogen at 25°C. Samples were taken for FACS analysis and protein and RNA preparation at the indicated times. (A) Flow cytometry analysis of h⁹⁰ wild type and h⁹⁰ cig1Δ cig2Δ puc1Δ mutants after nitrogen starvation at 25°C. (B) DAPI and calcofluor staining of cells before (t = 0h) and 4 h after (t = 4h) nitrogen starvation. Arrowheads show cells undergoing conjugation. (C) Rum1 protein and mRNA levels. Anti-tubulin antibodies and 18S rRNA were used as protein- and RNA-loading controls.

Figure 3

Rum1 protein persists for longer in the cig1Δ cig2Δ puc1Δ mutant than in the wild type. Wild-type and cig1Δ cig2Δ puc1Δ mutant cells were nitrogen starved for 8 h, and then nitrogen was added back to the culture. Samples were taken for protein extracts and flow cytometry at the indicated times. (A) Cdc13, rum1, and cdc2 protein levels in wild-type and cig1Δ cig2Δ puc1Δ cells. +N corresponds to cells growing in minimal medium. (B) Cdc2/cdc13 kinase assays. Cdc2/cdc13 complexes were immunoprecipitated with anti-cdc13 antibodies and assayed with the use of histone H1 as substrate. (C) Flow cytometry analysis of wild-type and cig1Δ cig2Δ puc1Δ mutant cells during the release from nitrogen starvation.

Figure 4

Rum1 protein levels oscillate throughout the cell cycle in cells lacking cig1, cig2, and puc1. wee1-50 cig1Δ cig2Δ puc1Δ mutant cells growing in minimal medium at 25°C (Async.) were synchronized by elutriation. A homogenous G1 population was selected and incubated at 25°C. Samples were taken every 40 min for protein extracts and FACS analysis. (A) Flow cytometry analysis. (B) Cdc13, rum1, and cdc2 protein levels throughout the cell cycle.

Figure 5

The cdc2/puc1 kinase can efficiently phosphorylate rum1 in vitro and is not inhibited by rum1. (A) Wild-type cells were grown to midexponential phase in minimal medium. Two milligrams of total protein extracts were immunoprecipitated with anti-cdc2, anti-cig1, and anti-puc1 antibodies. Protein kinase activity was measured with the use of rum1, rum1-A58A62 (A58A62), and histone H1 (H1) as substrates. The phosphorylated products were separated by 14% SDS-PAGE and exposed to autoradiography. Cdc2 immunocomplexes could phosphorylate p25^rum1 as efficiently as they could phosphorylate histone H1. Rum1 phosphorylation induced a band shift from 34 to 36 kDa that was not observed in the mutant rum1-A58A62. Cig1 and puc1 immunocomplexes induced a similar band shift to cdc2. (B) Wild-type fission yeast extracts were immunoprecipitated with anti-cdc13, anti-cig2, anti-cig1, and anti-puc1 antibodies. The immunoprecipitates were preincubated with different concentrations of rum1 protein and then assayed for histone H1 kinase activity. As negative controls (Δ), extracts of cig1Δ, cig2Δ, and puc1Δ were immunoprecipitated with anti-cig1, anti-cig2, and anti-puc1 antibodies and assayed for histone H1 kinase activity. IP, immunoprecipitate.

Figure 6

Deletion of the rum1⁺ gene suppresses the G1-delay phenotype of cig1Δ cig2Δ puc1Δ mutant cells. The indicated strains were grown in minimal medium to midexponential phase at 25°C. Samples were taken to determine the cell cycle distribution by flow cytometry.

Figure 7

Cells lacking rum1 do not delay progression through G1 upon nutritional shift-down experiments. Asynchronous cultures of the indicated strains were grown at 25°C in minimal medium containing 20 mM NH₄Cl supplemented with 0.5% yeast extract and then shifted to minimal medium containing 20 mM l-proline instead of NH₄Cl as a nitrogen source. A small population of G1 cells was detected in the wild type and the cig1⁺ deletion upon the shift. This population increased in cells deleted for cig2⁺ and puc1⁺. In the triple mutant cig1Δ cig2Δ puc1Δ, the percentage of cells in G1 increased to 80%. Deletion of rum1⁺ completely abolished this G1 population.