The Dcr2p phosphatase destabilizes Sic1p in Saccharomyces cerevisiae

Abstract

Initiation of cell division is controlled by an irreversible switch. In Saccharomyces cerevisiae degradation of the Sic1p protein, an inhibitor of mitotic cyclin/cyclin-dependent kinase complexes, takes place before initiation of DNA replication, at a point called START. Sic1p is phosphorylated by multiple kinases, which can differentially affect the stability of Sic1p. How phosphorylations that stabilize Sic1p are reversed is unknown. Here we show that the Dcr2p phosphatase functionally and physically interacts with Sic1p. Over-expression of Dcr2p destabilizes Sic1p and leads to phenotypes associated with destabilized Sic1p, such as genome instability. Our results identify a novel factor that affects the stability of Sic1p, possibly contributing to mechanisms that trigger initiation of cell division.

Figure 1. DCR2 over-expression leads to chromosome loss

Sectoring assay for chromosome loss, with cells carrying the indicated plasmids. Formation of red sectors indicates chromosome loss.

Figure 2. DCR2 over-expression destabilizes Sic1p

A, The levels of a GAL-induced Sic1p-Protein A fusion (Sic1p-TAG) were monitored by immunoblotting, from cells carrying the indicated plasmids after SIC1 synthesis was turned-off. The Ponceau-stained blots indicate loading. B, The relative half-life was determined from (A) from the band intensities. C, Over-expression of DCR2 allows a fraction of cln cells to complete one extra cell division. cln1,2Δ,GAL-CLN3 cells carrying the indicated plasmids were grown in galactose-containing medium, but they were then shifted to glucose-containing medium to turn CLN3 expression off. From nine independent experiments, the number of divisions the YEp-vector transformants were able to complete is shown on the x-axis, and the corresponding divisions from the paired YEp-DCR2 transformants is shown on the y-axis. The diagonal is shown to indicate where the data points should have fallen if there was no difference between the YEp-vector and YEp-DCR2 samples.

Figure 3. Over-expression of DCR2^DN leads to salt sensitivity

Growth of the indicated strains (all in the BY4741 background) was evaluated by spotting 10-fold serial dilutions of the cultures on solid media, as indicated. The plates were incubated at 30°C and photographed after 4 days.

Figure 4. Dcr2p targets Sic1p

A, SIC1-TAP cells carrying the indicated plasmids were grown as described in the text, and samples were subjected to SDS-PAGE either directly (bottom panel) or after the GAL-induced gene products fused to GST were precipitated with glutathione beads (top panels). For immunoblotting (IB) we used the indicated antibodies. B, Sic1p was precipitated with Co²⁺ beads from P_GAL-SIC1-TAG cells, which were arrested in G1 with α–factor and treated with high salt to promote Sic1p phosphorylation. Sic1p was then incubated with recombinant Dcr2p, Dcr2p^DN, or λ–PPase, in the presence (+) or absence (-) of phosphatase inhibitor cocktails, as indicated. The reactions were analyzed by SDS-PAGE and blotting, followed by phosphoprotein-specific detection (top), or immunoblotting with the PAP reagent (bottom).