Rapamycin-mediated G1 arrest involves regulation of the Cdk inhibitor Sic1 in Saccharomyces cerevisiae.

The rapamycin-sensitive (TOR) signalling pathway in Saccharomyces cerevisiae controls growth and cell proliferation in response to nutrient availability. Rapamycin treatment causes cells to arrest growth in G1 phase. The mechanism by which the inhibition of the TOR pathway regulates cell cycle progression is not completely understood. Here we show that rapamycin causes G1 arrest by a dual mechanism that comprises downregulation of the G1-cyclins Cln1-3 and upregulation of the Cdk inhibitor protein Sic1. The increase of Sic1 level is mostly independent of the downregulation of the G1 cyclins, being unaffected by ectopic CLN2 expression, but requires Sic1 phosphorylation of Thr173, because it is lost in cells expressing Sic1(T173A). Rapamycin-mediated Sic1 upregulation involves nuclear accumulation of a more stable, non-ubiquitinated protein. Either SIC1 deletion or CLN3 overexpression results in non-cell-cycle-specific arrest upon rapamycin treatment and makes cells sensitive to a sublethal dose of rapamycin and to nutrient starvation. In conclusion, our data indicate that Sic1 is involved in rapamycin-induced G1 arrest and that deregulated entrance into S phase severely decreases the ability of a cell to cope with starvation conditions induced by nutrient depletion or which are mimicked by rapamycin treatment.

Subcellular localization of the cyclin dependent kinase inhibitor Sic1 is modulated by the carbon source in budding yeast.

The cyclin dependent kinase inhibitor Sic1 and the cyclin Clb5 are essential regulators of the cyclin dependent kinase Cdc28 during the G1 to S transition in budding yeast. Yeast enters S phase after ubiquitin-mediated degradation of Sic1, an event triggered by Cln1, 2-Cdc28 mediated phosphorylation. We recently showed that Sic1 is involved in carbon source modulation of the critical cell size required to enter S phase. Here we show that the amount and sub-cellular localization of Sic1 are also carbon source-modulated. We identify a bipartite nuclear localization sequence responsible for nuclear localization of Sic1 and for correct cell cycle progression in a carbon-source dependent manner. Similarly to Cip/Kip proteins-Sic1 mammalian counterparts-Sic1 facilitates nuclear accumulation of its cognate cyclin, since cytoplasmic building-up of Clb5 is observed upon switching off expression of the SIC1 gene. Our data indicate a previously unrecognized inhibitor/activator dual role for Sic1 and put it among key molecules whose activity is regulated by their nuclear-cytoplasmic localization.