Gene Encodes an Importin-Implicated in the Nuclear Import of Cdc 25 in Schizosaccharomyces pombe

In Schizosaccharomyces pombe, the nuclear accumulation of Cdc25 peaks in G2 and is necessary for the proper timing of mitotic entry. Here, we identify the sal3 gene product as an importinhomolog that participates in the nuclear import of Cdc25. Loss of sal3 results in a cell cycle delay, failure to undergo G1 arrest under nitrogen-starvation conditions, and mislocalization of Cdc25 to the cytosol. Fusion of an exogenous classical nuclear localization sequence (cNLS) to Cdc25 restores its nuclear accumulation in a sal3 disruptant and suppresses the sal3 mutant phenotypes. In addition, we show that enhanced nuclear localization of Cdc25 at endogenous levels of expression advances the onset of mitosis. These results demonstrate that the nuclear translocation of Cdc25 is important for the timing of mitotic entry and that Sal3 plays an important role in this process. PROPER localization of gene products is necessary through the action of Wee1. A G2 cell size homeostasis for their normal function to ensure coordinated accheckpoint acting through Cdc25 monitors the size cess to regulators and substrates. Such is the case for threshold and triggers mitotic entry upon attainment elements involved in cell cycle control where subcellular of a critical cell size (Rupeš et al. 2001). Thus cells that localization can influence the progression of the cell have fulfilled the size requirement for mitosis show a cycle (reviewed in Takizawa and Morgan 2000; Yoneda rapid decrease in Y-15 phosphorylation as a conse2000). The mechanism of their regulated localization is quence of Cdc25 activation (Rupeš et al. 2001). therefore important in understanding cell cycle control. The localization of Cdc2, Cdc13, and Cdc25 fluctuates The fission yeast, Schizosaccharomyces pombe, is ideal for in the cell cycle, colocalizing in the nucleus at maximal studying this process since many of the cell cycle regulalevels from late G2 to mitotic metaphase and at minimal tory genes are well characterized and the consequences levels from mitotic anaphase to S phase (Booher et al. of perturbing their subcellular localization are easily 1989; Lopez-Girona et al. 1999; Decottignies et al. examined through molecular genetic and imaging tech2001). The coordinated nuclear accumulation of Cdc2niques. Cdc13 complexes and of Cdc25 results in Cdc2 activaThe crucial event for mitotic initiation is the activation tion in the nucleus and consequent mitotic entry. Conof a protein kinase complex consisting of catalytic and sistent with this model is the observation that nuclear regulatory cyclin B subunits encoded by the cdc2 and exclusion of Cdc25 by removal of a nuclear localization cdc13 genes, respectively (Simanis and Nurse 1986; sequence (NLS) results in a mitotic delay (Lopez-Girona Booher et al. 1989). The activity of the kinase is reguet al. 2001). lated by the phosphorylation state of tyrosine-15 (Y-15) Since Cdc25 is too large to diffuse through the nuclear on Cdc2 (Gould and Nurse 1989). Inhibitory phospore complex (NPC), the periodic accumulation of phorylation of Cdc2 Y-15 is carried out by the Wee1 and Cdc25 in the nucleus must be an active process. Nuclear Mik1 kinases (Lundgren et al. 1991; McGowan and transport is mediated by a family of importin(karyRussell 1993) while activation is dependent upon Y-15 opherin1)-related molecules consisting of importins dephosphorylation by the Cdc25 and Pyp3 phosphatases and exportins that transport proteins in and out of the (Gautier et al. 1991; Millar et al. 1992). Timing of nucleus, respectively (reviewed in Mattaj and Englmitotic entry and cell size at division is therefore highly meier 1998; Weis 1998). Three major steps involving sensitive to the gene dosage of the primary regulators, importininteractions are required for the nuclear Cdc25 and Wee1 (Russell and Nurse 1986, 1987a). import of proteins. First, proteins destined for the nuBelow the cell size threshold for mitotic entry Cdc2 is cleus contain a NLS that is recognized by importin. held inactive by Y-15 phosphorylation, predominantly NLSs are broadly divided into classical (cNLS) and nonclassical types. The former requires an adapter molecule (importin) to bridge the interaction between the 1Corresponding author: Department of Biology, Rm. 2443, Biosciences cargo protein and importin, and the latter involves Complex, Queen’s University, Kingston, Ontario K7L 3N6, Canada. E-mail: youngpg@biology.queensu. ca direct binding of importinto its cargo protein. ImGenetics 162: 689–703 (October 2002)