The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates events associated with the M to G1 transition. We investigated the function of two S. cerevisiae proteins related to the MEN proteins Mob1p and Dbf2p kinase. Previous work indicates that cells lacking the Dbf2p-related protein Cbk1p fail to sustain polarized growth during early bud morphogenesis and mating projection formation (Bidlingmaier, S., E.L. Weiss, C. Seidel, D.G. Drubin, and M. Snyder. 2001. Mol. Cell. Biol. 21:2449–2462). Cbk1p is also required for Ace2p-dependent transcription of genes involved in mother/daughter separation after cytokinesis. Here we show that the Mob1p-related protein Mob2p physically associates with Cbk1p kinase throughout the cell cycle and is required for full Cbk1p kinase activity, which is periodically activated during polarized growth and mitosis. Both Mob2p and Cbk1p localize interdependently to the bud cortex during polarized growth and to the bud neck and daughter cell nucleus during late mitosis. We found that Ace2p is restricted to daughter cell nuclei via a novel mechanism requiring Mob2p, Cbk1p, and a functional nuclear export pathway. Furthermore, nuclear localization of Mob2p and Ace2p does not occur in mob1–77 or cdc14–1 mutants, which are defective in MEN signaling, even when cell cycle arrest is bypassed. Collectively, these data indicate that Mob2p–Cbk1p functions to (a) maintain polarized cell growth, (b) prevent the nuclear export of Ace2p from the daughter cell nucleus after mitotic exit, and (c) coordinate Ace2p-dependent transcription with MEN activation. These findings may implicate related proteins in linking the regulation of cell morphology and cell cycle transitions with cell fate determination and development.

In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cellspecific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p-Cbk1p, Cbk1p-Kic1p, Kic1p-Tao3p, and Kic1p-Hym1p interactions, in addition to the previously documented Cbk1p-Mob2p and Cbk1p-Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p-and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p-Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p-Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes.

The genetics of the mating-type (MAT) locus have been studied extensively in Saccharomyces cerevisiae, but relatively little is known about how this complex system evolved. We compared the organization of MAT and mating-type-like (MTL) loci in nine species spanning the hemiascomycete phylogenetic tree. We inferred that the system evolved in a two-step process in which silent HMR͞HML cassettes appeared, followed by acquisition of the Ho endonuclease from a mobile genetic element. Ho-mediated switching between an active MAT locus and silent cassettes exists only in the Saccharomyces sensu stricto group and their closest relatives: Candida glabrata, Kluyveromyces delphensis, and Saccharomyces castellii. We identified C. glabrata MTL1 as the ortholog of the MAT locus of K. delphensis and show that switching between C. glabrata MTL1a and MTL1␣ genotypes occurs in vivo. The more distantly related species Kluyveromyces lactis has silent cassettes but switches mating type without the aid of Ho endonuclease. Very distantly related species such as Candida albicans and Yarrowia lipolytica do not have silent cassettes. In Pichia angusta, a homothallic species, we found MAT␣2, MAT␣1, and MATa1 genes adjacent to each other on the same chromosome. Although some continuity in the chromosomal location of the MAT locus can be traced throughout hemiascomycete evolution and even to Neurospora, the gene content of the locus has changed with the loss of an HMG domain gene (MATa2) from the MATa idiomorph shortly after HO was recruited.M ating in Saccharomyces cerevisiae is a choreographed fusion of two haploid cells with opposite mating types to form a diploid zygote. The mating type of a haploid cell is determined by its genotype at the mating-type (MAT) locus on chromosome III. The two variants of the MAT locus, MAT␣ and MATa, are referred to as idiomorphs rather than alleles because they differ in sequence, size, and gene content (1). Characterization of homothallic and heterothallic strains of S. cerevisiae by Winge and Lindegren (2) led to the discovery of genetic loci controlling homothallism and ultimately to the cassette model of mating-type switching proposed by Herskowitz and colleagues (3). The cassette model, which has been thoroughly verified by experimentation (4, 5), states that a haploid cell can switch genotype at the MAT locus (from MATa to MAT␣ or vice versa) by a gene-conversion process that involves replacing the genetic information at MAT with information copied from one of two silent cassette loci, HML␣ or HMRa. The gene conversion is initiated at a double-stranded DNA break made by the Ho endonuclease, encoded by the HO gene on chromosome IV, which cuts at a site that marks the boundary between the Y sequences unique to the MATa or MAT␣ idiomorphs and the shared Z sequence flanking them. HO is expressed only in cells that have budded once, which means that only mother cells switch mating type and neighboring cells in a colony can mate (4). Hence, most natural isolates of S. cerevisiae are diploid and phenotypic...

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G 1 by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G 1 transition to control cyclin-CDK inactivation and cytokinesis.During the transition from mitosis to G 1 , cytokinesis, disassembly of the mitotic spindle, chromatin decondensation, and DNA licensing must be precisely coordinated to ensure the genomic stability and viability of the cellular progeny (22,29,31,53,67). A major signal that controls these events is the degradation of mitotic cyclins and the inactivation of cyclindependent kinase (CDK) in late mitosis (52, 68). In Saccharomyces cerevisiae, mitotic cyclin degradation and CDK inactivation are regulated by a group of genes that constitute the mitotic exit network (MEN) (45,47). MEN genes encode four protein kinases (Cdc5p, Cdc15p, Dbf2p, and Dbf20p), Cdc14p phosphatase, a GTP binding protein (Tem1p), a GTP exchange factor (Lte1p), and Mob1p, which binds Dbf2p and Dbf20p (35,36,44,57,58,73,75). At the restrictive temperature, conditional alleles of the MEN genes cause cells to arrest in late mitosis with high levels of mitotic cyclin (33,48,58,66,69). The mitotic arrest of several MEN mutants can be suppressed by overexpression of CDK inhibitor SIC1 (18, 33), indicating that CDK inactivation is the major function of the MEN pathway. Indeed, a pivotal step in cyclin and CDK inactivation is mediated by the Cdc14p phosphatase, which is sequestered in the nucleolus during most of the cell cycle until it is released at the end of mitosis (3,60,72). Release of Cdc14p from the nuc...

In the absence of Cbk1 phosphorylation Ssd1-associated mRNAs are redirected from sites of polarized cell growth to stress granules and P-bodies.

Saccharomyces cerevisiae RAM is a conserved signaling network that regulates maintenance of polarized growth and daughter-cell-specific transcription, the latter of which is critical for septum degradation. Consequently, cells defective in RAM function (designated ramD) are round in morphology, form feeble mating projections, and fail to separate following cytokinesis. It was recently demonstrated that RAM genes are essential in strains containing functional SSD1 (SSD1-v), which encodes a protein of unknown function that binds the RAM Cbk1p kinase. Here we investigated the essential function of RAM in SSD1-v strains and identified two functional groups of dosage suppressors for ramD lethality. We establish that all ramD mutants exhibit cell integrity defects and cell lysis. All dosage suppressors rescue the lysis but not the cell polarity or cell separation defects of ramD cells. One class of dosage suppressors is composed of genes encoding cell wall proteins, indicating that alterations in cell wall structure can rescue the cell lysis in ramD cells. Another class of ramD dosage suppressors is composed of ZRG8 and SRL1, which encode two unrelated proteins of unknown function. We establish that ZRG8 and SRL1 share similar genetic interactions and phenotypes. Significantly, Zrg8p coprecipitates with Ssd1p, localizes similarly to RAM proteins, and is dependent on RAM for localization. Collectively, these data indicate that RAM and Ssd1p function cooperatively to control cell integrity and suggest that Zrg8p and Srl1p function as nonessential inhibitors of Ssd1p.

Saccharomyces cerevisiae Cbk1 is a LATS/Ndr protein kinase and a downstream component of the regulation of Ace2 and morphogenesis (RAM) signaling network. Cbk1 and the RAM network are required for cellular morphogenesis, cell separation, and maintenance of cell integrity. Here, we examine the phenotypes of conditional cbk1 mutants to determine the essential function of Cbk1. Cbk1 inhibition severely disrupts growth and protein secretion, and triggers the Swe1-dependent morphogenesis checkpoint. Cbk1 inhibition also delays the polarity establishment of the exocytosis regulators Rab-GTPase Sec4 and its exchange factor Sec2, but it does not interfere with actin polarity establishment. Cbk1 binds to and phosphorylates Sec2, suggesting that it regulates Sec4-dependent exocytosis. Intriguingly, Cbk1 inhibition causes a >30% decrease in post-Golgi vesicle accumulation in late secretion mutants, indicating that Cbk1 also functions upstream of Sec2-Sec4, perhaps at the level of the Golgi. In agreement, conditional cbk1 mutants mislocalize the cis-Golgi mannosyltransferase Och1, are hypersensitive to the aminoglycoside hygromycin B, and exhibit diminished invertase and Sim1 glycosylation. Significantly, the conditional lethality and hygromycin B sensitivity of cbk1 mutants are suppressed by moderate overexpression of several Golgi mannosyltransferases. These data suggest that an important function for Cbk1 and the RAM signaling network is to regulate growth and secretion via Golgi and Sec2/Sec4-dependent processes. INTRODUCTIONCell polarity is a common cellular feature among eukaryotic cells that is characterized by asymmetry in cell shape, protein distribution, and cellular function (Nelson, 2003;Pruyne et al., 2004). The establishment and maintenance of polarized growth are critical for the development and function of specialized cells, tissues, and organs. Polarized growth is particularly important for the development and function of neuronal cells, which can reach several meters in length, and for epithelial cells, which regulate homeostasis and maintain physical barriers between biological compartments (Arimura and Kaibuchi, 2005;Dow and Humbert, 2007;Tang, 2008). Dynamic changes in cell polarity are essential for cell migration during cell and tissue development. Polarized growth and morphogenesis are also important for yeast and filamentous fungi development (Harris, 2006).Saccharomyces cerevisiae is an excellent model organism for studying conserved mechanisms of polarized growth and cell division. Saccharomyces cerevisiae cells undergo polarized growth during cell division, mating, and pseudohyphae formation (Pruyne et al., 2004). Many of the evolutionarily conserved elements of cell growth and proliferation were first identified and characterized in yeast (Pruyne et al., 2004). Yeast polarity establishment involves the recruitment and activation of Cdc42 GTPase to a cortical landmark. Cdc42 regulates several polarity processes, including septin assembly and actin cytoskeleton polarization (Park and Bi, 2007). Po...

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=1798 KEY WORDSCdc14, Crm1, nuclear export signal, Ran, mitotic exit network, cytokinesis, nucleocytoplasmic shuttling Report Crm1-Mediated Nuclear Export of Cdc14 is Required for the Completion of Cytokinesis in Budding Yeast ABSTRACTThe mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In contrast, less is known about the mechanism by which Cdc14p is released from the nucleus to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.