Rho-type GTPases control many cytoskeletal rearrangements, but their regulation remains poorly understood. Here, we show that in S. cerevisiae, activation of the CDK Cdc28-Cln2 at bud emergence triggers relocalization of Cdc24, the GEF for Cdc42, from the nucleus to the polarization site, where it is stably maintained by binding to the adaptor Bem1. Locally activated Cdc42 then polarizes the cytoskeleton in a manner dependent on its effectors Bni1 and the PAK-like kinase Cla4. In addition, Cla4 induces phosphorylation of Cdc24, leading to its dissociation from Bem1 at bud tips, thereby ending polarized bud growth in vivo. Our results thus suggest a dynamic temporal and spatial regulation of the Cdc42 module: Cdc28-Cln triggers actin polarization by activating Cdc42, which in turn restricts its own activation via a negative feedback loop acting on its GEF Cdc24.

SCF-type (SCF: Skp1-Cullin-F-box protein complex) E3 ligases regulate ubiquitin-dependent degradation of many cell cycle regulators, mainly at the G1/S transition. Here, we show that SCF(Grr1) functions during cytokinesis by degrading the PCH protein Hof1. While Hof1 is required early in mitosis to assemble a functional actomyosin ring, it is specifically degraded late in mitosis and remains unstable during the entire G1 phase of the cell cycle. Degradation of Hof1 depends on its PEST motif and a functional 26S proteasome. Interestingly, degradation of Hof1 is independent of APC(Cdh1), but instead requires the SCF(Grr1) E3 ligase. Grr1 is recruited to the mother-bud neck region after activation of the mitotic-exit network, and interacts with Hof1 in a PEST motif-dependent manner. Our results also show that downregulation of Hof1 at the end of mitosis is necessary to allow efficient contraction of the actomyosin ring and cell separation during cytokinesis. SCF(Grr1)-mediated degradation of Hof1 may thus represent a novel mechanism to couple exit from mitosis with initiation of cytokinesis.

Site-specific activation of the Rho-type GTPase Cdc42p by its guanine-nucleotide exchange factor (GEF) Cdc24p is critical for the establishment of cell polarity. Here we show that binding of Cdc24p to the small GTPase Rsr1p/Bud1p is required for its recruitment to the incipient bud site. Rsr1p/Bud1p binds to the CH-domain of Cdc24p, which is essential for its function in vivo. We have identified a cdc24-mutant allele, which is specifically defective for bud-site selection. Our results suggest that Cdc24p is auto-inhibited by an intramolecular interaction with its carboxy-terminal PB1-domain. Rsr1p/Bud1p appears to activate the GEF activity of Cdc24p in vivo, possibly by triggering a conformational change that dissociates the PB1-domain from its intramolecular binding site. Genetic experiments suggest that Bem1p functions as a positive regulator of Cdc24p by binding to the PB1-domain of Cdc24p, thereby preventing its re-binding to the intramolecular inhibitory site. Taken together, our results support a two-step molecular mechanism for the site-specific activation of Cdc24p, which involves Rsr1p/Bud1p and the adaptor protein Bem1p.

Receptor-mediated cell polarization via heterotrimeric G-proteins induces cytoskeletal rearrangements in a variety of organisms. In yeast, Far1p is required for orienting cell growth towards the mating partner by linking activated Gβγ to the guanine-nucleotide exchange factor (GEF) Cdc24p, which activates the Rho-type GTPase Cdc42p. Here we investigated the role of Far1p in the regulation of Cdc24p in vivo. Using time-lapse microscopy of mating cells and artificial membrane targeting of Far1p, we show that Far1p is necessary and sufficient to recruit Cdc24p to the plasma membrane. Wild-type Far1p contains a PH-like domain, which is required for its membrane localization in vivo. Interestingly, expression of membrane-targeted Far1p causes toxicity, most likely by activating Cdc42p uniformly at the cell cortex. The ability of full-length Far1p to function as an activator of Cdc24p in vivo requires its interaction with Cdc24p and Gβγ. Our results imply that Gβγ not only targets Far1p to the correct site but may also trigger a conformational change in Far1p that is required for its ability to activate Cdc24p in vivo

The Caulobacter crescentus chromosomal clp locus contains the genes encoding the components of ClpXP, a multisubunit protease required for cell cycle progression in this organism. Here, we report the identification and characterization of cicA, a gene located between the clpX and clpP genes on the Caulobacter chromosome. cicA is a novel morphogene in C. crescentus and, like clpX and clpP, is essential for growth. A conditional cicA mutant stopped growth, but retained viability under restrictive conditions. In contrast, an increased concentration of CicA led to an immediate loss of the normal rod shape, an almost 10‐fold increase of the cell's volume and a cell division block. In parallel with this drastic morphological change, cells rapidly lost viability. Primary sequence analysis suggested that the cicA gene encodes a member of a large superfamily of phosphotransferases, that include phosphoserine phosphatases, the ATPase domain of P‐type ATPases and receiver domains of response regulators. Four conserved motifs of this protein family that have been implicated in the catalysis of phosphotransfer reactions were investigated by site‐directed mutagenesis and were found to be critical for in vivo function of CicA. Based on our observations, we postulate that CicA is involved in essential phosphotransferase reactions in Caulobacter and that increased activity of CicA has a deleterious effect on cell wall biosynthesis, morphogenesis and cell division.

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