| The polarization of the actin cytoskeleton is a prerequisite for bud formation in the yeast, Saccharomyces cerevisiae. Polarized actin cables are thought to act as ‘railroad tracks’ for the delivery of vesicles to the plasma membrane in the region of bud formation, thereby restricting growth to this site. Mutants like cdc42-1 (Adams et al., 1990) and cdc24-1 (Sloat et al., 1981) which cannot polarize their actin arrest as large, unbudded cells. These cells can, however, go through the cell cycle and become large and multinucleate with time.; The F-actin structures in yeast go through dramatic rearrangements that can be correlated with the different stages of the cell cycle. Progression through the cell cycle is controlled by the master regulatory kinase Cdc28p. Activation of Cdc28p by different cyclins has also been shown to be necessary for the changes in actin polarity to occur with the correct temporal kinetics (Lew and Reed, 1993). However, the targets of Cdc28p that lead to the cell cycle-dependent regulation of actin polarization are unknown.; Cdc42p is a protein that has been implicated in the actin polarity pathway due to its mutant phenotype. It is a small GTPase belonging to the Rho family of proteins (Johnson and Pringle, 1990), other members of which have also been shown to play a role in actin polarization. This led us to investigate whether Cdc42p would be a good candidate for regulation by the cell cycle, ultimately leading to changes in actin polarization in response to Cdc28p/cyclin activity.; We have tried to determine whether Cdc42p guanine nucleotide metabolism was regulated in response to Cdc28 kinase activity. Using total yeast lysates we found that exchange factor (GEF) activity towards Cdc42p increases in response to G1 cyclins, while, GAP (GTPase activating protein) activity increases in response to G2 cyclins. The increase in GEF activity can be attributed mainly to Cdc24p, the Cdc42p exchange factor, that also undergoes a cell cycle-dependent phosphorylation. This phosphorylation is dependent upon the formation of a complex containing the scaffold protein, Bem1p, the small GTPase, Cdc42p and the PAK, Cla4p. We have been unable to identify the GAPs that lead to the increase in GTPase activity in response to G2 cyclins. However, we have been able to demonstrate that Rga1p and Rga2p (two putative GAPs) do have GAP activity towards Cdc42p in vitro. |
