Detecting Actin Disorganization

Abstract

In the budding yeast Saccharomyces cerevisiae , defects in bud formation activate a morphogenesis checkpoint pathway that delays progression of the cell cycle through G 2 . Marquitz et al. tested yeast carrying individual mutations or deletions in all known genes involved in bud formation for defective activation of the morphogenesis checkpoint. Only bem1 and bem2 showed progression through the cell cycle and the formation of binucleate cells in the presence of the actin-disrupting agent Latrunculin-B. The bem1 mutants had multinucleated cells even in the absence of Latrunculin-B and thus were not a candidate for a checkpoint defect. Bem2p is a Rho and Cdc42 guanosine triphosphatase-activating protein (GAP). However, point mutations in Bem2p that compromised GAP activity did not exhibit defective checkpoint signaling. The checkpoint signaling activity appeared to be independent of the GAP activity and was localized to a hydrophobic stretch in the NH 2 -terminus of the protein. Progression through G 2 requires the dephosphorylation of the cyclin-dependent kinase Cdc28, and the balance of phosphorylated Cdc28 is maintained through the actions of the kinase Swe1p (which inhibits progression) and the phosphatase Mih1p (which stimulates progression). In the bem2 mutant cells the kinase Swe1p was stabilized and the Mpk1p kinase, which is thought to decrease the activity of the stimulating phosphatase Mih1p, was activated upon exposure to Latrunculin-B. Deletion of MIH did partially rescue the checkpoint defect in the bem2 mutants, suggesting that Mih1p may be involved in the Bem2p pathway. Finally, the levels of phosphorylated Cdc28p were elevated in bem2p mutant yeast and did not increase in response to Latrunculin-B, and thus the cells appeared unable to shift the balance of phosphorylated and unphosphorylated Cdc28p in response to disruption of the bud formation. A. R. Marquitz, J. C. Harrison, I. Bose, T. R. Zyla, J. N. McMillan, D. J. Lew, The Rho-GAP Bem2p plays a GAP-independent role in the morphogenesis checkpoint. EMBO J. 21 , 4012-4025 (2002). [Abstract] [Full Text]