Regulation of the Rho GTPase Cdc42 during cell polarization in budding yeast

Cell polarization is essential for processes such as cell migration and asymmetric cell division. Studies in many organisms have led to the elucidation of common elements in cell polarization. One element is the conserved Rho GTPase, Cdc42, which regulates cell polarity in most eukaryotic cells. This dissertation describes two studies that characterize mechanisms regulating Cdc42 and its guanine nucleotide exchange factor Cdc24 during polarization in budding yeast. Previous studies have postulated the existence of two feedback loops in yeast polarization networks. We characterized their role during bud formation. Using fluorescence microscopy, we show that disruption of either a feedback loop mediated by acto-myosin transport or another feedback loop mediated by the adapter protein Bem1 results in unstable polarization of Cdc42. When both feedback loops were disrupted, polarization of Cdc42 is abolished. Using photobleaching experiments, we show that many proteins are dynamically maintained at the polarized site. Together, these findings suggest that the polarized state is dynamic, and that robustness is conferred by two feedback loops working in parallel. The mechanistic details of Cdc24 activation at the onset of polarity are not clear. We asked whether multisite phosphorylation of Cdc24 plays a role in regulating Cdc24. We show through mass spectrometry analysis that Cdc24 may be phosphorylated on 35 sites. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that mutation of cyclin-dependent kinase and p21-activated kinase consensus sites does not confer any detectable growth or morphological defects. We do, however, observe a change in mobility shift of mutant Cdc24 proteins on SDS-PAGE. We conclude that lack of phosphorylation on these consensus sites has no overt functional consequence. A high level of redundancy in Cdc24 regulation is suggested by both the large number of phosphorylation sites, as well as inability to further perturb the phosphorylation mutant by introducing mutations of other polarity genes. Our studies have revealed a common theme of robustness and dynamicity in cell polarity processes. Rather than being hierarchical signaling pathways, polarity networks may instead be complicated and refined circuits, adapted over long periods of evolution to promote singular, efficient, and robust cell polarity.