Yeast cells can initiate bud formation at the G1/S transition in a cue-independent manner. Here, we investigate the dynamic nature of the polar cap and the regulation of the GTPase Cdc42 in the establishment of cell polarity. Using analysis of fluorescence recovery after photobleaching, we found that Cdc42 exchanged rapidly between the polar caps and cytosol and that this rapid exchange required its GTPase cycle. A previously proposed positive feedback loop involving actomyosin-based transport of the Cdc42 GTPase is required for the generation of robust cell polarity during bud formation in yeast. Inhibition of actin-based transport resulted in unstable Cdc42 polar caps. Unstable polarity was also observed in mutants lacking Bem1, a protein previously implicated in a feedback loop for Cdc42 activation through a signaling pathway. When Bem1 and actin were both inhibited, polarization completely failed. These results suggest that cell polarity is established through coupling of transport and signaling pathways and maintained actively by balance of flux.

BackgroundCell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process.Methodology/Principal FindingsHere, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology.Conclusions/SignificanceWe conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization.

In this study, we investigated on a systems level how complex protein interactions underlying cell polarity in yeast determine the dynamic association of proteins with the polar cortical domain (PCD) where they localize and perform morphogenetic functions. We constructed a network of physical interactions among >100 proteins localized to the PCD. This network was further divided into five robust modules correlating with distinct subprocesses associated with cell polarity. Based on this reconstructed network, we proposed a simple model that approximates a PCD protein's molecular residence time as the sum of the characteristic time constants of the functional modules with which it interacts, weighted by the number of edges forming these interactions. Regression analyses showed excellent fitting of the model with experimentally measured residence times for a large subset of the PCD proteins. The model is able to predict residence times using small training sets. Our analysis also revealed a scaffold protein that imposes a local constraint of dynamics for certain interacting proteins.budding yeast | modeling | network modularity T he study of many complex biological systems has reached a stage where much is known about the molecular components and their functional capacity and interactions. A challenge at hand is how to integrate this wealth of information to explain complex behaviors at the systems level. Cell polarity in the budding yeast Saccharomyces cerevisiae represents one such example (1-4). Functional analyses of the proteins involved have uncovered several subfunctions in the establishment and maintenance of cell polarity, including GTPase signaling, actomyosinbased transport, exocytic deposition of membrane components and cell wall materials, and endocytic recycling (1-4). Spatiotemporal coordination of these subfunctions leads to the morphogenesis of a bud required for cell division, or a shmoo-like projection required for mating.Existing data suggest that the polar cortical domain (PCD) is a dynamic assembly of loosely interacting components (5-8). Hence, "protein dynamics" in this study refers to the cycle of association and dissociation of polarity proteins with the PCD. Detailed analysis of Cdc42, a key regulator of cell polarity, has shown that the stability and shape of the PCD require balanced molecular flux and appropriately specified parameters governing the rates of Cdc42 dynamics (6, 9). A more challenging question is how a system composed of hundreds of different protein molecules associating and dissociating at different rates could function coherently and bring about precise morphogenetic outcomes. A basic assumption that we make to investigate this problem on a systems level is that the dynamics of the peripherally associated PCD proteins are governed by complex interactions.In this study, we first constructed a modularized network of physical interactions among all the known proteins localizing to and participating in the functions of the PCD. We then used an inverse fluorescence recovery...

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