Cell polarity is critical for the form and function of many cell types. During polarity establishment, cells define a cortical “front” that behaves differently from the rest of the cortex. The front accumulates high levels of the active form of a polarity-determining Rho-family GTPase (Cdc42, Rac, or Rop), which then orients cytoskeletal elements through various effectors to generate the polarized morphology appropriate to the particular cell type [1, 2]. GTPase accumulation is thought to involve positive feedback, such that active GTPase promotes further delivery and/or activation of more GTPase in its vicinity [3]. Recent studies suggest that once a front forms, the concentration of polarity factors at the front can increase and decrease periodically, first clustering the factors at the cortex and then dispersing them back to the cytoplasm [4–7]. Such oscillatory behavior implies the presence of negative feedback in the polarity circuit [8], but the mechanism of negative feedback was not known. Here we show that, in the budding yeast Saccharomyces cerevisiae, the catalytic activity of the Cdc42-directed GEF is inhibited by Cdc42-stimulated effector kinases, thus providing negative feedback. We further show that replacing the GEF with a phosphosite-mutant GEF abolishes oscillations and leads to the accumulation of excess GTP-Cdc42 and other polarity factors at the front. These findings reveal a mechanism for negative feedback and suggest that the function of negative feedback via GEF inhibition is to buffer the level of Cdc42 at the polarity site.

Establishment of cell polarity in animal and fungal cells involves localization of the conserved Rho-family guanosine triphosphatase, Cdc42, to the cortical region destined to become the “front” of the cell. The high local concentration of active Cdc42 promotes cytoskeletal polarization through various effectors. Cdc42 accumulation at the front is thought to involve positive feedback, and studies in the budding yeast Saccharomyces cerevisiae have suggested distinct positive feedback mechanisms. One class of mechanisms involves localized activation of Cdc42 at the front, whereas another class involves localized delivery of Cdc42 to the front. Here we show that Cdc42 activation must be localized for successful polarity establishment, supporting local activation rather than local delivery as the dominant mechanism in this system.

Regulation of the formin Bni1p by Cdc42p in yeast does not require direct interaction between Bni1p and Cdc42p. The Cdc42p effector Gic2p can bind both Bni1p and GTP-Cdc42p, providing a novel regulatory input.