Yeast Rgd3 is a phospho-regulated F-BAR-containing RhoGAP involved in the regulation of Rho3 distribution and cell morphology

Abstract

Polarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4dependent transport of secretory vesicles to sites of polarized growth. Over-expression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 overexpression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP2-enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho-state and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth.