ACTIN-BINDING VERPROLIN IS A POLARITY DEVELOPMENT PROTEIN REQUIRED FOR THE MORPHOGENESIS AND FUNCTION OF THE YEAST ACTIN CYTOSKELETON

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytosk eletal organization, endocytosis and mitochondrial protein distributio n and function. We show that verprolin is also required for bipolar bu d-site selection. Previously we reported that additional actin suppres ses the temperature-dependent growth defect caused by a mutation in VR P1. Here we show that additional actin suppresses all known defects ca used by vrp1-1 and conclude that the defects relate to an abnormal cyt oskeleton. Using the two-hybrid system, we show that verprolin binds a ctin. An actin-binding domain maps to the LKKAET hexapeptide located i n the first 70 amino acids. A similar hexapeptide in other acting-bind ing proteins was previously shown to be necessary for actin-binding ac tivity. The entire 70-amino acid motif is conserved in novel higher eu karyotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-local izes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location inde pendent of the actin cytoskeleton. Verprolin is a new member of the ac tin-binding protein family that serves as a polarity development prote in, perhaps by anchoring actin. We speculate that the effects of verpr olin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.