Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton

Abstract

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic 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-localizes 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 independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.

A Complex of Wiskott-Aldrich Syndrome Protein with Mammalian Verprolins Plays an Important Role in Monocyte Chemotaxis

“…Verprolin was originally identified as a yeast protein that is implicated in cell growth, cytoskeletal organization, and endocytosis (22). Three mammalian verprolins have been identified as proteins binding to the WASP N-terminal region (residues 1-170), termed WASP interacting protein (WIP) (23); WIP and CR16 homologous protein (WICH) (24) or WIP-related protein (WIRE) (25); and CR16 (26) (see Fig.…”

“…2, 4, 14, and 16) or a scrambled control (lanes 1, 3, 13, and 15). Cells were cotransfected with siRNAs for WIP and WICH/WIRE (lanes 6,8,10,12,18,20,22,24) or scrambled controls (lanes 5, 7, 9, 11, 17, 19, 21, and 23). A, Total RNA was prepared from the transfectants, and transcription of each gene was analyzed by RT-PCR.…”

Requirement for a Complex of Wiskott-Aldrich Syndrome Protein (WASP) with WASP Interacting Protein in Podosome Formation in Macrophages

“…Two independent groups identified WICH/WIRE simultaneously. Verprolin was originally identified as a yeast protein essential for cell polarization (13). These three mammalian verprolins share the same domain organization that consists of the verprolin homologous (V H ) domain, a proline-rich domain, and a WASP binding (WB) domain (9 -12, 14).…”

Verprolin: A cool set of actin‐binding sites and some very HOT prolines

“…The vrp1D cells are temperature-sensitive, that is, they are viable at 24 8C, but not at elevated temperature (e.g., 37 8C). In contrast, wild-type (VRP1) yeast cells are viable at both temperatures (2)(3)(4). The majority of vrp1D cells remain viable for a few hours and continue to grow in size at 37 8C but are unable to divide (5).…”

“…1). The verprolin-Las17p and verprolin-type I myosin complexes function in assembly of the branched F-actin that comprises actin patches and colocalise with actin patches (3,4,26,27).…”