POS-WED-091

Structure-function analysis of yeast Wiskott-Aldrich Syndrome Protein

MA Hahn, AV Trinh, H Mack and AL Munn

School of Medical Science and Molecular Basis of Disease Program, Menzies Health Institute Queensland, Griffith University (Gold Coast campus), Parklands Dr., Southport, Queensland, 4222, Australia

Wiskott-Aldrich Syndrome Protein (WASP) and WASP-Interacting Protein (WIP) are human proteins that form a complex that directs the actin cytoskeletal rearrangements required for cell motility, cell-cell adhesion, endocytosis and cytokinesis. Budding yeast (Saccharomyces cerevisiae) has functional homologues of both WASP (Las17p) and WIP (Vrp1p). Yeast gene knock-out mutants deficient in Vrp1p (vrp1Δ) are viable at 24 °C but not at 37 °C (i.e. temperature-sensitive) and defective in endocytosis and cytokinesis. High copy number of the LAS17 gene (resulting in Las17p over-expression) suppresses the growth defects of vrp1 mutant cells. The aim of this study is to determine the mechanism of this high-copy-number suppression. Multiple fragments of Las17p were expressed under the control of the LAS17 promoter carried on a yeast high-copy-number (2µ) vector in vrp1Δ gene knockout cells (devoid of Vrp1p) and tested for ability to suppress the growth defect. An N-terminal fragment of Las17p (aa 1-150) that binds Vrp1p, a large proline-rich central fragment (aa 151-530) that binds Src Homology 3 (SH3) domains and a C-terminal fragment (aa 531-633) that contains a WASP Homology 2 (WH2) domain that binds actin were individually insufficient for suppression, however a fragment containing both the central region and C-terminal region was sufficient for suppression. Within the central region aa 302-370 are required for suppression and within the C-terminal region the WH2 actin-binding domain and a second sequence of as yet unknown function are required for efficient suppression. Current efforts aim to elucidate the protein interactions responsible for suppression. The authors acknowledge the financial support of ARC Discovery Project grant (DP110100389).