In vitro and in vivo analyses of mutations that alter the target-binding of Abp1 SH3 domains.
Jennifer Haynes (1), Bianca Garcia (2), Arianna Rath (2), Alan Davidson (2), Brenda Andrews (1)
(1) Molecular and Medical Genetics, University of Toronto, 1 King's College Cir, Toronto, ON, M5S 1A8, CANADA;
(2) Department of Biochemistry, University of Toronto
Actin-binding protein 1 (Abp1) is one of numerous proteins involved in the actin cytoskeleton regulation and endocytosis that colocalize to cortical actin patches. Abp1 contains a C-terminal Src-homology 3 (SH3) domain, which is required for mediating multiple protein-protein interactions with other cortical actin patch proteins. We are investigating Abp1's role as a regulator of the actin cytoskeleton, specifically focusing on how it exerts its function through SH3-mediated protein-protein interactions. Using tryptophan fluorescence spectroscopy, we determined the affinity of the Abp1 SH3 domain for proline-rich peptide sequences derived from target proteins Srv2, Ark1 and Prk1. We have identified SH3 point mutants that affect the binding for some or for all of the target peptides. Our goal is to determine the relationship between in vitro binding activity and in vivo function. Using purified Abp1 SH3 domains, we show that SH3 specificity mutants have different binding affinities for target proteins in yeast extracts. Consistent with our in vitro binding studies, we found that Abp1 SH3-dependent localization of Ark1-GFP to cortical patches is differentially affected in Abp1 SH3 specificity mutants. We also show that an SH3 point mutant that has essentially no in vitro binding activity is still able to provide the essential function of the Abp1 SH3 domain in a subset of genetic backgrounds in which it is required.
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