AFL1 is a phosphoinositide phosphate- and actin-binding protein

At14a-Like 1 (AFL1) is highly induced during low water potential stress and remains at high levels during stress acclimation. AFL1, and the closely related At14a, are plant-specific proteins that have limited similarity to mammalian actin- and membrane-associated proteins. Previous research indicated that manipulation of AFL1 expression affects actin cytoskeleton dynamics and endocytic trafficking (as measured by uptake of membrane dye FM4-64). However, it has remained unclear whether this is a direct activity of AFL1 or an indirect effect. We found that AFL1 specifically bound actin filaments as well as the phosphoinositide phosphates (PIPs) phosphoinositide-3-monophosphate [PI(3)P], PI(5)P, and the diphosphate PI(3,5)P2 in co-sedimentation and PIP strip membrane assays, respectively. Interestingly, these binding activities were mediated by the same site within the C-terminal domain of AFL1. Mutation of a single amino acid in the AFL1 C-terminal domain was sufficient to disrupt both actin filament and PIP binding in vitro and to disrupt accumulation of the mutated protein in transgenic plants. We also found that the central hydrophobic region of AFL1 was required for AFL1 co-localization with actin filaments and plasma membrane. Mutation of AFL1 and At14a using genome editing confirmed that loss of these proteins reduced growth during low water potential stress and resulted in less extensive actin filament arrays and disrupted FM4-64 uptake. Together these observations indicate that AFL1 can directly participate in cytoskeleton organization and membrane dynamics via PIP and actin filament binding.