Compartmentalization and actin binding properties of ABP‐50: The elongation factor‐1 alpha of Dictyostelium

Open Access
Publisher Website
Google Scholar
Abstract
ABP‐50 is the elongation factor‐1 alpha (EF‐1 alpha) of Dictyostelium discoideum (Yang et al.: Nature 347:494–496, 1990). ABP‐50 is also an actin filament binding and bundling protein (Demma et al.: J. Biol. Chem. 265:2286–2291, 1990). In the present study we have investigated the compartmentalization of ABP‐50 in both resting and stimulated cells. Immunofluorescence microscopy shows that in addition to being colocalized with F‐actin in surface extensions in unstimulated cells, ABP‐50 exhibits a diffuse distribution throughout the cytosol. Upon addition of cAMP, a chemoattractant, ABP‐50 becomes localized in the filopodia that are extended as a response to stimulation. Quantification of ABP‐50 in Triton‐insoluble and‐soluble fractions of resting cells indicates that 10% of the total ABP‐50 is recovered in the Triton cytoskeleton, while the remainder is in the soluble cytosolic fraction. Stimulation with cAMP increases the incorporation of ABP‐50 into the Triton cytoskeleton. The peak of incorporation of ABP‐50 at 90 sec is concomitant with filopod extension. Immunoprecipitation of the cytosolic ABP‐50 from unstimulated cells using affinity‐purified polyclonal anti ABP‐50 results in the coprecipitation of non‐filamentous actin with ABP‐50. Purified ABP‐50 binds to G‐actin with a Kd of approximately 0.09 μM. The interaction between ABP‐50 and G‐actin is inhibited by GTP but not by GDP, while the bundling of F‐actin by ABP‐50 is unaffected by guanine nucleotides. We conclude that a significant amount of ABP‐50 is bound to either G‐ or F‐actin in vivo and that the interaction between ABP‐50 and F‐actin in the cytoskeleton is regulated by cheniotactic stimulation.