Compartmentalization and actin binding properties of ABP-50: the elongation factor-1 alpha of Dictyostelium.
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 microM. 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 chemotactic stimulation.
Dharmawardhane
S
S
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
Demma
M
M
Yang
F
F
Condeelis
J
J
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002633:Chemotaxis; D000242:Cyclic AMP / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004023:Dictyostelium / Q000737:chemistry*; D005656:Fungal Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011233:Precipitin Tests; D015800:Protozoan Proteins
C073748:ABP-50 protein, Dictyostelium; D000199:Actins; D005656:Fungal Proteins; D008840:Microfilament Proteins; D010445:Peptide Elongation Factors; D015800:Protozoan Proteins; D000242:Cyclic AMP