Binding and assembly of actin filaments by plasma membranes from Dictyostelium discoideum.
The binding of native, 125I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. In the presence of gelsolin, the amount of actin bound at saturation to three different membrane preparations was 80, 120, and 200 micrograms/mg of membrane protein. The respective concentrations of actin at half-saturation were 8, 12, and 18 micrograms/ml. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. In kinetic experiments, actin added as monomers bound to membranes at a rate of 0.6 microgram ml-1 min-1, while pre-polymerized actin bound at a rate of 3.0 micrograms ml-1 min-1. Even in the absence of phalloidin, actin bound to membranes at concentrations well below the normal critical concentration. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. We conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins.
M A
MA
Schwartz
E J
EJ
Luna
D000199:Actins / Q000378:metabolism*; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D003599:Cytoskeleton / Q000378:metabolism*; D004023:Dictyostelium / Q000378:metabolism*; D018260:Gelsolin; D007382:Intermediate Filaments / Q000378:metabolism*; D007700:Kinetics; D008840:Microfilament Proteins / Q000502:physiology; D010590:Phalloidine; D012235:Rhodamines; D013194:Staining and Labeling
D000199:Actins; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D018260:Gelsolin; D008840:Microfilament Proteins; D012235:Rhodamines; D010590:Phalloidine