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Binding And Assembly Of Actin Filaments By Plasma Membranes From Dictyostelium Discoideum, M. A. Schwartz, Elizabeth J. Luna
Binding And Assembly Of Actin Filaments By Plasma Membranes From Dictyostelium Discoideum, M. A. Schwartz, Elizabeth J. Luna
Elizabeth J. Luna
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 …
Domain Analysis Of Supervillin, An F-Actin Bundling Plasma Membrane Protein With Functional Nuclear Localization Signals, J. D. Wulfkuhle, I. E. Donina, N. H. Stark, Robert K. Pope, Kersi N. Pestonjamasp, M. L. Niswonger, Elizabeth J. Luna
Domain Analysis Of Supervillin, An F-Actin Bundling Plasma Membrane Protein With Functional Nuclear Localization Signals, J. D. Wulfkuhle, I. E. Donina, N. H. Stark, Robert K. Pope, Kersi N. Pestonjamasp, M. L. Niswonger, Elizabeth J. Luna
Elizabeth J. Luna
A growing number of actin-associated membrane proteins have been implicated in motile processes, adhesive interactions, and signal transduction to the cell nucleus. We report here that supervillin, an F-actin binding protein originally isolated from bovine neutrophil plasma membranes, contains functional nuclear targeting signals and localizes at or near vinculin-containing focal adhesion plaques in COS7-2 and CV1 cells. Overexpression of full-length supervillin in these cells disrupts the integrity of focal adhesion plaques and results in increased levels of F-actin and vinculin. Localization studies of chimeric proteins containing supervillin sequences fused with the enhanced green fluorescent protein indicate that: (1) the amino …
F-Actin Binds To The Cytoplasmic Surface Of Ponticulin, A 17-Kd Integral Glycoprotein From Dictyostelium Discoideum Plasma Membranes, L. J. Wuestehube, Elizabeth J. Luna
F-Actin Binds To The Cytoplasmic Surface Of Ponticulin, A 17-Kd Integral Glycoprotein From Dictyostelium Discoideum Plasma Membranes, L. J. Wuestehube, Elizabeth J. Luna
Elizabeth J. Luna
F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of …