Direct binding of F-actin to ponticulin, an integral plasma membrane glycoprotein.
We have developed an 125I-labeled F-actin blot overlay assay for the identification of F-actin-binding proteins after transfer to nitrocellulose from SDS-polyacrylamide gels. Two major F-actin-binding proteins from Dictyostelium discoideum, a cytoplasmic 30 kDa protein and a 17 kDa integral membrane protein, and two minor membrane polypeptides of 19 kDa and 15 kDa were detected by this method. Using F-actin affinity and immunoaffinity chromatography, the 17 kDa polypeptide was identified as ponticulin, a previously described actin-binding glycoprotein from D. discoideum plasma membranes (Wuestehube, L.J., and Luna, E.J., [1987]: J. Cell Biol. 105:1741-1751). The binding of F-actin to ponticulin on blots is specific because unlabeled F-actin competes with 125I-labeled F-actin and because G-actin does not bind. Nitrocellulose-bound ponticulin displays binding characteristics similar to those of purified plasma membranes in solution, e.g., F-actin binding is sensitive to high salt and to elevated temperatures. Under optimal conditions, 125-I-labeled F-actin blot overlays are at least as sensitive as are immunoblots with an antibody specific for ponticulin. When blotted onto nitrocellulose after 2-D gel electrophoresis, all isoforms of ponticulin and of the 19 kDa and 15 kDa polypeptides appear to bind F-actin in proportion to their abundance. Thus the actin-binding activies of these proteins do not appear to be regulated by modifications that affect isoelectric point. However, the actin-binding activity of nitrocellulose-bound ponticulin is diminished when the protein is exposed to reducing agents, suggesting an involvement of disulfide bond(s) in ponticulin function. The 125I-labeled F-actin blot overlay assay also may enable us to identify F-actin-binding proteins in other cell types and should provide a convenient method for monitoring the purification of these proteins.
Chia
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Cell Biology Group, Worchester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Hitt
A L
AL
Luna
E J
EJ
D000199:Actins / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008562:Membrane Glycoproteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011869:Radioligand Assay
D000199:Actins; D002352:Carrier Proteins; C034037:F-actin-binding proteins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D003208:Concanavalin A