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Article
Direct binding of F-actin to ponticulin, an integral plasma membrane glycoprotein
Women’s Health Research Faculty Publications
  • C. P. Chia
  • Anne L. Hitt
  • Elizabeth J. Luna, University of Massachusetts Medical School
UMMS Affiliation
Department of Cell Biology
Date
1-1-1991
Document Type
Article
Subjects
Actins; Binding, Competitive; Carrier Proteins; Cell Fractionation; Cell Membrane; Cell Movement; Chromatography, Affinity; Concanavalin A; Cytoskeleton; Dictyostelium; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Isoelectric Focusing; Membrane Glycoproteins; Microfilament Proteins; Protein Conformation; Radioligand Assay
Abstract
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.
Rights and Permissions
Citation: Cell Motil Cytoskeleton. 1991;18(3):164-79. Link to article on publisher's site
Related Resources
Link to article in PubMed
PubMed ID
2060029
Citation Information
C. P. Chia, Anne L. Hitt and Elizabeth J. Luna. "Direct binding of F-actin to ponticulin, an integral plasma membrane glycoprotein" Vol. 18 Iss. 3 (1991) ISSN: 0886-1544 (Print)
Available at: http://works.bepress.com/lunae/19/