Ponticulin, a developmentally-regulated plasma membrane glycoprotein, mediates actin binding and nucleation.
Ponticulin is a 17,000-dalton transmembrane glycoprotein that is involved in the binding and nucleation of actin filaments by Dictyostelium discoideum plasma membranes. The major actin-binding protein isolated from these membranes by F-actin affinity chromatography, ponticulin also binds F-actin on blot overlays. The actin-binding activity of ponticulin in vitro is identical to that observed for purified plasma membranes: it resists extraction with 0.1 N NaOH, is sensitive to high salt concentrations, and is destroyed by heat, proteolysis, and thiol reduction and alkylation. A cytoplasmic domain of ponticulin mediates binding to actin because univalent antibody fragments directed against the cytoplasmic surface of this protein inhibit 96% of the actin-membrane binding in sedimentation assays. Antibody specific for ponticulin removes both ponticulin and the ability to reconstitute actin nucleation activity from detergent extracts of solubilized plasma membranes. Levels of plasma membrane ponticulin increase 2- to 3-fold during aggregation streaming, when cells adhere to each other and are highly motile. Although present throughout the plasma membrane, ponticulin is preferentially localized to some actin-rich membrane structures, including sites of cell-cell adhesion and arched regions of the plasma membrane reminiscent of the early stages of pseudopod formation. Ponticulin also is present but not obviously enriched at phagocytic cups of log-phase amebae. These results indicate that ponticulin may function in vivo to attach and nucleate actin filaments at the cytoplasmic surface of the plasma membrane. A 17,000-dalton analogue of ponticulin has been identified in human polymorphonuclear leukocyte plasma membranes by immunoblotting and immunofluorescence microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
Luna
E J
EJ
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Wuestehube
L J
LJ
Chia
C P
CP
Shariff
A
A
Hitt
A L
AL
Ingalls
H M
HM
D000199:Actins / Q000378:metabolism*; D002352:Carrier Proteins / Q000502:physiology*; D004023:Dictyostelium / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D008562:Membrane Glycoproteins / Q000502:physiology*; D008840:Microfilament Proteins / Q000502:physiology*
D000199:Actins; D002352:Carrier Proteins; C034037:F-actin-binding proteins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins