The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.
Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.
Chia
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Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Shariff
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Savage
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Luna
E J
EJ
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004023:Dictyostelium / Q000648:ultrastructure; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D066298:In Vitro Techniques; D008840:Microfilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D014461:Ultracentrifugation
D000199:Actins; D002352:Carrier Proteins; C034037:F-actin-binding proteins; D005656:Fungal Proteins; D008840:Microfilament Proteins