Structure, subunit topology, and actin-binding activity of the Arp2/3 complex from Acanthamoeba.
The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actin-related proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of approximately 13 x 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 microM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actin-rich regions of Acanthamoeba.
Mullins
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Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. dmullins@welchlink.welch.jhu.edu
Stafford
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Pollard
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D000048:Acanthamoeba / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003285:Contractile Proteins; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D005455:Fluorescent Antibody Technique; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008970:Molecular Weight; D051304:Profilins; D015800:Protozoan Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D014461:Ultracentrifugation
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D003285:Contractile Proteins; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D051304:Profilins; D015800:Protozoan Proteins