Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

doi:10.1083/jcb.136.2.331

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© The Rockefeller University Press, 0021-9525/1997//331 $5.00
The Journal of Cell Biology, Volume 136, Number 2, , 1997 331-343

Article

Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

R. Dyche Mullins^*, Walter F. Stafford, and Thomas D. Pollard^*

^* Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and Boston Biomedical Research Institute, Boston, Massachusetts 02134

The Arp2/3 complex, first isolated from Acanthamoeba castellaniby affinity chromatography on profilin, consists of seven polypeptides;two actinrelated 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 coefficientof 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, indicatingthe purified complex contains one copy each of seven polypeptides.In electron micrographs, the complex has a bilobed or horseshoeshape with outer dimensions of $~$ 13 x 10 nm, and mathematicalmodels of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a batteryof cross-linkers of different spacer arm lengths and chemicalreactivities identify the following nearest neighbors withinthe complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3and either p18 or p19; and p19 and p14. By fluorescent antibodystaining with anti-p40 and -p35, the complex is concentratedin the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular tothe leading edge. Purified Arp2/3 complex binds actin filamentswith a K_d of 2.3 µM and a stoichiometry of approximatelyone complex molecule per actin monomer. In electron micrographsof negatively stained samples, Arp2/3 complex decorates thesides 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 accountsfor the localization of complex subunits to actinrich regionsof Acanthamoeba.

Abbreviations used in this paper: Arps, actin-related proteins; BMH, bismaleimidohexane; DSG, disuccinimidyl glutarate; DSP, dithiobis(succinimidyl propionate); DST, disuccinimidyl tartarate; EDC, 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide hydrochloride; NHS, N-hydroxysuccinimide.

Address all correspondence to R. Dyche Mullins, Department ofCell Biology and Anatomy, Johns Hopkins University School ofMedicine, 725 N. Wolfe St., Baltimore, Maryland 21205. Tel.:(410) 955-5672. Fax: (410) 955-4129. E-mail: dmullins{at}welchlink.welch.jhu.edu

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