Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba
Open Access
- 27 January 1997
- journal article
- Published by Rockefeller University Press in The Journal of cell biology
- Vol. 136 (2), 331-343
- https://doi.org/10.1083/jcb.136.2.331
Abstract
The Arp2/3 complex, first isolated from Acanthamoeba castellani by 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 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 ∼13 × 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 K d of 2.3 μM 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 actinrich regions of Acanthamoeba.Keywords
This publication has 36 references indexed in Scilit:
- The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton.The Journal of cell biology, 1996
- Actin-related protein 1 and cytoplasmic dynein-based motility - what's the connection?Trends in Cell Biology, 1996
- Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba.The Journal of cell biology, 1995
- Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin.The Journal of cell biology, 1994
- New yeast actin-like gene required late in the cell cycleNature, 1992
- Preparation of single molecules and supramolecular complexes for high-resolution metal shadowingJournal of Ultrastructure Research, 1983
- Rotary shadowing of extended molecules dried from glycerolJournal of Ultrastructure Research, 1980
- Trinodular structure of fibrinogenJournal of Molecular Biology, 1979
- Polarity of actin at the leading edge of cultured cellsNature, 1978
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976