Purification and architecture of the ubiquitous Wave complex.
The Wave proteins are major activators of the Arp2/3 complex. The ubiquitous Wave-2 is required for actin polymerization at the leading edge of migrating cells. Here we purify Wave-2 from HeLa cells. Five proteins, Sra, Nap, Wave-2, Abi, and Hspc, are copurified, indicating that they form a tight complex. These proteins are only present in the complexed form, with the exception of Hspc, which displays a free pool. We reconstitute the Wave-2 complex by cotranslating in vitro the five subunits and use this system together with specific immunoprecipitations to study the molecular architecture of the complex. The complex is organized around a core of Nap and Abi. Sra is a peripheral subunit recruited on the Nap side, whereas the Wave and Hspc subunits are recruited on the Abi side of the core.
Gautreau
Alexis
A
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Ho
Hsin-yi H
HY
Li
Jiaxu
J
Steen
Hanno
H
Gygi
Steven P
SP
Kirschner
Marc W
MW
D000595:Amino Acid Sequence; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry; D051300:Wiskott-Aldrich Syndrome Protein Family
D018076:DNA, Complementary; D008840:Microfilament Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; C120152:WASF2 protein, human; D051300:Wiskott-Aldrich Syndrome Protein Family