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Cytokines in immune regulation
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Published 18 January 2005. doi:10.1083/jcb.200408177
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 168, Number 2, 315-328
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Article

 Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function

Adam C. Martin1, Xiao-Ping Xu2, Isabelle Rouiller2, Marko Kaksonen1, Yidi Sun1, Lisa Belmont3, Niels Volkmann2, Dorit Hanein2, Matthew Welch1, and David G. Drubin1

1 Barker Hall, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
2 The Burnham Institute, La Jolla, CA 92037
3 Cytokinetics, Inc., South San Francisco, CA 94080

Correspondence to David G. Drubin: drubin{at}socrates.berkeley.edu

Contributions of actin-related proteins (Arp) 2 and 3 nucleotide state to Arp2/3 complex function were tested using nucleotide-binding pocket (NBP) mutants in Saccharomyces cerevisiae. ATP binding by Arp2 and Arp3 was required for full Arp2/3 complex nucleation activity in vitro. Analysis of actin dynamics and endocytosis in mutants demonstrated that nucleotide-bound Arp3 is particularly important for Arp2/3 complex function in vivo. Severity of endocytic defects did not correlate with effects on in vitro nucleation activity, suggesting that a critical Arp2/3 complex function during endocytosis may be structural rather than catalytic. A separate class of Arp2 and Arp3 NBP mutants suppressed phenotypes of mutants defective for actin nucleation. An Arp2 suppressor mutant increased Arp2/3 nucleation activity. Electron microscopy of Arp2/3 complex containing this Arp2 suppressor identified a structural change that also occurs upon Arp2/3 activation by nucleation promoting factors. These data demonstrate the importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for maintenance of cortical actin cytoskeleton cytoarchitecture.

Abbreviations used in this paper: Arp, actin-related protein; FRET, fluorescence resonance energy transfer; LY, Lucifer yellow; NBP, nucleotide-binding pocket; NPF, nucleation promoting factor; WASP, Wiskott-Aldrich syndrome protein.


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