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

Adam C Martin·Xiao-Ping Xu·Isabelle Rouiller·Marko Kaksonen·Yidi Sun·Lisa Belmont·Niels Volkmann·Dorit Hanein·Matthew Welch·David G Drubin
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Abstract

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.

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Actin-Related Protein 2Actin-Related Protein 3ActinsAdenosine TriphosphateBinding SitesCarrier ProteinsCytoskeletal ProteinsCytoskeletonEndocytosisGreen Fluorescent ProteinsImage Processing, Computer-AssistedIsoquinolinesMicrofilament ProteinsMicroscopy, ElectronMicroscopy, FluorescenceModels, BiologicalModels, MolecularMutationNucleotidesPhenotypePolymersProtein ConformationSaccharomyces cerevisiaeSaccharomyces cerevisiae ProteinsSuppression, GeneticWiskott-Aldrich Syndrome ProteinJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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Adam C Martin, Xiao-Ping Xu, Isabelle Rouiller, Marko Kaksonen, Yidi Sun, Lisa Belmont, Niels Volkmann, Dorit Hanein, Matthew Welch, David G Drubin. 2005-01-17. Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function.. https://doi.org/10.1083/jcb.200408177

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