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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Actin-Related Protein 2
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Actin-Related Protein 3
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Actins / genetics
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Actins / metabolism
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Actins / physiology*
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Adenosine Triphosphate / analogs & derivatives
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Adenosine Triphosphate / metabolism
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Binding Sites / genetics
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Cytoskeletal Proteins / metabolism
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Cytoskeleton / metabolism
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Cytoskeleton / physiology
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Endocytosis / physiology
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Green Fluorescent Proteins / genetics
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Image Processing, Computer-Assisted
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Isoquinolines / metabolism
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Microfilament Proteins / genetics
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Microfilament Proteins / metabolism
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Microscopy, Electron
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Microscopy, Fluorescence
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Models, Biological
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Models, Molecular
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Mutation
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Nucleotides / metabolism*
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Phenotype
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Polymers / metabolism
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Protein Conformation
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae / physiology*
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Saccharomyces cerevisiae / ultrastructure
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
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Saccharomyces cerevisiae Proteins / physiology*
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Suppression, Genetic
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Wiskott-Aldrich Syndrome Protein
Substances
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ABP1 protein, S cerevisiae
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ARP2 protein, S cerevisiae
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ARP3 protein, S cerevisiae
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Actin-Related Protein 2
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Actin-Related Protein 3
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Actins
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Carrier Proteins
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Cytoskeletal Proteins
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Isoquinolines
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LAS17 protein, S cerevisiae
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Microfilament Proteins
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Nucleotides
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Polymers
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SLA1 protein, S cerevisiae
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SLA2 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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Wiskott-Aldrich Syndrome Protein
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Green Fluorescent Proteins
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Adenosine Triphosphate
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lucifer yellow