Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function.
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.
Martin
Adam C
AC
Barker Hall, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Xu
Xiao-Ping
XP
Rouiller
Isabelle
I
Kaksonen
Marko
M
Sun
Yidi
Y
Belmont
Lisa
L
Volkmann
Niels
N
Hanein
Dorit
D
Welch
Matthew
M
Drubin
David G
DG
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism / Q000502:physiology; D004705:Endocytosis / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D007091:Image Processing, Computer-Assisted; D007546:Isoquinolines / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D010641:Phenotype; D011108:Polymers / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013489:Suppression, Genetic; D051316:Wiskott-Aldrich Syndrome Protein
C062612:ABP1 protein, S cerevisiae; C477632:ARP2 protein, S cerevisiae; C102245:ARP3 protein, S cerevisiae; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D007546:Isoquinolines; C480361:LAS17 protein, S cerevisiae; D008840:Microfilament Proteins; D009711:Nucleotides; D011108:Polymers; C082265:SLA1 protein, S cerevisiae; C082266:SLA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D051316:Wiskott-Aldrich Syndrome Protein; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; C017475:lucifer yellow