Arp2/3 ATP hydrolysis-catalysed branch dissociation is critical for endocytic force generation.
The Arp2/3 complex, which is crucial for actin-based motility, nucleates actin filaments and organizes them into y-branched networks. The Arp2 subunit has been shown to hydrolyse ATP, but the functional importance of Arp2/3 ATP hydrolysis is not known. Here, we analysed an Arp2 mutant in Saccharomyces cerevisiae that is defective in ATP hydrolysis. Arp2 ATP hydrolysis and Arp2/3-dependent actin nucleation occur almost simultaneously. However, ATP hydrolysis is not required for nucleation. In addition, Arp2 ATP hydrolysis is not required for the release of a WASP-like activator from y-branches. ATP hydrolysis by Arp2, and possibly Arp3, is essential for efficient y-branch dissociation in vitro. In living cells, both Arp2 and Arp3 ATP-hydrolysis mutants exhibit defects in endocytic internalization and actin-network disassembly. Our results suggest a critical feature of dendritic nucleation in which debranching and subsequent actin-filament remodelling and/or depolymerization are important for endocytic vesicle morphogenesis.
Martin
Adam C
AC
Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
Welch
Matthew D
MD
Drubin
David G
DG
D051377:Actin-Related Protein 2 / Q000235:genetics / Q000378:metabolism*; D051378:Actin-Related Protein 3 / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002384:Catalysis; D004705:Endocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C026742:plastin; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate