LETTERS 826 NATURE CELL BIOLOGY VOLUME 8 | NUMBER 8 | AUGUST 2006 Arp2/3 ATP hydrolysis-catalysed branch dissociation is critical for endocytic force generation Adam C. Martin 1 , Matthew D. Welch 1 and David G. Drubin 1,2 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. Actin-filament assembly generates forces that support processes such as cell motility and endocytosis 1,2 . Cycles of actin assembly and disas- sembly are driven, in part, by the free energy derived from actin ATP hydrolysis. ATP-bound actin polymerizes into filaments and subse- quent ATP hydrolysis and release of inorganic phosphate (P i ) destabi- lizes the filament, promoting depolymerization and recycling of actin subunits. As with actin, ATP binding by the Arp2 and Arp3 subunits of the Arp2/3 complex is critical for the function of this complex 3–6 . However, the function of ATP hydrolysis by the Arp2/3 complex is not clear. Arp2 hydrolyses ATP when a Wiscott-Aldrich syndrome protein (WASP)-family nucleation promoting factor (NPF) activates the Arp2/3 complex and promotes its association with the first actin monomer of the daughter filament 7 . However, depending on the experimental condi- tions, Arp2 ATP hydrolysis has been observed with half-lives ranging from 20 s (similar to the rate of nucleation) 7 to 800 s (similar to the rate of y-branch dissociation) 8 . Furthermore, studies using non-hydrolysable ATP analogues suggested that Arp2/3 ATP hydrolysis was essential for nucleation 3,4 , whereas studies using the ion Cr(III), which is thought to delay Arp2 P i release following hydrolysis, suggested a role for Arp2 P i release during y-branch dissociation or debranching 8 . Although Arp3 has not been observed to hydrolyse ATP, residues predicted to cata- lyse ATP hydrolysis are conserved in Arp3. Importantly, studies using non-hydrolysable ATP analogues and Cr(III) were unable to distinguish whether these molecules affect Arp2, Arp3 or both. In addition, the relevance of Arp2/3 ATP hydrolysis to actin-based force generation in a biological context has not yet been determined. Ideally, Arp2/3 ATP hydrolysis mutants would be used to dissect the in vitro mechanism and in vivo function of Arp2/3 ATP hydrolysis. We previously generated 26 mutants that altered conserved residues in the nucleotide-binding pockets of S. cerevisiae Arp2 and Arp3 (ref. 6). Two Arp2 mutants were hypothesized to decrease ATP hydrolysis. Here, we focus on arp2 H161A as this mutant resulted in a growth defect (see Supplementary Information, Fig. S1a), suggesting impaired Arp2/3 function. The homologous yeast actin histidine (H161) is predicted to activate a water molecule for direct nucleophilic attack on the ATP γ- phosphate 9 . Growth of the corresponding Arp3 mutant, arp3 H161A , was indistinguishable from wild type. However, arp3 H161A was synthetic lethal with arp2 H161A (data not shown), demonstrating that arp3 H161A also affects Arp2/3 complex function. The H161A mutant phenotypes were not due to reduced protein levels (data not shown), altered Arp2/3 complex subunit composition (see Supplementary Information, Fig. S1b) or a defect in ATP binding (see Supplementary Information, Fig. S1c). Analysis of Arp2/3 ATP hydrolysis during nucleation is complicated by the fact that actin, which is typically present at a 100-fold molar excess relative to the Arp2/3 complex, hydrolyses ATP soon after polymeriza- tion 1,2 . Therefore, an experimental strategy must be used to distinguish between actin, Arp2 and Arp3 ATP hydrolysis. We used an approach in which purified Arp2/3 complex containing Arp2 and Arp3 cova- lently bound to γ- 32 P-ATP was used to individually monitor Arp2 and Arp3 γ-phosphate cleavage 7,8 . Yeast Arp2 and Arp3 failed to hydrolyse ATP in the absence of either actin or a WASP-like NPF, Las17p (see Supplementary Information, Fig. S1d). However, the addition of actin and Las17p together resulted in an approximately 50% decrease in the Arp2 32 P-signal over 2 min (Fig. 1a, c). Although a decrease in the Arp3 32 P-signal was not observed, the possibility that crosslinking inactivates 1 16 Barker Hall, Dept. of Molecular and Cell Biology, University of California Berkeley, CA 94720-3202, USA. 2 Correspondence should be addressed to D.G.D. (e-mail: drubin@socrates.berkeley.edu) Received 28 April 2006; accepted 14 June 2006; published online 23 July 2006; DOI: 10.1038/ncb1443 © 2006 Nature Publishing Group