Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation

Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.     

Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1

Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.     

Identification of new genes ndr2 and ndr3 which are related to Ndr1/RTP/Drg1 but show distinct tissue specificity and response to N-myc

Ndr1 was isolated as a gene upregulated in N-myc mutant mouse embryos and is repressed by N-myc and c-myc. Consistent with Myc regulation, the same gene was also isolated as one sensitive to transformation (Drg1), and in addition as one induced under a few stress conditions (RTP). Two new genes, Ndr2 and Ndr3, were identified which encode proteins highly related to Ndr1/RTP/Drg1 and constitute the Ndr gene family. Ndr2 and Ndr3 are under spatio-temporal regulations distinct from Ndr1, and are not activated in N-myc mutants. When whole embryo RNA was analyzed, Ndr3 expression was already high at 9.5 days postcoitus (dpc), while expression of Ndr2 and Ndr1 became significant after 12.5 dpc and 13. 5 dpc, respectively. At 14.5 dpc, expression of these genes partially overlaps, but many tissues are unique to one of them. For instance, Ndr1 is strongly expressed in the liver and gut epithelium, Ndr2 in the ventricular zone throughout the CNS, and Ndr3 in the spinal cord and the thymus rudiment. Genes of the Ndr family probably have tissue-dependent allotments of the possibly related functions.     

Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae. Decoupling of intracellular function from ATPase activity and hexamerization

The AAA protein Drg1 from yeast was affinity-purified, and its ATPase activity and hexamerization properties were analyzed. The same parameters were also determined for several mutant proteins and compared in light of the growth characteristics of the corresponding cells. The protein from a thermosensitive mutant exhibited reduced ATPase activity and hexamerization. These defects were not reversed by an intragenic suppressor mutation, although this allele supported growth at the nonpermissive temperature. A different set of mutants was generated by site-specific mutagenesis intended to adjust the Walker A box of the D2 domain of Drg1p to that of the D1 domain. A S562G exchange in D2 produced a nonfunctional protein that did not hexamerize but showed above-normal ATPase activity. The C561T mutant protein, on the other hand, was functional but hexamerized less readily and had reduced ATPase activity. In contrast, the C561T/S562G protein hexamerized less than wild type but had much higher ATPase activity. We distinguished strong and weak ATP-binding sites in the wild type protein but two weak sites in the C561T/S562G protein, indicating that the stronger site resides in D2. These observations are discussed in terms of the inter-relationship of ATPase activity per se, oligomeric status, and intracellular function for AAA proteins.     

The Drug Diazaborine Blocks Ribosome Biogenesis by Inhibiting the AAA-ATPase Drg1

The drug diazaborine is the only known inhibitor of ribosome biogenesis and specifically blocks large subunit formation in eukaryotic cells. However, the target of this drug and the mechanism of inhibition were unknown. Here we identify the AAA-ATPase Drg1 as a target of diazaborine. Inhibitor binding into the second AAA domain of Drg1 requires ATP loading and results in inhibition of ATP hydrolysis in this site. As a consequence the physiological activity of Drg1, i.e. the release of Rlp24 from pre-60S particles, is blocked, and further progression of cytoplasmic preribosome maturation is prevented. Our results identify the first target of an inhibitor of ribosome biogenesis and provide the mechanism of inhibition of a key step in large ribosomal subunit formation.     

Crystal structure of the Ego1-Ego2-Ego3 complex and its role in promoting Rag GTPase-dependent TORC1 signaling

The target of rapamycin complex 1 (TORC1) integrates various hormonal and nutrient signals to regulate cell growth, proliferation, and differentiation. Amino acid-dependent activation of TORC1 is mediated via the yeast EGO complex (EGOC) consisting of Gtr1, Gtr2, Ego1, and Ego3. Here, we identify the previously uncharacterized Ycr075w-a/Ego2 protein as an additional EGOC component that is required for the integrity and localization of the heterodimeric Gtr1-Gtr2 GTPases, equivalent to mammalian Rag GTPases. We also report the crystal structure of the Ego1-Ego2-Ego3 ternary complex (EGO-TC) at 2.4 Å resolution, in which Ego2 and Ego3 form a heterodimer flanked along one side by Ego1. Structural data also reveal the structural conservation of protein components between the yeast EGO-TC and the human Ragulator, which acts as a GEF for Rag GTPases. Interestingly, however, artificial tethering of Gtr1-Gtr2 to the vacuolar membrane is sufficient to activate TORC1 in response to amino acids even in the absence of the EGO-TC. Our structural and functional data therefore support a model in which the EGO-TC acts as a scaffold for Rag GTPases in TORC1 signaling.     

Rickettsia parkeri invasion of diverse host cells involves an Arp2/3 complex, WAVE complex and Rho-family GTPase-dependent pathway

Rickettsiae are obligate intracellular pathogens that are transmitted to humans by arthropod vectors and cause diseases such as spotted fever and typhus. Although rickettsiae require the host cell actin cytoskeleton for invasion, the cytoskeletal proteins that mediate this process have not been completely described. To identify the host factors important during cell invasion by Rickettsia parkeri, a member of the spotted fever group (SFG), we performed an RNAi screen targeting 105 proteins in Drosophila melanogaster S2R+ cells. The screen identified 21 core proteins important for invasion, including the GTPases Rac1 and Rac2, the WAVE nucleation-promoting factor complex and the Arp2/3 complex. In mammalian cells, including endothelial cells, the natural targets of R. parkeri, the Arp2/3 complex was also crucial for invasion, while requirements for WAVE2 as well as Rho GTPases depended on the particular cell type. We propose that R. parkeri invades S2R+ arthropod cells through a primary pathway leading to actin nucleation, whereas invasion of mammalian endothelial cells occurs via redundant pathways that converge on the host Arp2/3 complex. Our results reveal a key role for the WAVE and Arp2/3 complexes, as well as a higher degree of variation than previously appreciated in actin nucleation pathways activated during Rickettsia invasion.     

Mapping the interactions between a RUN domain from DENND5/Rab6IP1 and sorting nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.     

Crystal structure of Rac1 bound to its effector phospholipase C-beta2

Although diverse signaling cascades require the coordinated regulation of heterotrimeric G proteins and small GTPases, these connections remain poorly understood. We present the crystal structure of the GTPase Rac1 bound to phospholipase C-beta2 (PLC-beta2), a classic effector of heterotrimeric G proteins. Rac1 engages the pleckstrin-homology (PH) domain of PLC-beta2 to optimize its orientation for substrate membranes. Gbetagamma also engages the PH domain to activate PLC-beta2, and these two activation events are compatible, leading to additive stimulation of phospholipase activity. In contrast to PLC-delta, the PH domain of PLC-beta2 cannot bind phosphoinositides, eliminating this mode of regulation. The structure of the Rac1-PLC-beta2 complex reveals determinants that dictate selectivity of PLC-beta isozymes for Rac GTPases over other Rho-family GTPases, and substitutions within PLC-beta2 abrogate its stimulation by Rac1 but not by Gbetagamma, allowing for functional dissection of this integral signaling node.     

The PRA1 gene family in Arabidopsis

Prenylated Rab acceptor 1 (PRA1) domain proteins are small transmembrane proteins that regulate vesicle trafficking as receptors of Rab GTPases and the vacuolar soluble N-ethylmaleimide-sensitive factor attachment receptor protein VAMP2. However, little is known about PRA1 family members in plants. Sequence analysis revealed that higher plants, compared with animals and primitive plants, possess an expanded family of PRA1 domain-containing proteins. The Arabidopsis (Arabidopsis thaliana) PRA1 (AtPRA1) proteins were found to homodimerize and heterodimerize in a manner corresponding to their phylogenetic distribution. Different AtPRA1 family members displayed distinct expression patterns, with a preference for vascular cells and expanding or developing tissues. AtPRA1 genes were significantly coexpressed with Rab GTPases and genes encoding vesicle transport proteins, suggesting an involvement in the vesicle trafficking process similar to that of their animal counterparts. Correspondingly, AtPRA1 proteins were localized in the endoplasmic reticulum, Golgi apparatus, and endosomes/prevacuolar compartments, hinting at a function in both secretory and endocytic intracellular trafficking pathways. Taken together, our data reveal a high functional diversity of AtPRA1 proteins, probably dealing with the various demands of the complex trafficking system.     

Function of Drg1/Rit42 in p53-dependent mitotic spindle checkpoint

Mutations in the Drg1/RTP/Rit42 gene are commonly identified in hereditary neuropathies of the motor and sensory systems. This gene was also identified as a p53 target gene and a differentiation-related, putative metastatic suppressor gene in human colon and prostate cancer. In this study, we show that the Rit42 protein is a microtubule-associated protein that localizes to the centrosomes and participates in the spindle checkpoint in a p53-dependent manner. When ectopically expressed and exposed to spindle inhibitors, Rit42 inhibited polyploidy in several p53-deficient tumor cell lines and increased the population of cells in mitotic arrest. Blocking endogenous Rit42 expression by small interfering RNA in normal human mammary epithelial cells resulted in the disappearance of astral microtubules, and dividing spindle fiber formation was rarely detected. Moreover, these cells underwent microtubule inhibitor-induced reduplication, leading to a polyploidy state. Our findings imply that Rit42 plays a role in the regulation of microtubule dynamics and the maintenance of euploidy.     

The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32

In yeast, the Ypt1 GTPase is required for ER-to-cis-Golgi and cis-to-medial-Golgi protein transport, while Ypt31/32 are a functional pair of GTPases essential for exit from the trans-Golgi. We have previously identified a Ypt1 guanine nucleotide exchange factor (GEF) activity and characterized it as a large membrane-associated protein complex that localizes to the Golgi and can be extracted from the membrane by salt, but not by detergent. TRAPP is a large protein complex that is required for ER-to-Golgi transport and that has properties similar to those of Ypt1 GEF. Here we show that TRAPP has Ypt1 GEF activity. GST-tagged Bet3p or Bet5p, two of the TRAPP subunits, were expressed in yeast cells and were precipitated by glutathione-agarose (GA) beads. The resulting precipitates can stimulate both GDP release and GTP uptake by Ypt1p. The majority of the Ypt1 GEF activity associated with the GST-Bet3p precipitate has an apparent molecular weight of > 670 kDa, indicating that the GEF activity resides in the TRAPP complex. Surprisingly, TRAPP can also stimulate nucleotide exchange on the Ypt31/32 GTPases, but not on Sec4p, a Ypt-family GTPase required for the last step of the exocytic pathway. Like the previously characterized Ypt1 GEF, the TRAPP Ypt1-GEF activity can be inhibited by the nucleotide-free Ypt1-D124N mutant protein. This mutant protein also inhibits the Ypt32 GEF activity of TRAPP. Coprecipitation and overexpression studies suggest that TRAPP can act as a GEF for Ypt1 and Ypt31/32 in vivo. These data suggest the exciting possibility that a GEF complex common to Ypt1 and Ypt31/32 might coordinate the function of these GTPases in entry into and exit from the Golgi.     

Involvement of the Arp2/3 complex and Scar2 in Golgi polarity in scratch wound models

Cell motility and cell polarity are essential for morphogenesis, immune system function, and tissue repair. Many animal cells move by crawling, and one main driving force for movement is derived from the coordinated assembly and disassembly of actin filaments. As tissue culture cells migrate to close a scratch wound, this directional extension is accompanied by Golgi apparatus reorientation, to face the leading wound edge, giving the motile cell inherent polarity aligned relative to the wound edge and to the direction of cell migration. Cellular proteins essential for actin polymerization downstream of Rho family GTPases include the Arp2/3 complex as an actin nucleator and members of the Wiskott-Aldrich Syndrome protein (WASP) family as activators of the Arp2/3 complex. We therefore analyzed the involvement of the Arp2/3 complex and WASP-family proteins in in vitro wound healing assays using NIH 3T3 fibroblasts and astrocytes. In NIH 3T3 cells, we found that actin and Arp2/3 complex contributed to cell polarity establishment. Moreover, overexpression of N-terminal fragments of Scar2 (but not N-WASP or Scar1 or Scar3) interfere with NIH 3T3 Golgi polarization but not with cell migration. In contrast, actin, Arp2/3, and WASP-family proteins did not appear to be involved in Golgi polarization in astrocytes. Our results thus indicate that the requirement for Golgi polarity establishment is cell-type specific. Furthermore, in NIH 3T3 cells, Scar2 and the Arp2/3 complex appear to be involved in the establishment and maintenance of Golgi polarity during directed migration.     

Scar1 and the related Wiskott–Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex

Background: The actin-related proteins Arp2 and Arp3 are part of a seven-protein complex which is localized in the lamellipodia of a variety of cell types, and in actin-rich spots of unknown function. The Arp2/3 complex enhances actin nucleation and causes branching and crosslinking of actin filaments in vitro; in vivo it is thought to drive the formation of lamellipodia and to be a control center for actin-based motility. The Wiskott–Aldrich syndrome protein, WASP, is an adaptor protein implicated in the transmission of signals from tyrosine kinase receptors and small GTPases to the actin cytoskeleton. Scar1 is a member of a new family of proteins related to WASP, and it may also have a role in regulating the actin cytoskeleton. Scar1 is the human homologue of Dictyostelium Scar1, which is thought to connect G-protein-coupled receptors to the actin cytoskeleton. The mammalian Scar family contains at least four members. We have examined the relationships between WASP, Scar1, and the Arp2/3 complex.Results: We have identified WASP and its relative Scar1 as proteins that interact with the Arp2/3 complex. We have used deletion analysis to show that both WASP and Scar1 interact with the p21 subunit of the Arp2/3 complex through their carboxyl termini. Overexpression of carboxy-terminal fragments of Scar1 or WASP in cells caused a disruption in the localization of the Arp2/3 complex and, concomitantly, induced a complete loss of lamellipodia and actin spots. The induction of lamellipodia by platelet-derived growth factor was also suppressed by overexpression of the fragment of Scar1 that binds to the Arp2/3 complex.Conclusions: We have identified a conserved sequence domain in proteins of the WASP family that binds to the Arp2/3 complex. Overexpression of this domain in cells disrupts the localization of the Arp2/3 complex and inhibits lamellipodia formation. Our data suggest that WASP-related proteins may regulate the actin cytoskeleton through the Arp2/3 complex.     

Identification of interaction partners and substrates of the cyclin A1-CDK2 complex

The CDK2-associated cyclin A1 is essential for spermatogenesis and contributes to leukemogenesis. The detailed molecular functions of cyclin A1 remain unclear, since the molecular networks involving cyclin A1-CDK2 have not been elucidated. Here, we identified novel cyclin A1/CDK2 interaction partners in a yeast triple-hybrid approach. Several novel proteins (INCA1, KARCA1, and PROCA1) as well as the known proteins GPS2 (G-protein pathway suppressor 2), Ku70, receptor for activated protein kinase C1/guanine nucleotide-binding protein beta-2-like-1, and mRNA-binding motif protein 4 were identified as interaction partners. These proteins link the cyclin A1-CDK2 complex to diverse cellular processes such as DNA repair, signaling, and splicing. Interactions were confirmed by GST pull-down assays and co-immunoprecipitation. We cloned and characterized the most frequently isolated unknown gene, which we named INCA1 (inhibitor of CDK interacting with cyclin A1). The nuclear INCA1 protein is evolutionarily conserved and lacks homology to any known gene. This novel protein and two other interacting partners served as substrates for the cyclin A1-CDK2 kinase complex. Cyclin A1 and all interaction partners were highly expressed in testis with varying degrees of tissue specificity. The highest expression levels were observed at different time points during testis maturation, whereas expression levels in germ cell cancers and infertile testes decreased. Taken together, we identified testicular interaction partners of the cyclin A1-CDK2 complex and studied their expression pattern in normal organs, testis development, and testicular malignancies. Thereby, we establish a new basis for future functional analyses of cyclin A1. We provide evidence that the cyclin A1-CDK2 complex plays a role in several signaling pathways important for cell cycle control and meiosis.     

ArhGAP12 plays dual roles in Stabilin-2 mediated efferocytosis: Regulates Rac1 basal activity and spatiotemporally turns off the Rac1 to orchestrate phagosome maturation

The rapid and precise clearance of apoptotic cells (efferocytosis) involves a series of phagocytic processes through which apoptotic cells are recognized, engulfed, and degraded within phagocytes. The Rho-family GTPases critically rearrange the cytoskeleton for these phagocytic processes, but we know little about the mechanisms by which regulatory proteins control the spatiotemporal activities of the Rho-family GTPases. Here, we identify ArhGAP12 as a functional GTPase-activating protein (GAP) of Rac1 during Stabilin-2 mediated efferocytosis. ArhGAP12 constitutively forms a complex with the phosphatidylserine receptor, Stabilin-2, via direct interaction with the downstream protein, GULP, but is released from the complex when Stabilin-2 interacts with apoptotic cells. When the phagocytic cup is closed and the apoptotic cell is surrounded by the phagosomal membrane, ArhGAP12 localizes to the phagocytic cup via a specific interaction with phosphatidylinositol-4,5-bisphosphate, which is transiently biosynthesized in the phagocytic cup. Down-regulation of ArhGAP12 results in sustained Rac1 activity, arrangement of F-actin, and delayed phagosome-lysosome fusion. Our results collectively suggest that ArhGAP12 carries dual roles in Stabilin-2 mediated efferocytosis: it binds to GULP/Stabilin-2 and switches off Rac1 basal activity and switches on the Rac1 by releasing itself from the complex. In addition, the spatiotemporal membrane targeting of ArhGAP12 inactivates Rac1 in a time-specific and spatially coordinated manner to orchestrate phagosome maturation. This may shed light on how other RhoGAPs spatiotemporally inactivate Rac or Cdc42 during phagocytosis by various cells, in different circumstances.     

HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex

Background  

V(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. The RSS contains highly conserved heptamer (consensus: 5'-CACAGTG) and nonamer (consensus: 5'-ACAAAAACC) motifs separated by either 12- or 23-base pairs of poorly conserved sequence. The high mobility group proteins HMGB1 and HMGB2 (HMGB1/2) are highly abundant architectural DNA binding proteins known to promote RAG-mediated synapsis and cleavage of consensus recombination signals in vitro by facilitating RSS binding and bending by the RAG1/2 complex. HMGB1/2 are known to recognize distorted DNA structures such as four-way junctions, and damaged or modified DNA. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element.     

ADP-ribosylation factors (ARFs) and ARF-like 1 (ARL1) have both specific and shared effectors: characterizing ARL1-binding proteins

Despite the 40-60% identity between ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins, distinct functional roles have been inferred from findings that ARLs lack the biochemical or genetic activities characteristic of ARFs. The potential for functional overlap between ARFs and ARLs was examined by comparing effects of expression on intact cells and the ability to bind effectors. Expression of [Q71L]ARL1 in mammalian cells led to altered Golgi structure similar to, but less dramatic than, that reported previously for [Q71L]ARF1. Two previously identified partners of ARFs, MKLP1 and Arfaptin2/POR1, also bind ARL1 but not ARL2 or ARL3. Two-hybrid screens of human cDNA libraries with dominant active mutants of human ARL1, ARL2, and ARL3 identified eight different but overlapping sets of binding partners. Specific interactions between ARL1 and two binding proteins, SCOCO and Golgin-245, are defined and characterized in more detail. Like ARFs and ARL1, the binding of SCOCO to Golgi membranes is rapidly reversed by brefeldin A, suggesting the presence of a brefeldin A-sensitive ARL1 exchange factor. These data reveal a complex network of interactions between GTPases in the ARF family and their effectors and reveal a potential for cross-talk not demonstrated previously.     

Activation of Arp2/3 complex by Wiskott-Aldrich Syndrome protein is linked to enhanced binding of ATP to Arp2

In response to signaling, the Arp2/3 complex (actin-related proteins 2 and 3 complex) is activated by binding the C-terminal (WA) domain of proteins of the Wiskott-Aldrich Syndrome family to promote the formation of a branched actin filament array, responsible for cell protrusion. The Arp2/3 complex exists in different structural/functional states: the inactive Arp2/3, the activated WA.Arp2/3 complex, the ternary G-actin.WA.Arp2/3 complex, which branches the filaments. This work addresses the role of ATP binding in Arp2/3 function. Using photo-cross-linking, hydrodynamic, and fluorescence techniques, we show that in the inactive Arp2/3 complex only one rapidly exchangeable ATP is tightly bound to Arp3 with an affinity of 10(8) m(-1). Upon activation of the Arp2/3 complex by WA, ATP binds to Arp2 with high affinity (10(7) m(-1)), implying that a large structural change of Arp2 is linked to Arp2/3 activation. ATP is rapidly exchangeable on Arp2 and Arp3 in WA.Arp2/3 and G-actin.WA.Arp2/3 complexes. ATP is not hydrolyzed in inactive Arp2/3, in WA.Arp2/3, nor in G-actin.WA.Arp2/3. Arp2 has a greater specificity than Arp3 for ATP versus ATP analogs. Using functional assays of actin polymerization in branched filaments, we show that binding of ATP to Arp2 is required for filament branching.