Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1.

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle - the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.

Characterization of guanine nucleotide exchange activity of DH domain of human FGD2.

FGD2, a member of FGD family, contains a Dbl homology domain (DH) and two pleckstrin homology domains segregated by a FYVE domain. The DH domain has been deduced to be responsible for guanine nucleotide exchange of CDC42 to activate downstream factors. Our aim was to build a prokaryotic expression system for the DH domain and to examine its guanine nucleotide exchange activity toward CDC42 in vitro. A recombinant vector, which was successfully constructed based on pGEX-6P-1, was employed to express the DH domain of human FGD2 (FGD2-DH) in E. coli BL21 (DE3). Purified FGD2-DH behaved as a homogeneous monomer with an estimated molecular weight that corresponded to the theoretical molecular weight and was predicted to be an α-helix protein by circular dichroism spectroscopy. FGD2-DH displayed weak guanine nucleotide exchange activity in vitro and very weak interactions with CDC42 following glutaraldehyde cross-linking.

Production of Phosphorylated Ric-8A proteins using protein kinase CK2.

Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that fold heterotrimeric G protein α subunits shortly after biosynthesis. Ric-8 proteins also act as test tube guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange. The GEF and chaperoning activities of Ric-8A are regulated by phosphorylation of five serine and threonine residues within protein kinase CK2 consensus sites. The traditional way that Ric-8A proteins have been purified is from Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tni) insect cells. Endogenous insect cell kinases do phosphorylate the critical regulatory sites of recombinant Ric-8A reasonably well, but there is batch-to-batch variability among recombinant Ric-8A preparations. Additionally, insect cell-production of some Ric-8 proteins with phosphosite alanine substitution mutations is proscribed as there seems to be interdependency of multi-site phosphorylation for functional protein production. Here, we present a method to produce wild type and phosphosite mutant Ric-8A proteins that are fully occupied with bound phosphate at each of the regulatory positions. Ric-8A proteins were expressed and purified from E. coli. Purified Ric-8A was phosphorylated in vitro with protein kinase CK2 and then re-isolated to remove kinase. The phosphorylated Ric-8A proteins were ∼99% pure and the completeness of phosphorylation was verified by chromatography, phos-tag SDS-PAGE mobility shifts, immunoblotting using phospho-site specific antibodies, and mass spectrometry analysis. E. coli-produced Ric-8A that was phosphorylated using this method promoted a faster rate of Gα subunit guanine nucleotide exchange than Ric-8A that was variably phosphorylated during production in insect cells.

The protein-binding N-terminal domain of human translation elongation factor 1Bß possesses a dynamic α-helical structural organization.

Translation elongation factor 1Bß (eEF1Bß) is a metazoan-specific protein involved into the macromolecular eEF1B complex, containing also eEF1Bα and eEF1Bγ subunits. Both eEF1Bα and eEF1Bß ensure the guanine nucleotide exchange on eEF1A while eEF1Bγ is thought to have a structural role. The structures of the eEF1Bß catalytic C-terminal domain and neighboring central acidic region are known while the structure of the protein-binding N-terminal domain remains unidentified which prevents clear understanding of architecture of the eEF1B complex. Here we show that the N-terminal domain comprising initial 77 amino acids of eEF1Bß, eEF1Bß(1-77), is a monomer in solution with increased hydrodynamic volume. This domain binds eEF1Bγ in equimolar ratio. The CD spectra reveal that the secondary structure of eEF1Bß(1-77) consists predominantly of α-helices and a portion of disordered region. Very rapid hydrogen/deuterium exchange for all eEF1Bß(1-77) peptides favors a flexible tertiary organization of eEF1Bß(1-77). Computational modeling of eEF1Bß(1-77) suggests several conformation states each composed of three α-helices connected by flexible linkers. Altogether, the data imply that the protein-binding domain of eEF1Bß shows flexible spatial organization which may be needed for interaction with eEF1Bγ or other protein partners.

Biochemical methods for studying kinetic regulation of Arf1 activation by Sec7.

The ADP ribosylation factor (Arf) family of small guanosine triphosphatases (GTPases) regulates vesicular transport at several locations within the cell, and is in turn regulated by guanine nucleotide exchange factors (GEFs) via a conserved catalytic domain, termed the Sec7 domain. The catalytic activity of the Sec7 domain is well characterized in the context of a few GEFs acting at the periphery of the cell. This chapter describes the techniques used to extend the biochemical analysis of activity to the much larger GEFs acting on the Arf family in the core secretory pathway, using the activity of Saccharomyces cerevisiae Sec7 on Arf1, regulating export from the trans-Golgi network, as a model. The complete methods for purification to near homogeneity of all proteins required, including several Sec7 constructs and multiple relevant small GTPases, are detailed. These are followed by methods for the quantification of the nucleotide exchange activity of Sec7 in a physiologically relevant context, including modifications required to dissect the signal integration functions of Sec7 as an effector of several other small GTPases, and methods for identifying stable Sec7-small GTPase interactions in the presence of membranes. These techniques may be extended to the analysis of similar members of the Sec7 GEF subfamily in other species and acting elsewhere in the secretory pathway.

Assaying the interaction of the Rab guanine nucleotide exchange protein Sec2 with the upstream Rab, a downstream effector, and a phosphoinositide.

Rabs are activated by guanine nucleotide exchange proteins, which are in turn controlled by complex regulatory mechanisms. Here we describe several different assays that have been used to delineate the mechanisms by which Sec2p, the exchange factor for the Rab Sec4p, is regulated. These assays assess the interaction of Sec2p with the upstream Rab, Ypt32p, a downstream Sec4p effector, Sec15p, and the lipid, phosphatidylinositol-4-phosphate.

Identification of CalDAG-GEFI as an intracellular target for the vicinal dithiol binding agent phenylarsine oxide in human platelets.

CalDAG-GEFI, a guanine nucleotide exchange factor activating Rap1, is known to play a key role in Ca2+-dependent glycoprotein (GP)IIb/IIIa activation and platelet aggregation. Although inhibition of CalDAG-GEFI could be a potential strategy for antiplatelet therapy, no inhibitor of this protein has been identified. In the present study, phenylarsine oxide (PAO), a vicinal dithiol blocker, potently prevented Rap1 activation in thrombin-stimulated human platelets without significantly inhibiting intracellular Ca2+ mobilisation and protein kinase C activation. PAO also prevented the Ca2+ ionophore-induced Rap1 activation and platelet aggregation, which are dependent on CalDAG-GEFI. In the biotin-streptavidin pull-down assay, CalDAG-GEFI was efficiently pull-downed by streptavidin beads from the lysates of biotin-conjugated PAO-treated platelets, suggesting that PAO binds to intracellular CalDAG-GEFI with high affinity. The above effects of PAO were reversed by a vicinal dithiol compound 2,3-dimercaptopropanol. In addition, CalDAG-GEFI formed disulfide-linked oligomers in platelets treated with the thiol-oxidant diamide, indicating that CalDAG-GEFI contains redox-sensitive thiols. In a purified recombinant protein system, PAO directly inhibited CalDAG-GEFI-stimulated GTP binding to Rap1. Using CalDAG-GEFI and Rap1-overexpressed human embryonic kidney 293T cells, we further confirmed that PAO abolished Ca2+-mediated Rap1 activation. Taken together, these results have demonstrated that CalDAG-GEFI is one of the targets of action of PAO, and propose an important role of vicinal cysteines for the functions of CalDAG-GEFI.

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles.

Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment.

Interaction proteomics identify NEURL4 and the HECT E3 ligase HERC2 as novel modulators of centrosome architecture.

Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as pericentriolar material. Although the mechanisms underpinning the control of centriole duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of pericentriolar material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of pericentriolar material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture.

Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system.

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.

RIC8 is a guanine-nucleotide exchange factor for Galpha subunits that regulates growth and development in Neurospora crassa.

Heterotrimeric (αßγ) G proteins are crucial components of eukaryotic signal transduction pathways. G-protein-coupled receptors (GPCRs) act as guanine nucleotide exchange factors (GEFs) for Gα subunits. Recently, facilitated GDP/GTP exchange by non-GPCR GEFs, such as RIC8, has emerged as an important mechanism for Gα regulation in animals. RIC8 is present in animals and filamentous fungi, such as the model eukaryote Neurospora crassa, but is absent from the genomes of baker's yeast and plants. In Neurospora, deletion of ric8 leads to profound defects in growth and asexual and sexual development, similar to those observed for a mutant lacking the Gα genes gna-1 and gna-3. In addition, constitutively activated alleles of gna-1 and gna-3 rescue many defects of Δric8 mutants. Similar to reports in Drosophila, Neurospora Δric8 strains have greatly reduced levels of G-protein subunits. Effects on cAMP signaling are suggested by low levels of adenylyl cyclase protein in Δric8 mutants and suppression of Δric8 by a mutation in the protein kinase A regulatory subunit. RIC8 acts as a GEF for GNA-1 and GNA-3 in vitro, with the strongest effect on GNA-3. Our results support a role for RIC8 in regulating GNA-1 and GNA-3 in Neurospora.

The RhoA GEF Syx is a target of Rnd3 and regulated via a Raf1-like ubiquitin-related domain.

BACKGROUND: Rnd3 (RhoE) protein belongs to the unique branch of Rho family GTPases that has low intrinsic GTPase activity and consequently remains constitutively active [1], [2]. The current consensus is that Rnd1 and Rnd3 function as important antagonists of RhoA signaling primarily by activating the ubiquitous p190 RhoGAP [3], but not by inhibiting the ROCK family kinases. METHODOLOGY/PRINCIPAL FINDINGS: Rnd3 is abundant in mouse embryonic stem (mES) cells and in an unbiased two-step affinity purification screen we identified a new Rnd3 target, termed synectin-binding RhoA exchange factor (Syx), by mass spectrometry. The Syx interaction with Rnd3 does not occur through the Syx DH domain but utilizes a region similar to the classic Raf1 Ras-binding domain (RBD), and most closely related to those in RGS12 and RGS14. We show that Syx behaves as a genuine effector of Rnd3 (and perhaps Rnd1), with binding characteristics similar to p190-RhoGAP. Morpholino-oligonucleotide knockdown of Syx in zebrafish at the one cell stage resulted in embryos with shortened anterior-posterior body axis: this phenotype was effectively rescued by introducing mouse Syx1b mRNA. A Rnd3-binding defective mutant of Syx1b mutated in the RBD (E164A/R165D) was more potent in rescuing the embryonic defects than wild-type Syx1b, showing that Rnd3 negatively regulates Syx activity in vivo. CONCLUSIONS/SIGNIFICANCE: This study uncovers a well defined Rnd3 effector Syx which is widely expressed and directly impacts RhoA activation. Experiments conducted in vivo indicate that Rnd3 negatively regulates Syx, and that as a RhoA-GEF it plays a key role in early embryonic cell shape changes. Thus a connection to signaling via the planar cell polarity pathway is suggested.

A modified tandem affinity purification technique identifies that 14-3-3 proteins interact with Tiam1, an interaction which controls Tiam1 stability.

The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 has important functions in multiple cellular processes including proliferation, apoptosis and adherens junction maintenance. Here we describe a modified tandem affinity purification (TAP) technique that we have applied to specifically enrich Tiam1-containing protein complexes from mammalian cells. Using this technique in conjunction with LC-MS/MS mass spectrometry, we have identified additional Tiam1-interacting proteins not seen with the standard technique, and have identified multiple 14-3-3 family members as Tiam1 interactors. We confirm the Tiam1/14-3-3 protein interaction by GST-pulldown and coimmunoprecipitation experiments, show that it is phosphorylation-dependent, and that they colocalize in cells. The interaction is largely dependent on the N-terminal region of Tiam1; within this region, there are four putative phospho-serine-containing 14-3-3 binding motifs, and we confirm that two of them (Ser172 and Ser231) are phosphorylated in cells using mass spectrometry. Moreover, we show that phosphorylation at three of these motifs (containing Ser60, Ser172 and Ser231) is required for the binding of 14-3-3 proteins to this region of Tiam1. We show that phosphorylation of these sites does not affect Tiam1 activity; significantly however, we demonstrate that phosphorylation of the Ser60-containing motif is required for the degradation of Tiam1. Thus, we have established and proven methodology that allows the identification of additional protein-protein interactions in mammalian cells, resulting in the discovery of a novel mechanism of regulating Tiam1 stability.

In vitro GEF and GAP assays.

Small GTPases act as tightly regulated molecular switches governing a large variety of critical cellular functions. Their activity is controlled by two different biochemical reactions, GDP/GTP exchange and GTP hydrolysis. These very slow reactions require catalysis in cells by two kinds of regulatory proteins. While the guanine nucleotide exchange factors (GEFs) activate small GTPases by stimulating the slow exchange of bound GDP for the cellularly abundant GTP, GTPase-activating proteins (GAPs) accelerate the slow intrinsic rate of GTP hydrolysis by several orders of magnitude, leading to inactivation. There are a number of methods that can be used to characterize the specificity and activity of such regulators, to understand the effect of binding on the protein structure, and, ultimately, to obtain insights into their biological functions. This unit describes (1) detailed protocols for the expression and the purification of small GTPases and the catalytic domains of GEFs and GAPs; (2) preparation of nucleotide-free and fluorescent nucleotide-bound small GTPases; and (3) methods for monitoring of the intrinsic and GEF-catalyzed nucleotide exchange as well as intrinsic and GAP-stimulated GTP hydrolysis.

3D structure of a binary ROP-PRONE complex: the final intermediate for a complete set of molecular snapshots of the RopGEF reaction.

Guanine nucleotide exchange factors (GEFs) catalyze the activation of GTP-binding proteins (G proteins) in a multi-step reaction comprising intermediary complexes with and without nucleotide. Rho proteins of plants (ROPs) are activated by novel RopGEFs with a catalytic PRONE domain. We have previously characterized structures of GDP-bound ROP and a ternary complex between plant-specific ROP nucleotide exchanger (PRONE) and ROP including loosely bound GDP. Now, we complete the molecular snapshots of the RopGEF reaction with the nucleotide-free ROP-PRONE structure at 2.9 A. The binary complex surprisingly closely resembles the preceding ternary intermediate including an unusually intact P-loop in the G protein. A striking difference is the prominent contact of the invariant P-loop lysine to a conserved switch II glutamate in ROP, favoring a key role of this interaction in driving out the nucleotide. The nucleotide-free state is supported by additional interactions involving the essential WW-motif in PRONE. We propose that this GEF region stabilizes the intact P-loop conformation, which facilitates re-association with a new nucleotide and further promotes the overall exchange reaction. With our novel structure, we provide further insights into the nucleotide exchange mechanism and present a first example of the complete GEF reaction at a molecular level.

Interaction of phosphodiesterase 3A with brefeldin A-inhibited guanine nucleotide-exchange proteins BIG1 and BIG2 and effect on ARF1 activity.

ADP-ribosylation factors (ARFs) have crucial roles in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange proteins (BIG)1 and BIG2 catalyze the activation of class I ARFs by accelerating replacement of bound GDP with GTP. Several additional and differing actions of BIG1 and BIG2 have been described. These include the presence in BIG2 of 3 A kinase-anchoring protein (AKAP) domains, one of which is identical in BIG1. Proteins that contain AKAP sequences act as scaffolds for the assembly of PKA with other enzymes, substrates, and regulators in complexes that constitute molecular machines for the reception, transduction, and integration of signals from cAMP or other sources, which are initiated, propagated, and transmitted by chemical, electrical, or mechanical means. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated BIG1 and BIG2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. Concurrently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated BIG1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microdomains where its enzymatic activity limited cAMP concentration. There, cAMP-activated PKA phosphorylated BIG1 and BIG2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these BIG1 and BIG2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP effects with spatial and temporal specificity.

High-throughput screening for small-molecule inhibitors of LARG-stimulated RhoA nucleotide binding via a novel fluorescence polarization assay.

Guanine nucleotide exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein-coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytoskeletal changes, motility, growth, survival, and gene transcription. The leukemia-associated RhoGEF (LARG) is a member of the regulators of G-protein signaling homology domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide-binding assay using BODIPY-Texas Red-GTPgammaS (BODIPY-TR-GTPgammaS), the authors performed a 10,000-compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a nonfluorescent radioactive guanine nucleotide-binding assay measuring LARG-stimulated [( 35)S] GTPgammaS binding to RhoA, thus ruling out nonspecific fluorescent effects. All 5 compounds selectively inhibited LARG-stimulated RhoA [( 35)S] GTPgammaS binding but had little to no effect on RhoA or Galpha( o) [(35)S] GTPgammaS binding. Therefore, these 5 compounds should serve as promising starting points for the development of small-molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide-binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.

Molecular interaction of neurocalcin alpha with alsin (ALS2).

Membrane microdomains (MDs), or lipid rafts, are recently identified dynamic membrane domains on which various signal-transductions are performed. Intracellular Ca(2+)-binding proteins participate in the Ca(2+) signaling through interaction with various proteins. Neurocalcin alpha (NCalpha) is a member of neuronal calcium sensor (NCS) protein family and shows Ca(2+)-dependent binding to the cell membrane through N-terminal myristoyl moiety. Since NCalpha was identified as a Ca(2+)-dependent binding protein to neuronal MDs, its binding proteins may participate in the signal-transduction on the MDs. In an immunoprecipitate using anti-NCalpha antibody, alsin (ALS2), a protein product of one of the responsive genes for amyotrophic lateral sclerosis, was detected through LC-MS/MS. Specific antibody to alsin was produced and immunoprecipitation using this antibody showed co-sedimentation of NCalpha. Some part of alsin bound to brain-derived MD fraction in the presence of Ca(2+) ions and eluted out by the chelation of Ca(2+) ions, as in the case of NCalpha. Immunostaining of cultured neurons showed broad distribution of alsin and NCalpha, and membrane association of these proteins were increased through Ca(2+) loading by maitotoxin. These results suggest that alsin binds cell membrane in a Ca(2+)-dependent manner through NCalpha and regulates membrane dynamics.

Simultaneous electrophoretic analysis of proteins of very high and low molecular weights using low-percentage acrylamide gel and a gradient SDS-PAGE gel.

To be able to separate and analyze giant proteins and small proteins in the same electrophoretic gel, we have used a continuous SDS-PAGE gel formed by the combination of a low-percentage acrylamide gel and a gradient SDS-PAGE gel that we have named LAG gel. To get a good resolution for proteins of more than 200 kDa, we used an acrylamide/bisacrylamide ratio of 80:1 in the low-percentage acrylamide gel. To successfully resolve proteins in the 5-200 kDa range, we used a conventional 6-15% SDS-PAGE gradient gel with the standard acrylamide/bisacrylamide ratio of 40:1. We show that the LAG system can be successfully used in general applications of SDS-PAGE electrophoresis such as proteomics and immunobloting techniques. Thus, using this continuous LAG gel, it is possible to simultaneously analyze giant proteins, such as HERC1 and dynein, big proteins like clathrin heavy chain and small proteins like ARF. The LAG system has a good resolution, low cost, and high reproducibility. Moreover, to simultaneously analyze all proteins saves time. All these characteristics, together with the use of a standard apparatus found in any biochemistry laboratory, make the LAG system an easy tool to use.