Onsite GTP fuelling via DYNAMO1 drives division of mitochondria and peroxisomes

Mitochondria and peroxisomes proliferate by division. During division, a part of their membrane is pinched off by constriction of the ring-shaped mitochondrial division (MD) and peroxisome-dividing (POD) machinery. This constriction is mediated by a dynamin-like GTPase Dnm1 that requires a large amount of GTP as an energy source. Here, via proteomics of the isolated division machinery, we show that the 17-kDa nucleoside diphosphate kinase-like protein, dynamin-based ring motive-force organizer 1 (DYNAMO1), locally generates GTP in MD and POD machineries. DYNAMO1 is widely conserved among eukaryotes and colocalizes with Dnm1 on the division machineries. DYNAMO1 converts ATP to GTP, and disruption of its activity impairs mitochondrial and peroxisomal fissions. DYNAMO1 forms a ring-shaped complex with Dnm1 and increases the magnitude of the constricting force. Our results identify DYNAMO1 as an essential component of MD and POD machineries, suggesting that local GTP generation in Dnm1-based machinery regulates motive force for membrane severance. Mitochondria and peroxisomes require a dynamin-like GTPase to remodel membranes during division. Here the authors identify DYNAMO1, a nucleoside diphosphate kinase-like protein that generates a local source of GTP to promote constriction of the division machinery and produce daughter organelles.

M embrane fission is essential for the life of cells. During cell division, the cell membrane is cleaved at the equatorial plane. Cellular communication from hormone signaling to neurotransmission is supported by a process known as endocytosis. For proliferation of intracellular organelles such as mitochondria and peroxisomes, portions of their membrane are severed to generate daughter organelles. These fission reactions are essential for all intracellular membrane remodeling events and are mediated by the dynamin family of GTPase proteins 1 . These proteins polymerize and form a ring or spiral structure to constrict and pinch off the membrane 2 . Typical dynamin family members are Dnm1, which mediates division of mitochondria 3 and peroxisomes 4 , and dynamin isoforms that pinch off the neck of endocytic pits 5 . They are among the most powerful motor proteins and are capable of constricting membrane gaps as large as a few hundred nanometers in less than 1 min 6,7 . Because the diameters of mitochondrial and peroxisomal division planes are substantially larger than the neck of an endocytic pit, the magnitude of the constriction of in vitro Dnm1 (~50 nm) is fivefold higher than that of dynamin (~10 nm) 7 . Despite the high magnitude of its constriction, Dnm1 has a weak affinity for GTP and relatively high rate of GTP hydrolysis 8 as similar to dynamin 9 . Furthermore, high rate of GTPase activity is enhanced by polymerization (k cat = 50 min −1 ) 8 much higher than that of small GTPase (k cat = ∼8 × 10 −3 min −1 ) 10 . The fact that significant constriction mediated by Dnm1 requires high levels of GTP (~1 mM) 7 , indicates that efficient replenishment of GTP is needed to sustain the activity of assembly and constriction. However, how the functions of Dnm1 are energetically supported is unclear. Accessory proteins of Dnm1, such as Fis1, Mdv1, Mff, and MiDs, might enhance the kinetics of its GTP affinity and facilitate membrane constriction 11 . However, conservation of these proteins among eukaryotes is limited, even though Dnm1-mediated division of mitochondria and peroxisomes is common to almost all eukaryotic cells including those in animals, fungi, land plants, and algae 12 .
To search for a fundamental protein that potentially functions in energetic regulation and kinetics of Dnm1, proteomic analysis of the division machinery is required. For this approach, isolation of the division machineries is essential. However, most of the eukaryotic cells contain numerous numbers of mitochondria and peroxisomes that divide randomly, which makes it difficult to collect bulk of intermediate of dividing organelles. In contrast, a unicellular red alga, Cyanidioschyzon merolae (C. merolae), has advantages to conquer this problem because it contains only one peroxisome, mitochondrion, and plastid per cell. Moreover, division of these organelles can be highly synchronized by light/dark stimulation in C. merolae (Supplementary Figure 1) 13,14 . This feature enables bulk isolation of mitochondrial and peroxisomal division machineries [15][16][17] . The division machinery of mitochondria, called mitochondrial division (MD) machinery, has a ring-shaped electron-dense structure with a diameter of 150-1200 nm 12 . This machinery consists of an outer ring formed around the cytoplasmic side of the mitochondrial outer membrane and an inner ring formed around the matrix side of the inner membrane. The outer ring is a dynamin-based ring containing Dnm1 and skeletal polyglucan filaments, called the MD ring 16,18,19 . The inner ring is a remnant of a bacterial cell division apparatus containing FtsZ 20 that Opisthokonta have lost 21,22 . However, MD machinery is physically associated with plastid dividing (PD) machinery, implying that the isolated MD machinery fraction may be contaminated with plastid division-associated proteins 15,16,23 . In contrast, the division machinery of peroxisomes is formed at different times and sites than MD and PD machineries 17,24 . The peroxisome-dividing (POD) machinery with a diameter of 50-600 nm is composed of dynamin-based (DB) rings and skeletal filamentous rings formed at the cytoplasmic side of peroxisomal membranes 17 . The DB ring is structurally analogous to the outer ring of MD machinery and also contains Dnm1 17 . Moreover, the DB ring can be physically separated from the other components of POD machinery, which enables mapping of candidate proteins to specific structures.
By using the unicellular red alga, C. merolae, 17-kDa nucleoside diphosphate kinase-like protein, dynamin-based ring motiveforce organizer 1 (DYNAMO1) is identified as a fundamental component of MD and POD machineries. DYNAMO1 directly binds to Dnm1 and localizes on the Dnm1-based ring structure in the machineries. DYNAMO1 generates GTP from ATP and GDP and this enzyme activity is essential to sustain Dnm1-mediated fission of mitochondrion and peroxisome. Furthermore, in vitro analysis demonstrates that DYNAMO1 enhances constriction force generated by Dnm1-based rings. Thus, energy source for the constriction of MD and POD machineries is most likely generated locally around the membrane scission sites during the division of mitochondrion and peroxisome.

Results
Identification of DYNAMO1 from isolated POD machinery. We investigated the molecular identity of Dnm1-based machinery by proteomic analysis of the POD machinery-enriched fraction (Fig. 1a, b). To prepare sufficient amounts of POD machinery, we improved existing isolation methods, including treatment with 0.2% lauryldimethylamine-N-oxide (LDAO) instead of 0.8% n-octyl-β-D-glucoside (OG) 17,25 , to greatly increase the yield of Dnm1-positive rings (Fig. 1c, d; Supplementary Figure 2a and b). The LDAO-treated fraction of POD machinery contained spiral structures (Fig. 2d) similar to those formed by Dnml GTPase in MD machinery 15 . Thus, the LDAOtreated fraction of POD machinery possibly contained intact POD machinery with retention of its in vivo functions. To identify components of the DB ring, the solubilized fraction of isolated POD machinery was compared with the interphase fraction. Six bands distinctly enriched in the POD machinery fraction were analyzed by liquid chromatography and mass spectrometry (LC-MS/MS) and peptide mass fingerprinting using the complete genomic sequence of C. merolae 14,26 . Dnm1 was evidently identified in band 1 at~90 kDa (Supplementary Figure 2c; Supplementary Table 1), indicating enrichment of POD machinery. Among the detected candidate proteins, we found that the gene product of CML110C, a top-assigned protein in the most enriched band 5 (Supplementary Figure 2c; Supplementary  Table 1), bound to Dnm1 in vitro and in vivo (Supplementary Figure 2d-g), suggesting that CML110C is a novel 17 kDa component of the DB ring. CML110C, termed as DYNAMO1, contains a consensus motif of nucleoside diphosphate kinase (NDPK) (Supplementary Figure 2h) that forms various oligomeric structures, such as a hexamer, tetramer, and dimer, and catalyzes conversion of ATP to GTP [27][28][29] . Recombinant DYNAMO1 formed a tetramer (Supplementary Figure 2i-k) and generated GTP upon addition of ATP plus GDP (Fig. 1e, f). When the histidine residue at position 116 of DYNAMO1 in the consensus active site motif was replaced by aspartic acid (DYNAMO1 H116D), its GTP generation was abolished (Fig. 1e), demonstrating that DYNAMO1 indeed functions as a nucleoside diphosphate kinase. Because nucleoside diphosphate kinase is involved in dynamin-mediated endocytosis and OPA1-mediated mitochondrial fusion in mammalian cells 29,30 , we suggest that DYNAMO1 is involved in Dnm1-based organelle division machineries.
Localization of DYNAMO1 at the organelle division sites. To investigate whether DYNAMO1 is involved in organelle division, we immunocytochemically analyzed endogenous DYNAMO1 with an anti-DYNAMO1 antibody. DYNAMO1 was expressed continuously during the cell cycle and was detected in both fractions of organelles containing peroxisomes and mitochondria and cytosol (Supplementary Figure 3a and b). Notably, 15-25% of DYNAMO1 were detected in organelle fractions as compared to cytosolic fraction at the G2-M cell cycle phase, when mitochondria and peroxisomes divide (Supplementary Figure 3b-d). Therefore, DYNAMO1 is likely involved in not only peroxisomal, but also mitochondrial division. We next analyzed localization of DYNAMO1 and Dnm1 at each cell cycle phase (Fig. 2). DYNAMO1 was initially distributed in the cytosol during G1 phase and then partially accumulated at both edges of the mitochondrion during S phase when the inner FtsZ ring formed at the destined division site of each mitochondrion (Supplementary Figure 4a and b). Subsequently, DYNAMO1 was recruited to Dnm1-labeled division sites of mitochondria during G2-prophase (Fig. 2a, b). Immediately after mitochondrial division, puncta of DYNAMO1 and Dnm1 apparently disappeared from the mitochondria. Furthermore, DYNAMO1 was almost    Figure 5a). DYNAMO1 was colocalized with Dnm1 in MD machinery labeled with Mda1 and FtsZ, but not in PD machinery labeled with the PD ring component PDR1 (Fig. 3a). The DYNAMO1 and Dnm1 signal intensities in MD machinery were constant, regardless of the diameter of the ring (Supplementary Figure 5b), suggesting that DYNAMO1 is a fundamental component of MD machinery and functions together with Dnm1 during its entire constriction process. In the isolated POD machinery, DYNAMO1 was also colocalized with Dnm1, and the DYNAMO1 and Dnm1 signal intensities were constant, regardless of the diameter of the POD ring ( Fig. 3b; Supplementary Figure 5c). We further explored nanoscale localization of DYNAMO1 by whole-mount negative-staining immunoelectron microscopy (Fig. 3c). The ultrastructure of the DB ring was defined by a Dnm1-labeled curved amorphous string that was partially peeled away from a rigid ring-shaped skeletal filamentous ring upon treatment with a nonionic detergent 17 . DYNAMO1-labeled 10 nm immunogold particles were detected on the string of the DB ring, but not on the skeletal filamentous ring, indicating that DYNAMO1 localizes on the DB ring formed at the cytosolic side of the division plane. These results strongly suggest that DYNAMO1 is a component of MD and POD machineries and generates GTP on their respective Dnm1-based rings.
GTP-generating reaction of DYNAMO1 induces membrane fission. To understand molecular function of DYNAMO1 on the MD and POD machinery, genetic disruption of DYNAMO1, particularly abolishment of nucleoside diphosphate kinase activity is needed. Nucleotide diphosphate kinases form an oligomer, including DYNAMO1 (Supplementary Figure 2j and k), and various dominant negative forms have been reported [31][32][33] . Thus, we next suppressed DYNAMO1 functions by transient expression of DYNAMO1 H116D, a DYNAMO1 mutant defective for GTPgenerating activity (Fig. 1e). DYNAMO1-HA and DYNAMO1 H116D-HA were expressed at about twofold higher than the endogenous protein (Supplementary Figure 6a  Collectively, these data suggest that the GTPgenerating activity of DYNAMO1 is essential for complete constriction at organelle division sites, and that adequate amounts of DYNAMO1 appear to be required for Dnm1 recruitment. DYNAMO1 localized to the division sites of both organelles and the entire cytoplasm, suggesting two possibilities of the GTPproviding mechanism. One is local GTP generation at the Dnm1positive organelle division site and the other is direct regulation of mitochondrial and peroxisomal divisions using a GTP pool in the cytosol. Although DYNAMO1 was localized at organelle division sites and in the entire cytoplasm ( DYNAMO1 facilitates constriction of Dnm1-based ring. To further investigate mechanisms underlying DYNAMO1 functions in the constriction of mitochondria and peroxisomes, we established an in vitro constriction assay using whole-mount negativestaining electron microscopy. Upon incubation of recombinant DYNAMO1 with Dnm1 under various conditions, single strings (DYNAMO1-Dnm1 strings) were formed with a semi-ring structure and diameter of~200 nm (Fig. 5a). The strings . Dnm1 of C. merolae forms a tetramer with a diameter of~10 nm in offmembrane state in vitro 36 , and Dnm1 molecules in the division machineries are most likely recruited from membrane-free Dnm1 pool within cytosol 19,25 . Thus, the presence of DYNAMO1 likely induces conformational changes to organize Dnm1 molecules into a highly ordered ring-like structure or stabilizes a ring-like structure at the division site. Because DYNAMO1 showed a GTPgenerating activity when supplied with both ATP and GDP (Fig. 1e), we verified whether this enzymatic activity was required for the formation of DYNAMO1-Dnm1 strings. Upon addition of ATP and GDP, the morphology of DYNAMO1-Dnm1 strings was dramatically altered to a spiral shape with one or both tips constricted toward the inside of the string (Fig. 5c-f). The same result was observed with addition of GTP (Fig. 5d, g). In contrast, addition of GMP-PCP did not induce the constriction activity (Fig. 5d, h). Thus, constriction of DYNAMO1-Dnm1 strings depended on the GTPase activity of Dnm1. DYNAMO1-free Dnm1 strings showed little evidence of this structural alteration (Fig. 5d). Collectively, these data indicate that DYNAMO1 is involved in facilitating constriction of the string. When the DYNAMO1-Dnm1 string was replaced by a DYNAMO1 H116D-Dnm1 string (Supplementary Figure 9a), there was no constriction upon addition of ATP or GDP (Fig. 5d, i). However, the DYNAMO1 H116D-Dnm1 string was constricted upon addition of GTP (Fig. 5d, j). Thus, the DYNAMO1 function in constriction of DYNAMO1-Dnm1 strings was independent of the GTPgenerating activity. Because the GTPase activity of Dnm1 was higher in DYNAMO1-Dnm1 and DYNAMO1 H116D-Dnm1 strings, in which DYNAMO1 contained half the amount of Dnm1, compared with DYNAMO1-free Dnm1 strings (Supplementary Figure 10), efficient GTP hydrolysis may have generated strong constrictive forces in the DYNAMO1-Dnm1 strings. GTPase activities of Dnm1 and dynamin are increased in an assembly-dependent manner 8,9 . Therefore, DYNAMO1-mediated stabilization of Dnm1 strings likely facilitates GTPase activity of the rings. Taken together, whole-mount morphological observations demonstrated three different DYNAMO1 functions (Fig. 5k): (1) DYNAMO1 enhances formation of the highly ordered structure of Dnm1; (2) DYNAMO1 generates GTP from ATP and GDP adjacent to Dnm1 molecules; (3) DYNAMO1 constructs DYNAMO1-Dnm1 strings that will then be constricted upon hydrolysis of GTP by Dnm1.

Discussion
Here, we demonstrated that a novel component, DYNAMO1, of MD and POD machineries is an essential molecule for the division of both mitochondria and peroxisomes. We propose three functions of DYNAMO1 in driving these organelle divisions in our working model (Fig. 6). First, the DYNAMO1 molecule is a constituent of MD and POD machineries. DYNAMO1 forms a complex structure with Dnm1 and is involved in Dnm1 assembly at the division site (Phase 1). Second, DYNAMO1 generates GTP on the rings of MD and POD machineries, serving as the energy source most likely provided by consumption of cytosolic ATP for constriction of organelle division machineries (Phase 2). Finally, DYNAMO1 drives constriction of MD and POD machineries (Phase 3). The NDPK domain in DYNAMO1 is conserved among eukaryotes. In flies, dysfunction of nucleoside diphosphate kinase (NDPK/NME) identified in the Awd mutant enhances paralysis of the Shibire mutant, a temperature-sensitive mutant of dynamin 37 . In mammalian cells, an NDPK ortholog, NME1/NME2, physically interacts with dynamin at the clathrin-coated endocytic pit and facilitates receptor-mediated endocytosis 27,28 . Moreover, NME4 supports dynamin-related GTPase OPA1 that mediates inner mitochondrial membrane fusion 29,30 . Here, we showed that the NDPK ortholog DYNAMO1 is a component of the Dnm1based organelle division machinery of mitochondria and peroxisomes, which most likely supports local GTP generation to induce the constrictive force. Thus, local GTP generation by the dynamin family member during membrane remodeling is a conserved phenomenon among eukaryotes. Although dynamin family members require considerable amounts of GTP because of the high rate of GTP hydrolysis and lower affinity for GTP 7-9 , cellular free-GTP levels are low (150-300 μM) 38,39 . The local enrichment of GTP is potentially critical to provide a sufficient energy source to drive the supramolecular nanomachineries that influence the dynamic behavior of organelles and cellular structures.
Purification of DYNAMO1. For DYNAMO1 expression, the plasmid encoding GST-tagged DYNAMO1 used for antibody generation was employed as described above. For DYNAMO1 H116D expression, the GST-tagged DYNAMO1 plasmid was amplified with the same primers to generate the plasmid for transient expression of the H116D mutant described above, and the amplified fragment was cloned by infusion cloning. XL-1 Blue strain cells were transformed with these plasmids, cultured at 37°C for 12 h in 100 mL Luria Bertani (LB) medium, scaled up to 1 L LB medium, and incubated further at 37°C for 2 h and then at 18°C for 1 h. Isopropyl β-D-1 thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM, and cells were harvested after a further 12 h of incubation at 18°C by centrifugation at 1000×g for 10 min. Cell pellets were resuspended in 200 mL HEPES buffer (HB250) containing 250 mM NaCl, 20 mM HEPES-KOH, pH 7.5, 2 mM EGTA, 1 mM MgCl 2 , 1 mM DTT, and a cOmplete. After homogenization by sonication for 10 min, the supernatant was filtered and rotated at 4°C for 1 h with 1 mL glutathione-Sepharose 4B beads (GE Healthcare). The beads were collected, washed, and resuspended in 10 mL HB250. Then, the beads were loaded onto a 10 mL Poly-Prep Chromatography column (BioRad) and washed with HBs: 5 mL HB250, 10 mL HB100 containing 100 mM NaCl, and 20 mL cOmplete-free HB50 containing 50 mM NaCl. To remove GST-tag, Sepharose beads were treated with PreScission Protease (GE Healthcare) at 4°C for 2 days, and DYNAMO1 was eluted.  Fig. 6 A working model of DYNAMO1 functions during the division of mitochondrion and peroxisomes. Initially, DYNAMO1 is recruited to the division site of the mitochondrion, together with Dnm1 (Phase 1, prophase). DYNAMO1 is involved in Dnm1 assembly around the division site. Next, DYNAMO1 converts ATP to GTP on the ring of the MD machinery. A cytosolic ATP source is used for this reaction. Upon GTP generation, the MD machinery is constricted and GTPase Dnm1 pinches off mitochondrion. Moreover, the DYNAMO1-Dnm1 structure effectively generates a strong motive force for the constriction (Phase 2). MD machinery containing DYNAMO1 and Dnm1 is immediately disassembled (Phase 3). After mitochondrial division, DYNAMO1 functions in the POD machinery during meta-anaphase, in an analogous manner to that in mitochondrial division Purification of Dnm1. For Dnm1 expression, pGex6p-1Dnm1 25 was introduced into the Escherichia coli XL-1 Blue strain. Transformed XL-1 Blue strain cells were cultured at 37°C for 12 h in 100 mL of LB medium, scaled up to 1 L of LB medium, and further incubated at 37°C for 2 h and at 18°C for 1 h. IPTG was added to a final concentration of 0.1 mM, and cells were harvested after a further 8-h incubation at 18°C by centrifugation at 1000×g for 10 min. Cell pellets were resuspended in 100 mL of HB150. After homogenization by sonication, the supernatant was filtered and rotated at 4°C with 1 mL of glutathione Sepharose 4B beads (GE Healthcare) for 1 h. The beads were collected and resuspended in 10 mL of HB100. Then, beads were loaded onto a 10 mL Poly-Prep Chromatography column (BioRad) and washed with cOmplete-free HB75 and 20 mL of complete-free HDB50. After washing with HBs, Sepharose beads were treated with PreScission Protease (GE Healthcare) at 4°C for 2 days and the cleaved-off proteins were eluted.
Gel filtration assay of recombinant DYNAMO1. A total of 1 μg purified recombinant DYNAMO1 was analyzed by gel filtration on a Sepharose 6 10/300 GL column using an AKTA prime system (GE Healthcare). Gel filtration standards (BioRad Laboratories) including thyroglobulin (669 kDa), γ-globulin (158 kDa), and ovalbumin (45 kDa) were used to calculate the molecular mass of DYNAMO1. Partition coefficient K av was calculated by is the elution volume, V 0 is the void volume, and V c is the total geometric volume of the column, 24 mL. Phase contrast and immunofluorescence microscopies. C. merolae cells were fixed at −30°C for 5 min with 1% wt/vol paraformaldehyde and 10% DMSO dissolved in 100% methanol 42 . Phase contrast and immunofluorescence images were captured with a fluorescence microscope (BX51; Olympus). Immunofluorescence profiles were acquired with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
LC-MS/MS analysis of isolated POD machinery. Proteins in isolated POD machinery and interphase fractions were separated by SDS-PAGE and stained with silver stain (0.02% Na 2 S 2 O 3 , 0.1% AgNO 3 , and 0.04% formalin/2% Na 2 CO 3 ; Sigma-Aldrich). Signal intensities of silver-stained bands were compared using the ImageJ plot profile between POD machinery and interphase fractions. Distinct bands with signal intensity ratios higher than 1 of POD machinery to interphase fractions were cut out, reduced with DTT, alkylated with iodoacetamide, digested with trypsin, and analyzed by an ion-trap mass spectrometer equipped with a nano-LC electrospray ionization source at the Human Proteome Research Centre, Kyushu University, Japan 46 . The obtained peptides were dried and then dissolved in a solution containing 0.1% trifluoroacetic acid and 2% acetonitrile before nanoscale liquid chromatography (nanoLC)-MS/MS analysis with a system consisting of an LTQ Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific) coupled with a nanoLC instrument (Advance LC, Michrom BioResources) and HTC-PAL autosampler (CTC Analytics). Peptide separation was performed with an in-house pulled and fused silica capillary (internal diameter, 0.1 mm; length, 10 cm; tip internal diameter, 0.05 mm) packed with a C18 L-column material (average particle size, 3 μm) (Chemicals Evaluation and Research Institute). The mobile phases were 0.1% formic acid in water (A) and 100% acetonitrile (B). Peptides were eluted with linear gradients of 5-40% B for 20 min and 40-95% B for 1 min, and then kept at 95% B for 4 min at a flow rate of 300 nL/min. Collision-induced dissociation (CID) spectra were acquired automatically in a data-dependent scan mode with the dynamic exclusion option. Full MS spectra were obtained with Orbitrap in the mass/charge (m/z) range of 300-2000 with a resolution of 60,000 at m/z 400. The nine most intense precursor ions (minimum ion count threshold of 1000) in the full MS spectra were selected for subsequent ion-trap MS/MS analysis with the automated gain control (AGC) mode. The AGC was set to 1 × 10 6 for full MS and 1 × 10 4 for CID MS/MS. The normalized collision energy value was set to 35%. The lock mass function was activated to minimize mass error during analysis. Peak lists were generated by MSn.exe (Thermo Fisher Scientific) with a minimum scan/group value of 1 and compared with the in-house-curated target/decoy C. merolae genome database (http://merolae.biol.s.u-tokyo.ac.jp/) using the MASCOT algorithm (ver. 2.4.1). Trypsin was selected as the enzyme, the allowed number of missed cleavages was set at 1, and carbamidomethylation on cysteine was selected as the fixed modification. Oxidized methionine was searched as the variable modification. Precursor mass tolerance was 10 ppm and the tolerance of MS/MS ions was 0.8 Da.
The threshold used for peptide identification was a MASCOT score of ≥30 and a delta score (the difference between firstand second-assigned peptides) of ≥ 18. Using these criteria, the false positive rate was <1%. Contaminants of plastid genome-encoded abundant photosynthetic proteins with a MASCOT score of ≥100 were excluded from the data.
LC-ESI-MS/MS analysis of nucleotides. To measure DYNAMO1 activity for nucleotide exchange, 100 nM recombinant DYNAMO1 described above was mixed with nucleotides and incubated at 37°C for 3 min. The reaction mixture was immediately mixed with 1.25 × and 2.5 × volumes of chloroform and methanol, respectively. Subsequently, 2 mM raffinose, as an internal standard used for normalization between samples, was added, and all samples were lyophilized and dissolved in methanol:water (1:1 v/v). After centrifugation at 10,000×g for 5 min, the supernatant was evaporated at room temperature under nitrogen for 6 h and then subjected to LC-ESI-MS/MS analysis. LC-ESI-MS/MS was performed using a 4000 QTRAP quadrupole linear ion-trap hybrid mass spectrometer (AB Sciex) with an ACQUITY UPLC System (Waters). Samples were injected into an ACQUITY UPLC BEH C18 column (1 × 150 mm; Waters) and then directly subjected to ESI-MS/MS analysis. A 10 μL aliquot was separated by step gradient elution with mobile phases A (water, 0.1% formic acid and 0.028% ammonia) and B (20% acetonitrile, 0.1% formic acid, and 0.028% ammonia) at the following ratios: 95:5 (for 0-5 min), 0:100 (5-25 min), 0:100 (25-30 min), and 95:5 (30-40 min). The flow rate was 50 μL/min at 30°C. The source temperature was 400°C, the declustering potential was −85, and the collision cell exit potential was −11. The ion transitions at m/z 506 > 79, 426 > 79, 522 > 79, 442 > 79, and 503 > 179 for ATP, ADP, GTP, GDP, and raffinose, respectively, were used in the multiple reaction monitoring mode. Data were analyzed and quantified using Analyst software (AB Sciex). Nucleotides were purchased from Sigma-Aldrich.