Arl6ip1−/− mouse model mimicking HSP with severe spastic paralysis and gait abnormalities. Neuroinflammation was identified as a key pathophysiological change. AAV9-ARL6IP1 delivery reduced limb paraplegia and gait abnormality, making ARL6IP1 a potential target for HSP gene therapy.We established an  Arl6ip1 knockout (KO) mouse model was generated to represent the clinically involved frameshift mutations and mimicked the HSP phenotypes. Notably, in vivo brain histopathological analysis revealed demyelination of the axon and neuroinflammation in the white matter, including the corticospinal tract. In in vitro experiments, ARL6IP1 silencing caused cell death during neuronal differentiation and mitochondrial dysfunction by dysregulated autophagy. ARL6IP1 localized on mitochondria-associated membranes (MAMs) to maintain endoplasmic reticulum and mitochondrial homeostasis via direct interaction with LC3B and BCl2L13. ARL6IP1 played a crucial role in connecting the endoplasmic reticulum and mitochondria as a member of MAMs. ARL6IP1 gene therapy reduced HSP phenotypes and restored pathophysiological changes in the Arl6ip1 KO model. Our results established that ARL6IP1 could be a potential target for HSP gene therapy.ARL6IP1 is implicated in hereditary spastic paraplegia (HSP), but the specific pathogenic mechanism leading to neurodegeneration has not been elucidated. Here, we clarified the molecular mechanism of ARL6IP1 in HSP using in vitro and in vivo models. The   Arl6ip1 KO versus Arl6ip1 wild-type (WT) mouse models. We also investigated the body morphology and neurodegenerative phenotypes present in the Arl6ip1 KO mouse model to examine the role of ARL6IP1 in neuronal homeostasis. Furthermore, the therapeutic effect of targeted gene delivery was assessed for HSP gene therapy development.Hereditary spastic paraplegia (HSP), a genetically and symptomatically heterogeneous neurodegenerative disease, is classified as either uncomplicated  or complicated  types . To dateARL6IP1 mutations are strongly implicated in HSP; five families have been reported with complex forms of autosomal recessive (AR)-HSP  was reported in two families at the same locus  mice were generated by Arl6ip1 heterozygotes (Arl6ip1+/−) and identified by genotyping. Genotyping of KO alleles in mice is usually performed via PCR using specifically designed primers. For the genotyping of pups, toes were used for genomic DNA extraction and amplification. ARL6IP1 transcripts and protein were not detected in the brain tissues of Arl6ip1−/− mice (Arl6ip1+/+) mouse brain, including the olfactory bulb, cortex, hippocampus, and cerebellum, but it was not detected in the Arl6ip1−/− mice -HSP . Among tme locus . This nome locus . A schem−/− mice . ARL6IP1−/− mice . They sh+/+mice . At 9 mothe tail . To eval+/+ mice . Footprio of age  and 2. OARL6IP1 deficiency in the central nervous system (CNS) of Arl6ip1−/− mice, we assessed astrocyte-, oligodendrocyte-, and neuron-specific protein levels via immunofluorescence and western blot analyses of the cerebral cortex of Arl6ip1−/− and Arl6ip1+/+ mice. The levels were decreased in neurons  and mature oligodendrocytes , while the expression of GFAP, an active astrocyte marker, was increased in Arl6ip1−/− mice  were increased, whereas those of Arg-1, Cd163, and Igf-1 (M2 microglia markers) were decreased. However, the mRNA level of Ym-1, an M2 marker, was significantly increased  and Arl6ip1+/+ (WT) mice on embryonic days 12 and 13 (E12–13). After carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment to induce mitochondrial dysfunction, cells were observed in 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl carbocyanine iodide (JC-1) staining under a fluorescence microscope and quantified by determining the ratio between the red and green fluorescence  protein according to the expression of ARL6IP1 in ARL6IP1 KO MEFs. Levels of protein encoded by ATGs were significantly reduced in ARL6IP1 KO MEFs compared with those in WT, either in the condition of autophagy induction by CCCP treatment or in the absence of treatment -LC3B HeLa cells transfected with mCherry-ARL6IP1, which showed that the ARL6IP1-transfected group had markedly increased GFP-LC3B puncta compared with that in the control group following autophagy induction by CCCP, while reduced GFP-LC3B puncta following autophagy inhibition by wortmannin . To detereatment . The bimreatment . BiFC vireatment . Biochemreatment . LC3B bereatment . To deteproteins . Furtherproteins . In prevproteins  and induproteins . Also, iproteins . To iden with WT . In ARL6s change . These rARL6IP1 KO MEFs compared with that in WT  cycle dehydrogenase  activity  was performed on ARL6IP1 KO and WT MEFs. ARL6IP1 silencing significantly reduced the membrane contact site between ER and mitochondria, suggesting that ARL6IP1 plays a structural role in ER–mitochondria connectivity in MAM , in mammalian cells . To veriat in WT . Total cse in WT . Next, tse in WT . SPG31 ase in WT . Mitocho WT MEFs . The basse in WT . Mitophaat in WT ; therefoat in WT . Mitochoactivity . We asse KO MEFs . Mitocho WT MEFs . Lower cy in MAM .ARL6IP1 gene therapy, we constructed an AAV9 serotype with a chicken β-actin promoter that drives expression in Arl6ip1+/+ mice and estimated its expression in Arl6ip1−/− mice, an HSP model. Arl6ip1−/− mice start showing initial mild phenotypic manifestations at 3 mo progressively exacerbating at 5 and 7 mo, culminating in pronounced symptoms by 9 mo. To evaluate the therapeutic effects, we performed a stereotaxic injection of AAV9-ARL6IP1 into Arl6ip1−/− mice at 2 mo of age (8 wk old), even before they displayed any symptoms. After injection, two mice per group were euthanized; the genome copy number of the AAV9 virus was calculated in the mouse tissues at 7 and 90 d  has been reported in two separate cases of HSP  showed HSP phenotypes clearly. Although Nestin-Cre-crossed KO mice (Tm1b) showed a more severe pathogenic phenotype earlier than the cassette KO mice, this Tm1b could not be expanded in sufficient numbers for experimentation; therefore, all experiments were conducted using Tm1a mice (named Arl6ip1−/− mice). We confirmed that the expression of the neighboring genes of Arl6ip1 on chromosome 7 had no effect in Arl6ip1−/− mice (data not shown). Arl6ip1−/− mice showed gait abnormalities, irregular stride lengths, lower leg angles, and pathophysiology of the brain, which are typical HSP phenotypes , which is the most common cause of AR-HSP and extensive white matter abnormalities , have been discovered in mammals. RTNs possess a conserved C-terminal RTN homology domain characterized by two unusually long hydrophobic regions interrupted by a hydrophilic loop , namely lic loop . The hydlic loop . These plic loop . Some prmembrane ; these pmembrane . ARL6IP1membrane ; its depmembrane . It was membrane . InteresDrosophila model  under a 12/12-h light/dark cycle. All animal housing and experiments were conducted following the Institutional Animal Care and Use Committee Guidelines (KRIBB-AEC-18090) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). WT mice were used as the control group, and to minimize genetic variation, mice of the same age from the same inbred strain were utilized.Arl6ip1−/− mice, heterozygote Arl6ip1tm1a(EUCOMM) mice were purchased from European Conditional Knockout Mouse Mutagenesis (EUCOMM) and maintained in a pathogen-free housing in the animal facility. Offspring of C57BL/6N inbred females were analyzed using PCR with genomic DNA from mouse toes. PCR conditions were as follows: 5 min at 95°C, followed by 35 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and a final 5-min incubation at 72°C. The Arl6ip1 WT allele was detected with the WT primer, resulting in the amplification of a 350-bp fragment, whereas the mutant allele was targeted by the MT primer and amplified to a 120-bp fragment. Based on these results, homologous Arl6ip1−/− mice were selected, maintained, and utilized for behavioral and histopathological analyses. The primer sequences are provided in To generate ARL6IP1 (NM_198737.2) KO model, rat shARL6IP1 was produced by subcloning nt 142–160 (5′-GATGCTGATGGCTGACAAA-3′) into pSuper-GFP/neo (Oligoengine). ARL6IP1 expression constructs were generated using reverse transcription (RT)-PCR from ReNcell CX cells using specific primers and incorporated into various plasmid vectors as below. Mutated constructs were generated using plasmids containing the ARL6IP1 WT gene using primers with the desired mutation using PCR-based methods. The pAAV9-CAG-GFP plasmid was purchased from Addgene (Plasmid #37825). All the plasmid constructs were confirmed via sequencing; detailed information is provided in For the 2 at 37°C. ReNcell CX cells, derived from the cortical region of human fetal brain tissue  as an immortalized human progenitor cell line, were maintained in ReNcell NSC maintenance medium  supplemented with 20 ng/ml epidermal growth factor , 20 ng/ml basic fibroblast growth factor , and P/S. For neuronal differentiation of ReNcell CX cells, the ReNcell NSC maintenance medium was used for culturing after removing growth factors. To generate an ARL6IP1-Cas9 stable cell line, the ARMER CRISPR/Cas9 (sc-409837) and ARMER homology-induced repair  plasmids were purchased from Santa Cruz Biotechnology. HEK293 cells (3 × 105 cells/well in a six-well plate) were cotransfected with 2 μg ARMER CRISPR/Cas9 and 2 μg ARMER-HDR plasmids using the Transporter 5 transfection reagent . After 24 h, the HEK293 cells expressing GFP and red fluorescent protein were selected using 5 μg/ml puromycin dihydrochloride treatment  for 2 wk and confirmed using RT-qPCR and western blot analysis. The cells used in this experiment were tested for mycoplasma contamination prior to use using the CycleavePCR Mycoplasma Detection Kit .HEK293, HeLa, U2OS cell lines (KCTC), and GFP-LC3B HeLa (generated GFP-LC3B-expressing cell line) were maintained in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum  and penicillin–streptomycin  in a humidified incubator with 5% COArl6ip1−/− and Arl6ip1+/+ mice were euthanized by carbon dioxide (CO2) inhalation and the embryos were separated from their placentas and washed with Dulbecco’s phosphate-buffered saline  three times to remove the residual blood. The embryonic bodies were minced, washed with sterile PBS, treated with 0.25% trypsin-EDTA  at 37°C for 5 min to dissociate the cells, and passed through a 40-μm cell strainer  to eliminate clumps and debris. Then, the passaged cells were centrifuged and maintained in DMEM with 10% FBS and P/S in a humidified incubator with 5% CO2 at 37°C; the medium was changed routinely every 2–3 d; cells used in this study were between passages 2 and 4.For MEF culture, E12.5–13.5 HEK293 cells were transfected with the indicated plasmid constructs using the Transporter 5 transfection reagent and U2OS, or HeLa cells were transfected using Lipofectamine 3000 Reagent  following the manufacturer’s instructions. ReNcell CX cells were harvested, resuspended in BTX electroporation buffer , and transfected using the ECM830 electroporation system (BTX). Electroporation was performed using one pulse at a 70 V discharge for 30 ms.GAPDH mRNA levels, relative quantification of gene expression was performed using the 2−ΔΔCT method  from cells and tissues. cDNA was synthesized from 1 μg of total RNA using a Verso cDNA Synthesis Kit  according to the manufacturer’s instructions. For RT-qPCR, the reaction mixture was prepared in a total volume of 20 µl containing 2 µl of template, 0.8 µl of each primer , and 10 µl of 2× Brilliant III SYBR Ultra-Fast qPCR Master Mix (Agilent). PCR was performed according to the manufacturer’s instructions  as follows: 95°C for 3 min, followed by 40 cycles of 5 s at 95°C and 10 s at 60°C. Melting curve analysis was performed under the following conditions: 30 s at 95°C, 30 s at 65°C, and 30 s at 95°C. After normalization against T method . The priBriefly, total RNA was isolated from cells and tissues using an RNeasy mini kit (Qiagen). cDNA was synthesized using a Verso cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The mRNA expression of target genes was analyzed by RT-PCR using specific primers. The PCR was conducted at 95°C for 3 min, followed by 25 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min, in a T100 PCR thermal cycler (BioRad). The primer sequences are provided in ARL6IP1 WT and KO MEFs (2–5 × 107 cells/assay) at passage 3 were lysed in PBST via sonication and centrifuged at 13,000 × g at 4°C for 10 min. Cleared cell lysates were prepared from equal amounts of ARL6IP1 WT and KO MEFs for the quantitative analysis of intracellular metabolites. These assays were performed using commercial acetyl-CoA  and α-ketoglutarate assay kits  according to the manufacturer’s instructions. The levels of intracellular acetyl-CoA and α-ketoglutarate were measured by converting a nearly colorless probe to a fluorescent product using a Spectra Max i3x microplate reader (Molecular Devices) at excitation and emission wavelengths of 535 and 587 nm, respectively. Detailed information regarding the assay kits used is provided in ARL6IP1 WT and KO MEFs (2–5 × 107 cells/assay) at passage 3, mitochondrial cholesterol content was determined using a total cholesterol assay kit , according to the manufacturer’s instructions. Detailed information regarding the assay kit used is provided in After isolation of mitochondrial lysates from ARL6IP1 WT and KO MEFs were determined using the Luminescent ATP Detection Assay Kit , according to the manufacturer’s protocol. Briefly, MEFs at passage 3 were seeded at a density of 5 × 104 cells/well in 24-well plates (SPL Life Sciences) for 24 h, lysed, and analyzed using a luminescence plate reader . Detailed information regarding the assay kits used is provided in Intracellular ATP levels in 5 cells/well) at passage 2 were seeded in XF24 cell culture microplates . After 24 h, the cells were incubated in 1 ml of XF DMEM supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate for 1 h at 37°C in a non-CO2 incubator. The mitochondrial respiration was measured using oligomycin (1.5 μM), carbonyl cyanide-p-trifluoromethoxy phenylhydrazone , and a mixture of rotenone/antimycin A  using an XFe24 analyzer.Cellular respiration (oxygen consumption rate [OCR] and extracellular acidification rate [ECAR]) was assessed using an XFe24 Extracellular Flux analyzer (Seahorse Bioscience). Briefly, MEFs , according to the manufacturer’s protocol. MEFs at passages 2–3 were cultured at a density of 3 × 104 cells in a confocal dish  for 24 h and then incubated with 100 nM Mtphagy Dye at 37°C for 30 min. After washing with PBS, the cells were treated with 10 μM CCCP for 6 h, incubated with 1 μM Lyso Dye at 37°C for 30 min, and washed with PBS. Colocalization of Mtphagy and Lyso Dye was observed using the fluorescence microscope  and CellSens imaging software (Olympus).Mitophagy was detected in ARL6IP1 KO mice  were anesthetized by intraperitoneal injection with avertin  and perfused with ice-cold PBS until the liver became clear (∼30 min) and then with a fixation solution  for 20 min at a low speed (2 ml/min). The spinal cord was dissected and fixed in the same solution overnight at 4°C. The fixed spinal cord was cut into 1-mm-thick sections, washed with ice-cold DPBS at 4°C, and fixed with 1% osmium tetroxide solution (Sigma-Aldrich) for 1 h at 4°C. To observe the contact surface between the mitochondria and ER, MEFs (3 × 106 cells) were grown in 10-cm dishes (>90% confluency) and fixed with fixation solution  overnight at 4°C. Cells scraped into ice-cold PBS were harvested via centrifugation at 1,500 × g for 2 min. The pelleted cells were uniformly coated with 2% agarose via gentle vortexing and fixed with 1% osmium tetroxide solution for 1 h at 4°C.To confirm the myelin morphology of the spinal cord via TEM, WT or Fixated cells and tissues were dehydrated with increasing concentrations of ethanol, infiltrated with propylene oxide, and embedded in EMbed-812 resin (EMS). Polymerization was performed at 60°C for 48 h and the sections were obtained using an ultracut EM UC7 ultramicrotome (Leica). The ultrathin sections (70 nm) were stained with uranyl acetate and Reynold’s lead citrate (Sigma-Aldrich), observed using FEI Tecnai G2 Spirit Twin TEM (FEI), and captured with NanoSprint 12 sCMOS camera (AMT Imaging). All images were obtained using ImageJ v1.57 .E. coli cells. Recombinant adenovirus packaging and amplification were performed in HEK293 cells (KCTC). For AAV production, we used a large-scale AAV9 packaging service and purified AAV9 for in vivo experiments from Vigene Biosciences.For adenovirus production using the AdEasy system (Agilent), a recombinant adenovirus plasmid was obtained by homologous recombination between the pCMV-Shuttle vector (Agilent), encoding the target gene and the adenovirus genome vector in BJ5183 16 and 1 × 1023 copies/ml for the qPCR standard curve. qPCR mixtures were prepared to a total volume of 20 µl with 2 µl of a template (plasmid DNA), 400 nM of each primer, and 10 µl of 2× Brilliant III SYBR Ultra-Fast qPCR Master Mix (Agilent) in a 96-well optical plate (Agilent). PCR was performed using EGFP and SV40 polyA primers according to the manufacturer’s instructions  as follows: an initial denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, and annealing or extension at 60°C for 30 s. The R2, slope, and intersection values were calculated from standard curves using the EGFP and the SV40 polyA primers. The primer sequences are provided in R2, slope, and intersection values are described in For the standard curve, circular and singly cut linear DNA plasmids were used as qPCR standards, and DNA concentrations were measured using Nanodrop spectrophotometers . The plasmid was diluted to the range between 1 × 10Cells were lysed on ice using RIPA buffer . For protein extraction, tissues were placed in ice-cold PRO-PREP buffer  containing protease inhibitor cocktail , homogenized, and treated with benzonase nuclease . The whole-cell or tissue lysates were separated on 12 or 15% SDS-PAGE gel and then transferred onto PVDF membranes (Merck Millipore). The membranes were incubated with the primary antibodies in PBST  overnight at 4°C. The membrane was then incubated with a secondary antibody in PBST-containing 0.5% skim milk for 1 h at 25°C. Proteins were visualized using a chemiluminescence kit .g for 1 min for buffer removal. The microarray result was detected by scanning with an Axon GenePix 4000A Microarray Scanner (Molecular Devices) and acquired using GenePix Pro6.0 software (Molecular Devices).Protein microarrays were performed using a HuProt human proteome microarray version 4.0 . The protein chip was blocked with 2% BSA in PBST (1× PBS with 0.1% Tween 20) on an orbital shaker at 4°C for 2 h. After washing three times with PBST, 9 μg His tagged ARL6IP1 was treated on the microarray and incubated overnight at 4°C. The microarray was incubated with 1 μg streptavidin-fluorescence (Alexa-Fluor 532) for 1 h at 4°C. After three washes with PBST, the array was centrifuged at 600 × 6 cells) were lysed in NET gel buffer  by sonication, and cell lysates cleared by centrifugation  were incubated with 2 µg/reaction of anti-ARL6IP1, anti-LC3B, and anti-p62 antibodies in PBST overnight at 4°C and immobilized onto Protein A magnetic beads  for 2 h at 4°C with gentle rotation. The precipitates or bead-bound proteins were washed thoroughly with PBST three times and analyzed using western blotting.Whole-cell lysates and reaction mixtures after the in vitro assay were incubated with glutathione Sepharose 4B beads  for 4 h at 4°C with gentle rotation. For immunoprecipitation assays, cells  for 3 h at 37°C. Cells were lysed in lysis buffer  by sonication, centrifuged at 15,000 × g at 4°C for 20 min, and then the supernatants were incubated with glutathione Sepharose 4B (Sigma-Aldrich) for 3 h at 4°C. The bead–protein complexes were immediately washed with 5–10 column volumes of PBS and eluted in buffer . Six His-tagged proteins were produced in BL21DE3 E. coli and purified following the manufacturer’s instruction (Qiagen). Bacterial cells were grown at 37°C to reach an OD600 nm of 0.6 and then induced by incubation with 1 mM IPTG for 24 h at 20°C. Cells were lysed in lysis buffer  by sonication, and cleared supernatants via centrifugation were incubated with Ni-NTA agarose  for 3 h at 4°C. The bead–protein complexes were washed in washing buffer  and eluted in elution buffer . Purified GST or His proteins were dialyzed in dialysis buffer  overnight at 4°C.For the purification of GST fusion proteins, GST proteins were produced in BL21DE3 ARL6IP1 and LC3-like genes were subcloned into pGEX4T1 and pET28a, respectively. The purified recombinant proteins from E. coli (each 0.5 μg protein/reaction) were incubated in PBST overnight at 4°C on a rotating platform. After incubation, the bound proteins were pulled down using glutathione Sepharose 4B beads (Sigma-Aldrich) for 3 h at 4°C, washed thrice with PBST, and analyzed using western blotting with the indicated antibodies.For the in vitro binding and pull-down assays, 6 cells), and the whole-cell lysate was carefully layered on the top of the gradient and centrifuged at 100,000 g rpm at 4°C for 16 h. Fractions of density gradient layers (#1–#12) were collected and analyzed using western blotting.OptiPrep Density Gradient Medium  was diluted to 30% in ice-cold PBS, while 30% iodixanol solution (high density) was put in one chamber connected through a stopcock to a second chamber; 5% iodixanol solution (low density) was placed in the second chamber. The iodixanol solution flowed from the first chamber into the second, while a continuous gradient was formed by gravity in polypropylene centrifugation tubes (Hitachi). Cells were lysed on ice using RIPA buffer (500 μl per 5 × 10ARL6IP1 KO and WT MEFs (2 × 107 cells) were harvested, lysed in lysis buffer , and centrifuged at 7,000 × g for 10 min at 4°C. The supernatant was then mixed with an equal volume of OptiPrep (60% wt/vol iodixanol). Iodixanol gradients were generated using OptiPrep solution and lysis buffer and layered as follows: 2.2 ml at 30%, 2.2 ml at 25%, 2.2 ml at 20%, 1.8 ml at 15%, 1.8 ml at 10%. Additionally, 0.45 ml represented 5%, and 0.45 ml represented 0%. The gradients were centrifuged at 150,000 × g for 3 h at 4°C, and subsequently, 13 fractions (each 0.85 μl) were collected from the top and analyzed using western blotting.Flotation analysis was performed as previously described . BrieflyThe pBiFC-VN155 (#27097) and pBiFC-VC155 (#22011) plasmids were purchased from Addgene; recombinant plasmids encoding ARL6IP1 and ATGs were cloned into the pBiFC-VN155 or pBiFC-VC155 plasmids for the BiFC assay. Detailed information on the plasmid constructs is provided in Apoptosis was detected with Muse Annexin V & Dead Cell Reagent  using a Muse Cell Analyzer (Merck Millipore). ReNcell CX cells were transduced with adenovirus encoding ARL6IP1-shRNA or scrambled-shRNA at 200 multiplicity of infection (MOI) for 48 h and cultured in ReNcell NSC maintenance medium for 2 d. Cells were then harvested, stained with annexin V and 7-AAD according to the manufacturer’s instructions, and analyzed using a Muse Cell Analyzer. Apoptosis and cell death rates were reported as percentages of the total cell number.4 cells/well) at passages 3 and 5 were seeded in 96-well black-bottom plates coated with poly-L lysine . After 24 h of incubation, the number of senescent cells was determined based on β-galactosidase activity using the Senescence β-Galactosidase Activity Assay Kit , according to the manufacturer’s protocol. The fluorescence intensity  was detected using a Spectra Max i3x microplate reader (Molecular Devices) equipped with the Max Pro software.For measurement of MEF senescence, MEFs (2 × 105 cells/well) in 6-well plates (SPL Life Sciences), and after 24 h, the cells were transduced with an adenovirus encoding ARL6IP1 or LacZ at 100 MOI for 48 h. The cells were harvested and the autophagy induction ratio was determined following the manufacturer’s instructions.Autophagy was determined using the Muse RFP-LC3 Reporter Autophagy Assay Kit and Muse Cell Analyzer (Merck Millipore). HeLa cells were seeded (2 × 10ARL6IP1 WT and KO MEFs were seeded (5 × 104 cells) in confocal dishes , treated with 10 µM CCCP (Sigma-Aldrich) for 6 h, and incubated with 1 μM JC-1 for 20 min at 37°C. JC-1 staining images were obtained using a fluorescence microscope  and CellSens imaging software (Olympus).MMP was determined using a JC-1 probe  according to the manufacturer’s instructions. MEFs were cultured in 96-well black bottom plates (Corning Inc.) coated with poly-L lysine overnight, treated with CCCP for 6 h, and incubated with 100 μl of 1 μM JC-1 per well for 20 min at 37°C. To quantify the monomers and aggregates of JC-1, the fluorescence was measured using a Spectra Max i3x microplate reader (Molecular Devices) at excitation and emission wavelengths of 485 and 535 nm, respectively, for JC-1 monomer and at 485 and 595 nm, respectively, for JC-1 aggregates.2+ concentration was visualized using Rhod-2 AM , a fluorescent calcium indicator. Briefly, ARL6IP1 WT and KO MEFs at passage 3 were seeded (5 × 104 cells) in a confocal dish , cultured for 24 h, and incubated with Rhod-2 AM (4.5 µM) and MitoTracker Green FM  in complete medium for 30 min at 37°C. After replacing with fresh medium, live cells were imaged using a fluorescence microscope  and CellSens imaging software (Olympus).The mitochondrial CaCells or frozen tissue sections were fixed with 4% PFA overnight at 4°C, washed thrice in PBS, and permeabilized with 0.5% Triton X-100 in PBS for 15 min at 25°C. Thereafter, blocking was performed with 2% normal horse serum in PBS for 1 h at 25°C. Cells were incubated with primary antibodies, described as For immunofluorescence staining of primary rat hippocampal neurons, hippocampal neurons at 9 d after transfection were fixed, permeabilized, and incubated with anti-EGFP antibody and fluorophore-conjugated secondary antibodies . Individual neurons were imaged using confocal laser scanning microscopy (LSM800) at 200×. Primary and secondary dendrites were quantified as previously described .8 cells) at passages 2–3 were lysed in lysis buffer  by sonication and centrifuged at 600 × g at 4°C for 5 min. The pellets were collected as the nuclear fraction. The supernatants were centrifuged at 10,000 × g at 4°C for 10 min to separate the crude mitochondrial fraction and the pellet containing the cytosolic/microsomal fractions. The supernatants were further centrifuged at 20,000 × g at 4°C for 1 h to collect the microsomal fractions. The crude mitochondrial fraction was resuspended in mitochondrial suspension buffer  and separated via Percoll gradient ultracentrifugation to obtain the pure mitochondria and MAM fraction. The protein concentration from each subcellular fraction was measured via absorbance at 280 nm, calculated using a Nanodrop spectrophotometer, and analyzed using western blotting.Isolation of mitochondria and MAM fractions from cells was performed as previously described . BrieflyFor footprint analysis, mice paws were put with nontoxic water-based ink (front paws in red and hind paws in blue ink), and subsequently, the mice were allowed to walk down an enclosed runway lined with white paper. The trials were repeated three to five times per mouse, and hind stride, hind sway, and stance length (the distance between the left and right forelimb stride) were measured in the middle part of each run.Hindlimb reflexes were observed in the hindlimb position after grasping the tail for 10 s and scored on a scale of 0–3 with a degree of splaying outward from the abdomen. A score of 0 represents hindlimbs fully spread and moving; 1, a retraction toward the abdomen for >50% of one hindlimb; 2, a retraction toward the abdomen for >50% of both hindlimbs; and 3, both hindlimbs fully retracted to the abdomen.For the collection of the adult mouse brain or spinal cord tissues, mice were anesthetized by intraperitoneal injection using avertin  and perfused with ice-cold PBS to flush the blood and blood cells from the brain, followed by perfusion with 4% PFA solution for fixation. Thereafter, the organs were fixed in 4% PFA overnight at 4°C and immersed in 30% sucrose for 72 h for osmotic dehydration. Fixed tissues were embedded in Tissue-Tek OCT compound , sectioned at 5 μm using a cryostat , and analyzed using immunofluorescence staining with appropriate antibodies. Detailed information on antibodies used is provided in http://atlas.brain-map.org), and reference images were generated from an adult mouse 3D corona averaged template.Individual mouse brain atlas was downloaded from the Allen Brain Atlas  were collected from the hepatic portal vein and serum was obtained via centrifugation at 7,500 × g for 5 min at 4°C. All CSF and serum samples were aliquoted and stored at −80°C until use.Mice were anesthetized using avertin  and the CSF was immediately collected from the cisterna magna (5–10 μl CSF/25 g mouse). The pooled CSF samples were centrifuged at 7,500 × n = 8/group) was measured using a Total Neurofilament-L Kit , according to the manufacturer’s instructions. Then absorbance was measured at 450 nm using a Spectra Max i3x microplate reader (Molecular Devices).CSF and serum samples were analyzed using a proteome profiler mouse cytokine array kit  according to the manufacturer's instructions. NF-L level in CSF and serum  containing B-27 supplement  and MEM nonessential amino acid solution .Cortical neurons were isolated from the E14.5 mouse brain and then dissociated by trypsinization . Clump tissues were removed by a 40-μm cell strainer, and cells were centrifuged at 150 × Primary hippocampal neurons were prepared as previously described . HippocaSholl analysis was performed employing the modified method as described previously . Individ13 Vg/ml, each ARL6IP1 and GFP) per the primary motor cortex (M1) was delivered using an injection syringe . An injection was regulated by a syringe pump  at a constant volume of 0.2 μl/min , respectively. After the injection, the needle was kept in place for 5 min to minimize the upward flow of the virus solution. The coordinates for injection of the motor cortex region were calculated as anterior–posterior; −0.7 or −1.4, medial–lateral; +1.4, and dorsal–ventral; +1.6 relative to the bregma = 0.Mice were anesthetized by intraperitoneal injection using avertin  and fixed in a stereotaxic frame (David Kopf Instruments). The injection position was marked and the skull was drilled. Then, 1 μl AAV9 virus  or mean ± SEM (n > 10).All statistical analyses were performed with GraphPad Prism 7.0 software. The Student’s Arl6ip1−/− mice model. Arl6ip1−/− mice. Arl6ip1−/− or Arl6ip1+/+ mice after AAV9 gene delivery. Arl6ip1−/− mice versus Arl6ip1+/+ mice, respectively.All animal housing and experiments conducted were in accordance with the KRIBB Institutional Animal Care and Use Committee Guidelines (KRIBB-AEC-18090). The study involved no human participants.Table S1provides a summary of clinical features with ARL6IP1 mutations.Click here for additional data file.Table S2lists the antibodies used in this study.Click here for additional data file.Table S3lists reagents and kits list used in this studyClick here for additional data file.Table S4shows the primer list for genotyping of C57BL/6N-Arl6ip1(tm1a) mice.Click here for additional data file.Table S5lits plasmid constructs used in the study.Click here for additional data file.Table S6shows the primer list for RT-qPCR.Click here for additional data file.Table S7shows the primer list for conventional RT-PCR.Click here for additional data file.Table S8shows the primer sets used in qPCR for titration of AAV genome copy numbers.Click here for additional data file.Table S9lists R-square, slope, and intersection values of the qPCR.Click here for additional data file.Table S10lists titration of AAV9 genome copy number using SV40 primer to mouse tissues.Click here for additional data file.SourceData F2is the source file for Fig. 2.Click here for additional data file.SourceData F3is the source file for Fig. 3.Click here for additional data file.SourceData F4is the source file for Fig. 4.Click here for additional data file.SourceData F5is the source file for Fig. 5.Click here for additional data file.SourceData F7is the source file for Fig. 7.Click here for additional data file.SourceData FS1is the source file for Fig. S1.Click here for additional data file.SourceData FS2is the source file for Fig. S2.Click here for additional data file.SourceData FS3is the source file for Fig. S3.Click here for additional data file.SourceData FS4is the source file for Fig. S4.Click here for additional data file.SourceData FS5is the source file for Fig. S5.Click here for additional data file.


This system efficiently produces H2 (14μmol h–1) and O2 (6.9 μmolh–1) at an ideal molar ratio of 2:1 in an aqueoussolution driven by light, resulting in a remarkably high apparentquantum yield of 6.06% at 420 nm under neutral conditions. This exceptionalselective H2 and O2 production is due to thepreferential adsorption of iodide (I–) on Na0.56WOx3– and iodate (IO3–) on WO3, which is evidencedby both experiments and density functional theory calculation. Thepresent liquid Z-scheme in the presence of efficient shuttle moleculespromises a separated H2 and O2 evolution byapplying a dual-bed particle suspension system, thus a safe photochemicalprocess.The stoichiometric water splitting using a solar-drivenZ-schemeapproach is an emerging field of interest to address the increasingrenewable energy demand and environmental concerns. So far, the reportedZ-scheme must comprise two populations of photocatalysts. In the presentwork, only tungsten oxides are used to construct a robust Z-schemesystem for complete visible-driven water splitting in both neutraland alkaline solutions, where sodium tungsten oxide bronze (Na A single photocatalyst with appropriatecocatalysts for pure water splitting has thus met with very limitedsuccess due to stringent bandgap requirements.6 Inparallel, there are many studies on the half-reaction of either protonreduction or water oxidation in the presence of an efficient but costlychemical scavenger. Typically, BiVO47 or WO38 for wateroxidation and C3N49 or Rh dope SrTiO310 for protonreduction were reported. A Z-scheme, also known as a dual photoexcitationsystem, akin to natural photosynthesis comprising H2 evolutionphotocatalyst (HEP) and O2 evolution photocatalyst (OEP),should be more efficient and economical for overall water splittingthan the single photocatalyst with complicated cocatalysts.12 It offers an extended choice of semiconductor materials with a narrowbandgap for both half-reactions, enabling it to achieve high solar-to-hydrogenconversion efficiency (STH). Furthermore, it has been predicted thata maximum of 12% STH can be achieved using a single absorber whereasit can be upraised significantly to 22% for Z-scheme-based systemsdue to much better visible light harvesting.13 Typically, HEPs with the more negative conduction band (CB) potentialconcerning proton reduction (0.0 V vs NHE) and OEPs with the morepositive valence band (VB) potential concerning water oxidation (1.23V vs NHE) are suitable for H2 and O2 evolutions,respectively. The semiconductor particulate suspension-based Z-schemesystem utilizing soluble redox mediator has been highly focused asit is much more simple and cost-effective than the solid Z-scheme,14 and more importantly promises to produce H2 and O2 separately in a dual-bed particle suspensionsystem on a large scale, guaranteeing a safer chemical process comparedwith others, e.g., a solid Z-scheme.17Hydrogen  and Pt-TiO2 (rutile) for H2 and O2 evolutions,respectively, in the presence of IO3–/I– redox mediator, while under ultraviolet (UV)light.20 Subsequently, several narrow-bandgapsemiconductors viz. cation-doped SrTiO3, graphitic carbonnitride (g-C3N4), Sm2Ti2S2O5, etc., as HEPs, and BiVO4,WO3, H2WO4, AgNbO3, TaON,etc., as OEPs have been explored and implemented in a Z-scheme systemto split water into stoichiometric amounts of H2 and O2.22 Notably, surface-modifiedoxynitrides were described to improve the water splitting significantlyby absorbing visible light effectively and suppressing the chargecarrier recombination; however, oxynitrides are self-photocorrosive,leading to poor photostability.23 In anotherreport, a heterojunction based on oxynitrides (Pt-loaded MgTa2Ox6– Ny/TaON) as a HEP was shown to suppress charge carrier recombination,resulting in a drastic enhancement in overall water splitting witha benchmark apparent quantum yield (AQY) of 3.4% at 420 nm when consideringa 2-electron process for H2 production, in combinationwith PtOx/WOx as an OEP in the presence of IO3–/I– as redox mediator.24 Similarly,the AQY of a Z-scheme photocatalytic system at 420 nm was often measured.29 Though substantial advances have been made so far, a significantbreakthrough is yet to come. Hence, intensive research is underwayby developing new materials30 with specialsurface properties, high crystallinity, fewer defects,31 spatial separation of reduction and oxidationsites, cocatalyst loading,32 controlledmorphology, heterojunction formation, etc., to achieve a high AQY.Several reports have successfully demonstrated the conceptof dualsemiconductor photocatalysts suspended in an aqueous solution containingredox couples such as Fe0.56WO x3–) as the HEP and two-dimensional(2D) WO3 nanosheets as the OEP in the presence of IO3–/I– redox couple. Tungstenoxide-based materials have been widely studied but only as photoanodesor water oxidation photocatalysts due to their conduction band beingtoo positive to meet proton reduction.34 Moreover, WO3 absorbs ∼12% of thesolar spectrum and possesses a moderate hole-diffusion length (∼150nm) in addition to impressive electron mobility (∼12 cm2 V–1 s–1).34 Numerous stable nonstoichiometric WO3 materials could be realized by altering the lattice oxygen vacanciesto attain interesting optoelectronic/catalytic properties. To thebest of our knowledge, a Z-scheme system composed of one type of oxidephotocatalysts for visible-driven-pure water splitting has not beenreported. Very recently upraising the CB position of WOx to a more negative potential was shown, e.g., bycreating oxygen vacancies35 or surfacedefects engineering.36 Here, we prepareda new and stable WOx-based HEP, Na0.56WOx3–. Interestingly,it can be coupled with WO3 nanosheets as an OEP to forma robust Z-scheme, which demonstrates efficient pure water splittingunder light irradiation. Compared with the bulk materials, WO3 nanosheets provide more exposed active sites due to theirsheet-like morphology. Such a novel Z-scheme generates H2 and O2 stoichiometrically, demonstrating a high AQY of6.06% at 420 nm, under light irradiation and ambient conditions.Herein, we have developed a new tungsten oxide-only Z-scheme (WOZ)system for overall water splitting, in which both the HEP and OEPare tungsten oxides, i.e., sodium tungsten oxide bronze  , matching well with the standard pattern (PDF No. 01-75-0237).The Na content present in the annealed NaxWOx3– was determined to be around0.56 using a microwave plasma-atomic emission spectrometer, whichis also verified using the experimentally (XRD) measured lattice parameter(ao) according to the 38 so this sampleis denoted Na0.56WOx3–.NaFigure S2 representstransmission electronmicroscopy (TEM) images of the as-synthesized HEP, indicating thatthe sample is composed of interconnected spheres with dimensions around100–150 nm. The high-resolution TEM (HRTEM) image of the as-synthesizedsample shows the absence of crystallinity, demonstrating the amorphousnature. After annealing, the amorphous sample becomes crystalline,which is obvious from the TEM image , and the Pt nanoparticles distribution on the surface of the Na0.56WOx3– reveals the successfulcocatalyst loading. Furthermore, the new crystalline Na0.56WOx3– lattice was identified as(100), and the photodeposited Pt nanoparticles show (200) lattice. The elemental mapping of Na0.56WOx3– shows the uniformdistribution of Na, W, and O elements (0.56WOx3– were assessedusing X-ray photoelectron spectroscopy (XPS). The survey spectrumis shown in Figure S5. 0.56WOx3–. As shown in 7/2 and 4f5/2) in the W-4fspectra of Na0.56WOx3– unveils the presence of tungsten in multiple oxidation states. Forinstance, it shows peaks at binding energy (BE) values around 33.3and 35.4 eV, 34.8 and 36.9 eV, and 35.5 and 37.6 eV. The first setof doublets corresponds to the presence of W4+, while thesecond and third doublets disclose the presence of W5+ andW6+ species, respectively. The deconvoluted O-1s XPS consistsof three peaks at BE values 530.4, 531.6, and 533.1 eV37 (2–) surrounded by W atoms in the Na0.56WOx3– lattice. The originof the peak at 531.6 eV is due to oxygen (OH–) (theadsorbed OH–) in the regions of oxygen vacancies(due to W5+/W4+ species) within the Na0.56WOx3– matrix. In these oxygen-deficientsites, OH groups are known to bond with metal cations to compensatefor the charge. The peak located at 533.1 eV could be due to the presenceof adsorbed H2O on the surface of Na0.56WOx3–.37 Thedeconvoluted Na-1s XPS shows a single peak at 1071.2 eV .2.1.23 nanosheets as an OEP in the Z-scheme system wereobtained by thermal annealing of 2D tungstic acid (WO3·H2O) under an air atmosphere at 500 °C for orthorhombic (Pmnb) hydrated WO3 withstrong diffraction peaks for (111), (020), and (131) planes, in agreementwith the earlier report.402D WOP21/n) WO3 nanosheets is observed, corresponding to the standardpattern (PDF No: 00-043-1035). It confirms the transformation of orthorhombicWO3·H2O into monoclinic WO3 dueto annealing . This phenomenonis further verified by Raman spectroscopy, as shown in 3·H2O show three peaks at ∼642, ∼820,and ∼946 cm–1, corresponding to the firstW–O–W, [ν1(W–O–W)], second W–O–W[ν2(W–O–W)], and terminal W=O stretchingvibrations, respectively. The sharp peaks at ∼642 and ∼946cm–1 confirm that the as-synthesized nanosheetsare hydrated WO3 (WO3·H2O).The Raman spectra of WO3 nanosheets /Iν(W=O) can provide information about x in WO3·xH2O as suggestedearlier.40 As expected the ratio is lowerfor WO3·H2O, while it is higher for WO3 nanosheets, validating the dehydration of WO3·H2O. The layered morphology of WO3·H2O nanosheets is evident from the TEM images, as shown in Figure S7. The EDX spectra validate that thematerial is mainly made of W and O elements. 3, which shows the presence of Pt nanoparticles on the surface ofthe WO3 mixture composed of nanosheets and nanoparticles(denoted WO3 nanosheets afterward). The deconvoluted W-4fXPS of WO3 shown in 3. The presence of W+6 also confirms the formation of WO3 upon annealing of WO3·H2O, complementingXRD, and Raman measurements.After annealing it in the air at 500 °C for3 h, the formationof monoclinic  = EAg/AgCl + EAg/AgCl0. The WO3 has a CB potential of +0.2 V, whichis consistent with the reported42 and theNa0.56WOx3– has CB at−0.2 V. To validate it further, we have tested the CB potentialof the Na0.56WOx3– andWO3 using the Mott–Schottky method , and the observed results agree withthe photocurrent onset potential measurement. In addition, we haveperformed a control experiment to test the water oxidation propertyof Na0.56WOx3– in thepresence of IO3– and observed a tinyamount of O2 evolved (0.3 μmol h–1), 60 times less active compared to WO3 (17.9 μmolh–1). Such a small O2 evolution rateof Na0.56WOx3– revealsthe less positive VB potential of Na0.56WOx3– compared to WO3. Thus, it is essentialto engineer a Z-scheme composed of Na0.56WOx3– and WO3 for complete water splitting.Accordingly, the energy band diagram was drawn, as shown in 3 is too positive to reduce protons into H2, whichis essential to form an efficient Z-scheme system as it is close tothe VB of the HEP when coupling Na0.56WOx3– with WO3. The deep VBM of WO3 nanosheets and negative CBM of Na0.56WOx3– suggest that it is highly suitable for water oxidation,and reduction reactions, respectively. More importantly, these well-matchedband positions promise a new Z-scheme composed of tungsten oxides.UV–Visdiffuse reflectance spectroscopy (DRS) was used to disclose the bandgapof the investigated photocatalysts. The optical absorbance spectraof Na2.20.56WOx3–-based materials for H2 production, experimentswere performed in aqueous solutions containing NaI (pH ∼7.0)as a hole scavenger under Xe lamp irradiation, and the results areshown in 0.56WOx3– can produce H2 without any cocatalysts,which is otherwise difficult with WO3-based materials dueto the inappropriate CB position concerning the H2 evolutionpotential, suggesting that both NWO and Na0.56WOx3– can be used as a photocatalyst for H2 production from water. Both NWO (amorphous) and Na0.56WOx3–  photocatalystsproduce H2 at a rate of 2.2 and 2.5 μmol h–1, respectively. Subsequently, the effect of pH on H2 evolutionwas studied to optimize the condition at which NWO-based materialsyield higher rates of H2 production. As shown in Figure S11, the Na0.56WOx3– shows better activity than NWO from neutral to weaklyalkaline solution, where the H2 evolution rate was obtainedfrom the first 6 h run. Furthermore, we have tested the chemical stabilityof Na0.58WOx3– for H2 evolution at pH 8.5 and 10.5 by running four 6 h reactioncycles . The initial rate ofH2 evolution does not change significantly at both pH values,but from the third cycle onward, the H2 evolution ratestarts to reduce and saturates in the fourth cycle only at pH 10.5.This suggests that the present HEP is highly stable under a weaklyalkaline condition but not stable at a strong alkaline condition fora prolonged run. Hence, well-crystallized Na0.56WOx3– was selected for further studies.To improve the activity, 3wt%-Pt  particles were loadedon the surface of Na0.58WOx3– by photodeposition , the best activity wasobserved at pH 7.0. This is anticipated as the cocatalysts extractthe electron efficiently and minimize the recombination with holes.The H2 evolution reaction in the presence of I– ions at pH 7.0 can be represented by the following equations.To verify the photocatalytic ability of Na0.56WOx3– for prolonged H2 evolutionwas investigatedby measuring the H2 evolution for 21 h (composed of threeconsecutive 7 h runs) and no significant change in the H2 evolution rate is observed, as shown in 0.56WOx3– under neutral pH. We furthervalidated the stability of the HEP by testing the compositional changesusing XPS after three consecutive photocatalytic runs, as shown in Figure S13. No obvious changes were observedin the deconvoluted spectra of O-1S and Na-1S; however, a small reductionin the W4+ signal was observed, while the signals for W5+ and W6+ are stable. The identical H2 evolution suggests that W5+ and W6+ are thespecies dominating water reduction under the present experimentalconditions. Furthermore, the XRD spectra of the 3%Pt-Na0.56WOx3– before and after the photocatalysisshow  no significant changes,which again confirms the robustness of the HEP under neutral pH.The stability of the 3%Pt-Na2.32 evolutionhalf-reaction using WO3 under a full-arc condition in watercontaining IO3– ions as a sacrificialelectron acceptor. As expected, a significant amount of O2 evolution was observed at ∼pH 8.5 using both WO3·H2O and WO3, but monoclinic WO3 nanosheets show better activity than orthorhombic WO3·H2O , leading to an O2 evolution rate of 17.9 μmol h–1, which issuperior to cocatalyst-free WO3 (3.8 μmol h–1) and WO3·H2O (2.2 μmol h–1). The present Pt-WO3 water oxidation activity is comparableto the best activity reported previously,45 thanks to theadvantages of nanosheet morphology. The O2 evolution usingPt-WO3 from weakly alkaline water containing IO3– ions can be represented by the following equations.Subsequently, we investigated the photocatalytic O WO3·H2O 4b. There3–), no O2 evolution is observed. The H2 evolution activityof the Pt-WO3 was also monitored using I– as the hole scavenger to examine the suitability of WO3 for a potentially efficient Z-scheme for water splitting. As anticipated,negligible H2 evolution is observed , in agreement with the CB position of WO3 measured in 3 is highly selective for the water oxidationhalf-reaction. The stability of Pt-WO3 nanosheets for prolongedO2 evolution at pH 8.5 was examined for 21 h (composedof 3 consecutive 7-h runs), and no significant decrease in O2 evolution rate is observed, as shown in 3 is a robustphotocatalyst under weakly alkaline conditions. In addition, no changeswere observed in the XRD of Pt-WO3 before and after photocatalysis, which further ensures stability.Furthermore, we also tested the stability of WO3 at pH10.5 and observed no change in the beginning of the reaction, similarto the HEP, but over a prolonged run, the O2 evolutionrate was significantly reduced . This strongly suggests that the present OEP is stable only underweak alkaline pH and not stable in strong alkaline solutions.In the absence of a photocatalyst oran electron acceptor  has been found to produce H2 andO2 gases simultaneously with a rate of 14 μmol h–1 (2800 μmol h–1 g–1) and 6.9 μmol h–1 (1380 μmol h–1 g–1), respectively, at a stoichiometricmolar ratio of 2:1  activity at pH10.5 and observed no stoichiometric gas evolution at a 1:1 mass ratio was also measured, as shown in Figure S17. Again, the simultaneous productionof H2 and O2 with a rate of 7.4 and 3.6 μmolh–1 is monitored, and the gas amount produced remainsalmost linear increase with time. The H2 production activityof the Na0.56WOx3– hasbeen tested under half-reaction conditions at higher wavelengths using600 and 650 nm monochromatic filters. The obvious H2 evolutionwas observed under half-reaction conditions with a rate of 0.9 and0.27 μmol h–1, respectively , but under Z-scheme working conditions, pure watersplitting was not observed (generation of both H2 and O2) as WO3 is silent at these very long wavelengths.The AQY for H2 production from water using the proposedWOZ system was assessed three times and the average was reported.At 420 nm, an AQY of 6.06% for water splitting was determined by considering the 2-electron processinvolved for one molecule of H2 evolution . The H2 and O2 evolution rates under420 nm monochromatic irradiation are shown in Figure S20. The Z-scheme photocatalytic activity of the presentworks was compared with the reported representative studies (Table S2).The temporal gas evolution is shown in ss ratio1, run 9,ss ratio1, run 10ss ratio1,run 112.52 production in the presence of hole scavenger I– . One can see that the H2 is produced on the 3%Pt-Na0.56WOx3– but not on the 0.5%Pt-WO3 nanosheets,proving that the CB of WO3 nanosheets is not appropriatefor proton reduction while the CB of Na0.56WOx3– is negative enough to reduce the proton to H2  might compete with water oxidation by consuming the photogeneratedholes from the OEP. We examined this by adding 1 mM I– anions at 3 h during the ongoing water oxidation half-reaction inan aqueous solution (pH ∼8.5) containing 5 mM IO3. Interestingly, no influence was observed by the extra I– anions on the water oxidation reaction , strongly indicating that the nanosheets OEP can produceO2 highly selectively even in the presence of efficienthole scavenger I–. To disclose the reason behindthis, we have studied the adsorption behavior of IO3– and I– anions onto the OEP by measuringtheir concentrations before and after the addition of 0.5%Pt-WO3 powder to the solution under dark conditions driven by the photogenerated electrons on 3%Pt-Na0.56WOx3–, HEP. As shown in Figure S22, we added two distinct concentrations of IO3– (100 μM and 1 mM) after 3 h duringthe ongoing water reduction half-reaction and noticed no significantchange upon the addition of 100 μM of IO3–. However, a substantial deceleration of H2 evolutionwas observed when adding 1 mM IO3–. Theseresults suggest that at higher IO3– concentrations,the photogenerated electrons from the HEP are also consumed by IO3– ions, in good agreement with the earlierreport20 where a similar phenomenon wasobserved.As shownin – and IO3– on 3%Pt-Na0.56WOx3–. As shownin – and IO3– anionsare adsorbed on the surface of the HEP, but I– ionsshow relatively high preference, which is also proved by our XPS measurementof the used photocatalyst and is crucial for the Z-scheme water splitting. The adsorption behaviorof I– and IO3– anionson bare Na0.56WOx3– wasalso undertaken . It is obviousthat the I– ions are preferentially adsorbed especiallyat low concentrations. Therefore, the sufficient adsorption of I– on Na0.56WOx3– enables to accept the photogenerated holes, reducing the recombinationwith photogenerated electrons and hence promoting the selective H2 evolution. However, beyond 1 mM concentration, IO3– adsorption is dominated. Hence, in the presenceof excess IO3– ions, H2 evolutionis limited, which agrees well with our control experiment . We also found out that only O2 is produced in the WOZ with only the IO3– mediator . Therefore, controlling the concentration of IO3– ions in the WOZ is one key issue, which fromanother angle proves that the initial addition of only I– mediator instead of both I– and IO3– mediators is of importance as demonstrated inour experimental design. We also found that continuous productionof H2 and O2 for a prolonged time in our WOZis stable and reproducible and the concentration of I– and pH were observed to be constant, demonstrating a highly selectiveand efficient Z-scheme system. A similar experimental study was reportedpreviously48 in which the selective adsorptionof IO3– anion on WO3 (OEP)was observed, leading to the selective O2 evolution reaction.To understand further, we observed the competitiveadsorption behaviorof Imediator 1, run 3,catalyst S13aand 3Pt4 and Na0.625WO2.875Pt4 is detailed in the Supporting Information. IO3– and I– wereadsorbed onto each catalyst at two sites: over the platinum clusterand directly onto the surface of the catalyst. The optimized catalystmodels, as well as the adsorption models, can be seen in Figures S24–S31. Adsorption energies arecalculated using E represents the enthalpy, A is the adsorbent,B is the adsorbate, and AB is the complex structure.To consolidate the adsorption behavior observed experimentally,the redox molecules′ adsorption on photocatalysts was alsostudied by DFT calculation. The description of the model for WO3Pt4, iodate is more easily adsorbed than iodide . For the Na0.625WO2.875Pt4 catalyst, it was found that both iodate and iodidewere adsorbed onto the catalyst; however, iodide was adsorbed to agreater degree onto the catalyst. In each case, iodate adsorbed morepreferentially onto the Pt cluster for subsequent reduction reactions,while iodide adsorbed more easily onto the catalyst surface for subsequentoxidation reactions. It should be noted that for both catalysts, therelaxation of iodate led to the stretching of the I=O bondsin every case, even when constraints were applied and, in most cases,the O atom dissociated. Overall, the DFT calculation results suggestthat the preferential adsorption of IO3– on OEP and I– on HEP would be the reason for theenhanced selective H2 and O2 evolution usingthe present Z-scheme.The calculated adsorption energiesfor each tested adsorption siteare shown in 0.56WOx3– and 0.5%Pt-WO3 powders in an aqueous solution to produce H2 andO2, simultaneously. However, no gas evolution is noted by mixing 3%Pt-Nais noted1, run 113–/I–. Upon irradiation, photoexcited electrons from Pt-Na0.56WOx3– transfer to Ptactive sites, where proton reduction occurs to produce H2, and the corresponding holes oxidize I– ions intoIO3–, which gets regenerated back toI– by the photogenerated electrons on Pt-WO3, while photogenerated holes on Pt-WO3 performwater oxidation to generate O2, resulting into a completecycle.Based on the above results, we proposed the reactionpathway forthe new WOZ system schematically in 33–/I– redox couple.Such a design enables separately and readily optimization of eachphotocatalyst and the cocatalyst. So the stepwise independent H2 and O2 evolution half-reactions optimization wascarried out to minimize/eliminate undesirable reactions. The bandposition measurements indicate a matched electronic structure forthe new Z-scheme. Then, for the first time, the WO3-basedZ-scheme has been validated for visible-driven complete water splittingby a double excitation mechanism. This novel particulate WOZ exhibits6.06% AQY at 420 nm when considering the electron number for H2 production as 2. Furthermore, I– was foundto be favorably adsorbed on Na0.56WOx3– and IO3– on WO3 nanosheets by both experimental runs and theoretical modeling,being crucial for an efficient Z-scheme system. The low cost and ensuredstability of tungsten oxides are also highly appropriate to subseqentscaling up.In summary, we have demonstratedthe original tungsten oxides-onlysuspension Z-scheme for efficient photocatalytic pure water splittingunder the full-arc and visible light conditions in the presence ofthe IO44.12WO4·2H2O), sodiumborohydride (NaBH4), sodium iodide (NaI), and sodium iodate(NaIO3) were purchased from Sigma Aldrich and used as received.All solutions were prepared using deionized (DI) water. Other chemicalsused for experiments were purchased from commercial sources and usedwithout further purification.Sodium tungstatedihydrate (Na4.2xWOx3–) was synthesized by reducing the sodium tungstatedihydrate (Na2WO4·2H2O) usingsodium borohydride (NaBH4).49 Briefly, 25 mL of 2.4 M NaBH4 prepared in ice-cold waterat pH >10  was added dropwise to 25mL of 0.24 M Na2WO4·2H2O solution(pH decreased to 6.5) under stirring. Simultaneously, 5 M HCl wasadded dropwise to the stirring solution to maintain the pH of ∼6.5because the addition of NaBH4 would raise the pH due tothe formation of NaOH and NaBO2. This mixture was stirredcontinuously for 2 h, resulting in a dark brown precipitate, whichwas kept undisturbed for 2 h for settling down the precipitate. Then,the suspension was centrifuged to separate the precipitate for 10min at 9000 rpm (each run) and the residue was washed several timeswith water to remove unreacted precursors, followed by drying at 70°C for 2 h in a vacuum oven. To obtain crystalline NaxWOx3–, thesynthesized sample was annealed at 800 °C for 3 h (denoted Na0.56WOx3–) under a N2 atmosphere in a tube furnace.Sodium tungstenoxide bronze (Na4.33·H2O) nanosheetswere synthesized as per the earlier report.40 Briefly, 120 mM Na2WO4·2H2O was prepared in 40 mL of DI water, and 4 mL of HCl was added ina dropwise manner while stirring for 1 h. The obtained pale greensuspension was then centrifuged for 10 min at 9000 rpm (each run)to separate the precipitate, and the residue was washed several timeswith water to remove the unreacted precursors, followed by dryingin an air oven at 70 °C overnight. The as-synthesized WO3·H2O nanosheets were annealed at 500 °Cfor 3 h in a muffle furnace at a heating rate of 10 °C min–1 to obtain monoclinic WO3 nanosheets.Two-dimensional(2D) tungstic acid (WO4.40.56WOx3– (HEP) surface and 0.5wt%Pt was loaded on WO3 nanosheets (OEP) using a photodeposition method by continuousfull-arc irradiation of the 10% aqueous methanol solution containinga H2PtCl6·6H2O precursor for1 h using a 300 W Xe lamp (TrusTech PLS–SXE 300/300UV). Later, the reaction mixture was centrifuged, followed by washingseveral times to remove the unreacted Pt precursors. Then, the residuewas dried at 80 °C for 12 h under vacuum. The HEP photocatalystwas immediately used after the centrifugation.A 3wt%Ptcocatalyst was loaded onto the Na4.52SO4 (pH ∼7.0)solution was used as the electrolyte in the presence of 10% methanolas a hole scavenger. The photocatalyst film was prepared by dispersing20 mg of the photocatalyst in a 5.4 mL solution comprising water:ethanolat a 4:1 (v/v) ratio and 0.4 mL of Nafion (5% solution), followedby 1 h ultrasonication. 50 μL of the obtained slurry was drop-castonto the precleaned fluorine-doped tin oxide (FTO) conducting substrate,followed by drying at 60 °C in an oven before the electrochemicaltests. The area of the photocatalyst thin film was 1 × 1 cm2, whereas the size of the FTO substrate was 1 × 2 cm2. The catalyst loading amount was ca. 3.7 mg mL–1.The X-ray diffraction(XRD) patterns were obtained using a STOE STADI-P diffractometer withMo Kα as the X-ray radiation source. UV–Vis diffuse reflectancespectra (DRS) of the HEP and OEP powders were collected using an AgilentCary 5000 spectrophotometer fitted with an integrating sphere usingstandard barium sulfate powder as a reference. TEM measurements wereperformed using a JEOL2100 TEM, operated at 200 kV. Samples for TEMmeasurements were prepared by drop-casting the dilute dispersionsof the photocatalysts onto the carbon-coated copper grids. X-ray photoelectronspectroscopy (XPS) measurements were undertaken using a Kratos AxisSUPRA machine using monochromated Al-Kα irradiation as a sourceof X-rays. XPS data analysis was performed using Casa XPS software.Shirley/Touguard methods were used for background corrections. Ramanspectra were measured on a Renishaw inVia Raman microscope, usinga 532 nm excitation laser. The photoelectrochemical onset potentialmeasurements were recorded using linear sweep voltammetry by sweepingthe potential from +0.8 to −0.5 V vs Ag/AgCl in a conventionalthree-electrode  cell using an electrochemical analyzer (IVIUMTechnologies). A 0.1 M Na4.62 and O2 evolution half-reactions and their simultaneousproduction in a Z-scheme system were carried out in a custom-madeglass batch reactor with a top quartz window. The known amount ofphotocatalysts was loaded in the reactor containing 70 mL of waterand 5 mM NaI and dispersed well by ultrasonication for 30 min. ThepH of the solution was controlled by adding dilute solutions of H2SO4 and NaOH. The reactor was sealed and purgedwith high-purity Ar gas for 1 h to remove air/dissolved oxygen inthe solution and headspace. After baseline measurement (0 h), thereactor was irradiated using a 300 W Xe lamp (Newport). The reactorwas placed in a water bath during irradiation to maintain the reactiontemperature. The production of H2 and O2 gaseswas quantified at regular intervals using gas chromatography  equipped with a molecular 5A columnusing Ar as the carrier gas and N2 as the internal reference.The apparent quantum yield (AQY) was determined by performing photocatalyticexperiments at specific wavelengths by using an appropriate bandpassfilter. For AQY measurements, 60 mg of HEP and 30 mg of OEP were dispersedin 70 mL of water containing 5 mM NaI. The reactor was irradiatedthrough a 2 cm diameter aperture, enabling the central beam through,which is very reliable for the AQY analysis as highlighted by Domenet al.50 The light intensity of the lampwas measured at five different points to obtain an average intensityusing a calibrated photodiode coupled with an optical power meter. The measured light intensity with a 420 nmbandpass filter was 0.5 mW cm–2. The AQY was calculated using the following equation2 andO2 evolution reactions provided that there are 2 electronsrequired for one H2 molecule production and 4 holes forone O2 molecule production, respectively. The GC calibrationcurves for H2 and O2 are provided in Figure S32. Furthermore, the raw GC chromatogramsfor the Z-scheme water splitting, under a full-arc condition, correspondingto the gas evolution shown in Figure S33.The H4.7– and IO3– ions onthe surface of the HEP and OEP was studied by measuring their concentrationsusing UV–Vis absorption spectroscopy. The determination ofthe I– ion concentration is straightforward as itcan show the absorption peak at 225 nm without adding any reagent.However, IO3– shows absorption peaksat 288 and 352 nm only in the presence of an excess of I– and 0.2 M H2SO4. The calibration curves forI– and IO3– are shownin Figures S34 and S35, respectively. Wemonitored the peak at 352 nm to determine the concentration of IO3–.The adsorption ofI4.83– and I– ontoboth WO3Pt4 and Na0.625WO2.875Pt4. The structure of Na0.625WO2.875Pt4, through XRD analysis, was deemed to be structurallysimilar enough to the experimentally investigated Na0.56WOx3–. For the adsorption calculations,BIOVIA Materials Studio (MS) package was used with the generalizedgradient approximation (GGA) and Perdew–Burke–Ernzerhof(PBE) functional to account for the exchange-correlation energy.51 All calculations were spin-polarized, with theprojector augmented wave (PAW) pseudopotentials and the Van der Waalsinteractions were described by the Grimme DFT-D3 method.52 The Kohn–Sham equations were solved witha convergence criterion of 2.0e-5 eV/atom for energy and 0.05 eV Å–1. Additionally, k-point samplingwas carried out using the Monkhorst–Pack scheme with a (1 ×1× 1) grid, and a cutoff energy of 489.8 eV was used.Density functionaltheory (DFT) calculations were carried out to investigate the adsorptionof IO


Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks, the most severe form of DNA damage. The Rad51 protein is central to HR, but multiple auxiliary factors regulate its activity. The heterodimeric Swi5–Sfr1 complex is one such factor. It was previously shown that two sites within the intrinsically disordered domain of Sfr1 are critical for the interaction with Rad51. Here, we show that phosphorylation of five residues within this domain regulates the interaction of Swi5–Sfr1 with Rad51. Biochemical reconstitutions demonstrated that a phosphomimetic mutant version of Swi5–Sfr1 is defective in both the physical and functional interaction with Rad51. This translated to a defect in DNA repair, with the phosphomimetic mutant yeast strain phenocopying a previously established interaction mutant. Interestingly, a strain in which Sfr1 phosphorylation was blocked also displayed sensitivity to DNA damage. Taken together, we propose that controlled phosphorylation of Sfr1 is important for the role of Swi5–Sfr1 in promoting Rad51-dependent DNA repair. Taken together with the fact that a single DSB is sufficient to cause cell death, DSBs pose a particularly challenging threat to the cell. Homologous recombination (HR) is a major DSB repair mechanism that circumvents the problem of disrupted continuity by identifying another region in the genome that shares high sequence identity (t repair .The DNA ends at a DSB site are processed by nucleases to generate 3′-ended single-stranded DNA (ssDNA) tails that are first bound by the ssDNA-binding protein replication protein A (RPA) . RPA is Schizosaccharomyces pombe (The Swi5–Sfr1 (S5S1) auxiliary factor complex was first identified as a Rad51 regulator in the fission yeast es pombe . Biochemes pombe , 8. Homoes pombe , 10, andes pombe , 13, 14.S. pombe S5S1 demonstrated that it is an elongated heterodimer: the crystal structure of the C-terminal half of Sfr1 in complex with Swi5 (S5S1C) revealed that this truncated complex forms a coiled-coil, whereas the N-terminal half of Sfr1 (Sfr1N) was shown to be intrinsically disordered by circular dichroism (CD) spectroscopy and nuclear magnetic resonance analysis  also displayed DNA damage sensitivity, despite this mutant protein being biochemically comparable to wildtype. Taken together, we propose that phosphorylation negatively regulates the interaction of S5S1 with Rad51, providing a means to fine-tune Rad51 potentiation by S5S1.Here, we investigated the possibility that phosphorylation of Sfr1 regulates the interaction of S5S1 with Rad51. Mutation of the five S/T residues to phosphomimetic D residues severely impaired the binding of S5S1 to Rad51. Furthermore, the stabilization of Rad51 filaments by S5S1 was compromised by the phosphomimetic mutations, and this translated to a substantial reduction in the stimulation of Rad51-driven strand exchange. Importantly, these defects were comparable in magnitude to the S5S1-7A mutant, a bonafide interaction mutant . Yeast sA and B). Since the proteins were purified from Escherichia coli, wildtype S5S1 serves as the unphosphorylated sample. A variant in which five S/T residues were mutated to the phosphomimetic D residue serves as a proxy for the phosphorylated sample . Given that several of these S/T residues are situated in/around the two Rad51 interaction sites within Sfr1N (A), any defects observed for S5S1-PM could be explained by the loss of S/T residues that are important for the interaction with Rad51. To control for this, we also purified a variant in which the S/T residues of interest were mutated to A . Finally, a previously established Rad51 interaction mutant was also purified for comparison .To investigate if phosphorylation of Sfr1 is pertinent to the function of S5S1 in HR, we mutated several phosphorylation sites of Sfr1 and purified the protein in complex with Swi5 , A and Bin Sfr1N  Fig. 1AAA and B.mparison   . Sfr1N and Sfr1N-PM both show similar elution profiles from size-exclusion chromatography , and their CD spectra were characteristic of a disordered protein , as was previously observed for wildtype Sfr1N . Taken together, these results indicate that the global structure of S5S1 or the disordered state of Sfr1N are not obviously altered by the introduction of phosphomimetic mutations in Sfr1N.S/T residues located in intrinsically disordered domains are commonly targeted for phosphorylation, and in some cases, these phosphorylation events promote folding into structured domains , 26. To Fig. 1D) , as expeD) , 17. The protein   was mixed with S5S1 and complexes were precipitated using nickel resin, a significant amount of Rad51 was seen to coprecipitate with S5S1 (A). By contrast, substantially less Rad51 was observed when S5S1-7A was precipitated. Strikingly, a similar reduction in the coprecipitation of Rad51 was seen when S5S1-PM was employed, and this was not the case for S5S1-NON.Next, we sought to directly test whether the binding of Rad51 to S5S1 is affected by the phosphomimetic mutations in Sfr1. To this end, the hexahistidine tag at the N terminus of Sfr1 was utilized in pull-down assays. As expected, when purified Rad51 A was mixith S5S1 A. By cone.g., Rad52 in yeast and BRCA2 in humans) . Briefly, Rad51 filaments were formed on ssDNA that was annealed to a biotinylated oligonucleotide, and reactions were supplemented with S5S1. Following a short incubation, the Rad51–ssDNA complexes (precipitate) were separated from the unbound proteins (supernatant) using streptavidin-coated magnetic beads, and both fractions were analyzed by SDS-PAGE. Whereas a clear Sfr1 band was observed for S5S1, the Sfr1 band for S5S1-PM was barely detectable, corresponding to an approximately 5-fold reduction in the coprecipitation of S5S1 with Rad51 (C). A similar reduction was observed for S5S1-7A, the established Rad51 interaction mutant  . This wan per se . We inten per se B. Brieflth Rad51 C. A simin mutant . These ri.e., S5S1-7A) severely impaired the efficiency of filament stabilization, with several-fold more protein required to effectively stabilize filaments  formed in the presence of ATP-Mg2+ (A). We reasoned that, under these conditions, stabilization of Rad51-ssDNA filaments by S5S1 would result in increased filament length. Indeed, longer filaments were observed upon inclusion of S5S1, and there was a concomitant reduction in other Rad51 species, indicative of improved filament stability (B). The addition of either S5S1-PM or S5S1-7A seemingly resulted in an intermediate state, with some longer filaments observed (C and D). These qualitative observations were corroborated by measurements of filament length (E), which revealed that, while S5S1-PM and S5S1-7A were able to partially stabilize Rad51 filaments, they were unable to do so to the same extent as wildtype S5S1. These differences in filament stabilization were statistically significant, and the defect of S5S1-PM was indistinguishable from S5S1-7A.The formation of human RAD51-ssDNA filaments in the presence of ATP-Mgligomers . We obseATP-Mg2+ A. We reatability B. The adobserved , C and Dt length E, which S. pombe and mammals (A). Rad51 filaments were assembled on a biotinylated ssDNA oligonucleotide (100-mer) and supplemented with S5S1 before challenging them with purified Fbh1. The ssDNA was then precipitated with streptavidin-coated magnetic beads, and the contents of the precipitate (DNA bound) and supernatant (unbound) fractions were examined by SDS-PAGE. Although Fbh1 was purified in complex with its binding partner Skp1 (B), which improves the solubility of Fbh1, it was previously shown that the filament disruption activity of the Fbh1–Skp1 complex is dependent on the translocase activity of Fbh1 , confirming the DNA dependency of protein precipitation. In contrast to this negligible nonspecific binding, significant Rad51 was found in the precipitate when ssDNA was included, and this was largely lost when Fbh1 was added to the reaction, indicating efficient filament disruption by Fbh1. Wildtype S5S1 antagonized this disruption, consistent with previous observations (B and D), consistent with previous fluorescence anisotropy experiments (C and D). The magnitude of the defect for S5S1-PM was comparable to S5S1-7A (D), indicating that these phosphomimetic mutations in Sfr1 severely impair the stabilization of Rad51 filaments by S5S1.Minimal protein was precipitated in the absence of ssDNA B, confirrvations , whereasrvations , B and Deriments . Strikineriments , C and D S5S1-7A D, indicaA). In this assay, the Rad51 filament is formed on circular ssDNA (cssDNA) and homologous linear dsDNA (ldsDNA) is added. Rad51 drives the pairing of cssDNA and ldsDNA, forming joint molecules (JMs), which are the reaction intermediates. Following strand transfer over the length of the DNA, linear ssDNA and nicked circular DNA (NC) are generated as the reaction products. This assay requires the eukaryotic ssDNA-binding protein RPA (A).Next, we sought to examine whether the defect of S5S1-PM in filament stabilization translated to an impairment in the stimulation of Rad51-driven DNA strand exchange. A strand exchange assay with plasmid-sized DNA substrates was employed to directly test this A. In thitein RPA , which wtein RPA A.Figure B). The inclusion of substoichiometric amounts of S5S1 strongly stimulated Rad51 activity, with peak stimulation observed at approximately 0.5 μM (S5S1:Rad51 of 1:10). This stimulatory effect was diminished at higher concentrations, with significantly less NC observed at 2 μM . This is consistent with previous observations . Given that S5S1-NON showed a stimulation profile that is indistinguishable from wildtype, these results strongly suggest that the introduction of phosphomimetic mutations rather than the loss of S/T residues in Sfr1 compromises the stimulation of Rad51 by S5S1.We first confirmed that the hexahistidine tag introduced at the N terminus of Sfr1 did not affect the ability of S5S1 to stimulate Rad51-driven strand exchange . In the Fig. 5rvations , 8, 17. A). In the strand exchange assays conducted thus far, Rad51 filaments were allowed to form on ssDNA before the inclusion of S5S1, then RPA was added to facilitate the reaction. To more closely mimic the physiological condition, we changed the order of addition to precoat the ssDNA with RPA, then added Rad51 and S5S1. Even under these conditions, S5S1-7A and S5S1-PM were severely impaired for Rad51 stimulation, whereas S5S1-NON behaved similarly to wildtype S5S1 (B). Overall, the defect in strand exchange stimulation observed for S5S1-PM was very similar to S5S1-7A, though perhaps slightly less severe. Taken together, these results suggest that phosphorylation of Sfr1 at these five residues significantly impairs the ability of S5S1 to stimulate Rad51-driven DNA strand exchange.Since the results with S5S1-PM are reminiscent of previous observations with S5S1-7A, the established Rad51 interaction mutation, the two mutants were compared to each other side-by-side over a broader concentration range. Consistent with previous observations, S5S1-7A showed peak Rad51 stimulation at 2 to 5 μM, with only a marginal reduction in stimulation at 10 μM; this is in stark contrast to wildtype, where a substantial loss of stimulation was seen even at 5 μM A. In theype S5S1 B. OveralA) and dsDNA  significantly, although not to the same extent as wildtype S5S1. Importantly, S5S1-NON shifted DNA comparably to wildtype across all concentrations examined. These results indicate that the introduction of phosphomimetic residues mildly impairs DNA binding. Despite the clear difference in DNA binding between S5S1-7A and S5S1-PM, the fact that both mutants are similarly defective for filament stabilization ] or E substitutions [sfr1-PM(E)] at the five S/T residues of interest. A strain encoding the nonphosphorylatable mutant (sfr1-NON) was also constructed. These strains were then assayed for DNA damage sensitivity via a standard spot-test. S. pombe possesses an alternative ultraviolet light (UV) damage repair pathway that requires HR for the repair of endonuclease-induced DNA breaks; the response to UV damage is therefore a suitable measure of HR proficiency (sfr1+ gene was deleted (sfr1Δ), the phosphomutants showed normal or near-normal growth following acute exposure to UV, suggesting that DNA repair is not obviously perturbed in these strains (A).Our biochemical analyses suggested that phosphorylation of Sfr1 severely impairs the S5S1-Rad51 interaction. However, it remained unclear whether this constitutes a physiological mechanism to regulate recombinational DNA repair. To test this, we constructed ficiency . In cont strains A.Figure sfr1-7A mutant, it was previously shown that Rad55-Rad57 can suppress defects in the interaction of S5S1 with Rad51 , both phosphomimetic mutants showed similar sensitivity to sfr1Δ in the rad55Δ background (B). Interestingly, the sfr1-NON mutant also displayed a clear sensitivity to DNA damage in the rad55Δ background, although this was not as severe as the phosphomimetic mutants. Given that both phosphomimetic mutants phenocopied the previously established Rad51 interaction mutant , these results strongly suggest that phosphorylation serves to weaken the S5S1-Rad51 interaction and downregulate recombinational DNA repair, while also pointing toward a potentially complex regulatory mechanism in which abolishing phosphorylation is also detrimental to DNA repair.Given the biochemical defects observed for S5S1-PM, this result may seem surprising. However, through examination of the th Rad51 . With th on S5S1 . Unlike ous work , 35. Strous work A, both pckground B. IntereS. pombe expressing phosphomimetic Sfr1 showed defects in DNA repair, but only in the absence of Rad55-Rad57 ; the addition of negatively charged phosphate groups to Sfr1 likely masks the positively charged sites and impairs binding of S5S1 to Rad51, essentially mimicking the S5S1-7A mutant in which positive residues are neutralized via mutation to A.Given that the CD spectra of S5S1-PM and Sfr1N-PM were indistinguishable from wildtype, phosphorylation-induced conformational changes are unlikely to be responsible for the impaired binding of S5S1-PM to Rad51. We favor a scenario where the S5S1-Rad51 interaction involves multiple electrostatic contacts, and the negative charge imparted by phosphorylation, which is mimicked by phosphomimetic mutations, disrupts these contacts. This possibility is supported by several lines of evidence. First, the interaction of Sfr1N with Rad51 displayed clear sensitivity to salt; while Sfr1N coimmunoprecipitated with Rad51 at 25 mM NaCl, this interaction was significantly diminished at 50 mM NaCl . Second,Fig. 7sfr1-NON mutant. Alternatively, it is possible that phosphorylation of Sfr1 is required to promote the dissociation of S5S1 from Rad51 after strand invasion/exchange to prevent unproductive interactions that may hinder downstream steps of HR. Presently, it is not possible to distinguish between these two possibilities. Additional support for the involvement of CDK1 in regulating S5S1 comes from the observation that genetic interactions exist between sfr1+ and the CDK activating kinase csk1+, with the authors suggesting that Csk1 may act via CDK1 to influence HR  match the minimal consensus sequence for CDK1 (S/T-P) . By contuence HR . Given tS. pombe Swi5–Sfr1 : S109 and S165 were conserved in all species; S116 was partially conserved (a T residue in every other species); and T73 was only conserved in Schizosaccharomyces japonicus. Given that S109 and S165 were completely conserved within the Schizosaccharomyces genus, we postulate that these two residues are central to the phosphoregulation of Sfr1. It would be interesting to compare a variant of S5S1 in which only these two sites are mutated to phosphomimetic residues. When compared to mouse SWI5-SFR1, S24 was conserved and T73 was partially conserved (S in mouse) (B). Interestingly, S116 was replaced with a nonphosphorylatable A residue and S165 was replaced with a phosphomimetic E residue. No conservation was observed with human SWI5-SFR1, with the only notable observation being that S109 was replaced with a phosphomimetic E residue (C). It remains to be determined whether these similarities/differences are of functional significance.Within the rvations A: S109 an mouse) B. Intere residue C. It remS. pombe Rad51 is ubiquitylated by both Fbh1, an F-box family helicase, and Rrp1, a RING-domain containing SWI/SNF-family translocase  and swi5Δ (or sfr1Δ) single mutants compared with the severe sensitivity of the double mutant—which resembles the rad51Δ single mutant—was interpreted to mean that Rad55-Rad57 and Swi5–Sfr1 function in independent subpathways of HR  and sfr1-PM(E) phenocopy sfr1-7A provides further support for the existence of interplay between Rad55-Rad57 and S5S1. How Rad55-Rad57 suppresses the defects of S5S1-7A and S5S1-PM is unclear. Given that Rad55-Rad57 can interact with both Rad51 and S5S1, it was proposed that Rad55-Rad57 can function as a molecular bridge to increase the local concentration of S5S1-7A around Rad51, thereby allowing S5S1C to stimulate Rad51  that had been pretransformed with the pRARE2 plasmid (Novagen). Plasmids used are: pBA184, S5S1; pBA186, S5S1-NON; pBA187, S5S1-PM; and pBA197, S5S1-7A. For each S5S1 variant, 2 L of E. coli was grown to an A600 of 0.4 to 0.5, 1 mM of IPTG was added, and incubation was continued at 18 °C for ∼15 h at 170 rpm. Cells were then harvested by centrifugation. All subsequent steps were carried out at 4 °C. Cells (8–10 g wet weight) were resuspended in 50 ml of lysis buffer , lysed by sonication, and clarified by centrifugation . Cleared lysates were supplemented with 0.05% polyethyleneimine, incubated for 30 min with mixing, and precipitate was removed by centrifugation . Ammonium sulphate was slowly added to the supernatant (35% saturation), which was then incubated with mixing for 1 h. Precipitate was collected by centrifugation , resuspended in 50 ml of lysis buffer, and applied to a 5 ml cOmplete His-tag purification column (Roche). The column was then washed with ∼100 ml of lysis buffer, and bound proteins were eluted in 25 ml of lysis buffer supplemented with 300 mM imidazole. The eluate was dialyzed overnight against 1 L of H buffer  containing 200 mM KCl, diluted with 3× volumes of H buffer, and applied to a HiTrap Q column (5 ml). Proteins were eluted with a linear gradient . Peak fractions were combined, concentrated to ∼3 ml, applied to a 16/60 Superdex 200 pg gel filtration column, and developed in H buffer containing 200 mM KCl. Peak fractions were combined, concentrated, and flash-frozen in liquid nitrogen as small aliquots. Protein concentration was estimated by measuring A280 with a molar extinction coefficient of 12,490 M−1 cm−1.Rad51 and RPA were purified exactly as previously described . N-termiUp until ammonium sulphate precipitation, N-terminally hexahistidine-tagged Sfr1N (pBA198) and Sfr1N-PM (pBA200) were treated the same as S5S1. Ammonium sulphate precipitation was at 45% saturation. Following nickel affinity purification and dialysis, the sample was applied to a HiTrap Q column (5 ml) and recovered in the flow-through. The sample was then applied to a HiTrap Heparin column (5 ml) and eluted with a linear gradient . Peak fractions were concentrated, and Sfr1N was further purified by gel filtration, as for Swi5–Sfr1. Protein concentration was estimated by the bicinchoninic acid assay, with wildtype S5S1 serving as a standard. Other than the His-tag purification column, all columns are from Cytiva. Chromatograms for size-exclusion chromatography are shown in Global secondary structure was examined using a spectrometer  with a Peltier temperature controller (Quantum Northwest). Swi5–Sfr1 or Sfr1N (wildtype or mutants) were diluted to 7.35 μM in 20 mM potassium phosphate (pH 7.4) and transferred into a 1 mm path length quartz cuvette . Circular dichroism was recorded from 190 to 260 nM at 20 °C .A), 500 nM of Rad51 was mixed with 500 nM of S5S1 (wildtype or mutant) in pull-down buffer  on ice . Ten microliter was withdrawn and mixed with SDS-PAGE loading buffer (input). The remaining reaction was incubated at 30 °C for 15 min then on ice for 5 min. Ten microliter of cOmplete nickel resin (Roche) was added to each reaction, and hexahistidine-tagged proteins were immobilized on the resin by incubating with gentle agitation for 1 h at 4 °C. The resin was pelleted by brief centrifugation and washed twice with 400 μl of pull-down buffer. Bound proteins were eluted with 40 μl of SDS-PAGE loading buffer (37 °C 10 min 1300 rpm) and analyzed by SDS-PAGE.For the in-solution pull-down assay using nickel resin A, 500 nMC), 3.33 μM Rad51 was added to filament binding buffer  containing 10 μMnt of circular ssDNA  immobilized on magnetic Streptavidin-coated beads  through an oligonucleotide with a biotin moiety at its 5′ end . Filaments were allowed to form by incubating at 37 °C with gentle agitation for 5 min, then 3.33 μM S5S1 (wildtype or mutant) was added, and incubation was continued as before for a further 5 min. The solution was then separated into precipitate (DNA bound) and supernatant (unbound) fractions using a magnetic stand. Proteins were eluted from the resin in 1x SDS-PAGE loading buffer (37 °C 15 min 1300 rpm) and, along with the supernatant fraction, analyzed by SDS-PAGE. Bound proteins were quantified in FIJI  containing 1.5 μMnt of a 116-mer oligonucleotide  was supplemented with Rad51 (500 nM) and incubated at 37 °C for 5 min. Swi5–Sfr1  was then added, and incubation was continued for 10 min. Four microliters of the reaction was then applied to a carbon-coated 300 mesh copper grid (Agar Scientific) and fixed with 2% uranyl acetate. Images were captured using a Tecnai 12 electron microscope at 52,000× magnification, and filament length was measured using FIJI  containing 10 μMnt of a 5′-biotinylated 100-mer oligonucleotide  was supplemented with Rad51 (3.33 μM) and incubated for 7 min. The indicated concentration of a Swi5–Sfr1 variant was added, and reactions were incubated for a further 7 min. To disrupt filaments, Fbh1 (0.312 μM) was added and incubation continued for 10 min. Ten microliter of streptavidin-coated magnetic beads  was then added, followed by a further 15 min incubation (1300 rpm). A magnetic tube rack was then used to separate the beads from the supernatant. Beads were then resuspended in SDS-PAGE loading buffer and bound proteins eluted (10 min 1300 rpm). Both supernatant and precipitate fractions (in SDS-PAGE loading buffer) were heated (65 °C 5 min), then proteins were analyzed by SDS-PAGE and Coomassie staining. Following sufficient staining and destaining, gels were imaged using the ChemiDoc MP Imaging System (Bio-Rad). Quantification was performed in FIJI  was supplemented with 10 μMnt cssDNA . Proteins were then added in the order indicated for each experiment, with concentrations described in respective figure legends. 10 μMnt ldsDNA  was added to initiate the strand exchange reaction. After 2 h, 1 μl of 200 μg/ml psoralen was added, and the reactions were exposed to 200 μJ/cm2 UV . Reactions were then deproteinized through the addition of 2.5 μl stop solution  and incubation for 15 min at 37 °C. Reactions were separated by agarose gel electrophoresis, and DNA was visualized by staining with SYBR Gold (Thermo Fisher Scientific). Images were captured using the ChemiDoc MP Imaging System (Bio-Rad). Quantification was performed in FIJI  containing 20 nM ssDNA with a Cy5 label at the 3′ end , or BA663 annealed to its unlabeled complementary strand (BA664), was supplemented with the denoted concentration of Swi5–Sfr1 (wildtype or mutants) in a 10 μl reaction. After a 15 min incubation at 37 ˚C, 2 μl of loading dye was added to the reaction, and samples were separated by agarose gel electrophoresis at 4 °C (0.8% gel in TAE buffer). Gels were imaged using a ChemiDoc MP Imaging System (Bio-Rad). Oligonucleotides were synthesized by Eurofins and purified by HPLC.DNA binding buffer  or exposed to the indicated dose of ultraviolet light, then left for 3 to 4 days until sufficient growth was observed. Growth was always at 30 °C. Strains used in Figure 7A are: BA196 (sfr1+-kanMX6), BA125 (sfr1::kanMX6), BA215 (sfr1-NON-kanMX6), BA240 (sfr1-PM[E]-kanMX6), and BA243 (sfr1-PM[D]-kanMX6). Strain used in Figure 7B are: BA53 (wildtype), BA194 (rad55::natMX6 sfr1+-kanMX6), BA126 (rad55::arg3 sfr1::kanMX6), BA252 (rad55::natMX6 sfr1-NON-kanMX6), BA260 (rad55::natMX6 sfr1-PM[E]-kanMX6), and BA256 (rad55::natMX6 sfr1-PM[D]-kanMX6). All strains are isogenic derivatives of BA53 (h-mat1M smt0 ura4-D18 leu1-32 his3-D1 arg3-D1).rotocols , and sporotocols . BrieflyThere are no data to be deposited in databases. All relevant data have been included in the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.