Abstract
ADCK4 mutations usually manifest as steroid-resistant nephrotic syndrome, and cause 48 coenzyme Q 10 (CoQ 10 ) deficiency. However, the function of ADCK4 remains obscure. 49 We investigated ADCK4 function using mouse and cell models. Podocyte-specific 50 Adck4 deletion in mice significantly reduced survival and caused severe focal 51 segmental glomerular sclerosis with extensive interstitial fibrosis and tubular atrophy, 52 which were prevented by treatment with 2,4-dihydroxybenzoic acid (2,4-diHB), an 53 analog of CoQ 10 precursor molecule. ADCK4 knockout podocytes exhibited significantly 54 decreased CoQ 10 level, respiratory chain activity, mitochondrial potential, and 55 dysmorphic mitochondria with loss of cristae formation, which were rescued by 2,4-diHB 56 treatment, thus attributing these phenotypes to decreased CoQ 10 levels. ADCK4 57 interacted with mitochondrial proteins including COQ5, and also cytoplasmic proteins 58 including myosin and heat shock proteins. ADCK4 knockout decreased COQ complex 59 levels, and the COQ5 level was rescued by ADCK4 overexpression in ADCK4 knockout 60 podocytes. Overall, ADCK4 is required for CoQ 10 biosynthesis and mitochondrial 61 function in podocytes. 62 63 65 Coenzyme Q (CoQ, ubiquinone), a lipophilic component located in the inner 66 mitochondrial membrane, Golgi apparatus, and cell membrane, plays a pivotal role in 67 oxidative phosphorylation (Stefely & Pagliarini, 2017). CoQ shuttles electrons from 68
Abstract
complexes I and II to complex III in the mitochondrial respiratory chain (Mitchell, 1975). 69
Introduction
Loss of ADCK4 caused mitochondrial defects in podocytes 221 To investigate the function of ADCK4 at the cellular level, we generated ADCK4 222 knockout (KO) cells of human podocytes and HK2 cells, which were originated from treatment reduced cell viability in both the control and ADCK4 KO podocytes, and findings suggested that the loss of ADCK4 caused CoQ deficiency and that podocytes 251 were more susceptible than HK-2 cells were. 252 To comprehensively understand the molecular changes induced by the KO of 253 ADCK4, we performed proteomic analysis and quantified protein abundance changes 254 by MS-based proteomics using isobaric tag for relative and absolute quantification 255 (iTRAQ) (Chung, Lagranha et al., 2015) in podocytes with and without AA treatment.
256
Proteomic characterization of the control and ADCK4 KO podocytes revealed more than 257 2,500 proteins, and 421 (16%) proteins were mitochondrial proteins. By Gene Ontology 258 (GO) analysis, using DAVID functional annotation tool (david.abcc.ncifcrf.gov), 259 differentially expressed proteins in the control and ADCK4 KO cells were divided into 260 the following three categories of GO annotations: biological process, cellular component, 261 and molecular function. The results indicated that proteins related to cellular defense 262 response were upregulated and those associated with cytokine production pathway 263 were downregulated in ADCK4 KO podocytes compared with those in the control cells 264 (Appendix Fig S7H) . In an injury situation, with the use of AA treatment, coenzyme were less affected by AA (Fig 4I) , AA-treated ADCK4 KO podocytes showed more 281 severe mitochondrial defects such as swollen and shortened cristae, and fewer inner 282 membranes (Fig 4J) . These results indicate that ADCK4 KO confers susceptibility to 283 cellular stress, such as autoxidation of polyunsaturated fatty acids. To examine 284 functional defects of mitochondria in ADCK4 KO cells, we measured the mitochondrial 285 membrane potential using the JC-10 dye, which is concentrated in the mitochondrial 286 matrix based on membrane polarization (Li, Yu et al., 2013) . The results revealed the 287 presence of inactive mitochondria in ADCK4 KO podocytes (Fig 4K) . This finding was 288 confirmed by another mitochondrial membrane potential assay using the potentiometric 289 probe tetramethylrhodamine methyl ester (TMRM)-based fluorimetric assay. TMRM 290 fluorescence intensity was significantly reduced in ADCK4 KO cells; however, 291 supplementation of 2,4-diHB partially restored the lowered mitochondrial membrane 292 potential (Fig 4L) . In contrast, mitochondrial membrane potential was not different between the control and KO in HK-2 cells (Appendix Fig S8I and J) . Therefore, the 294 loss of ADCK4 caused morphological and functional defects of mitochondria in cultured 295 podocytes by disrupting CoQ 10 biosynthesis.
296
As we previously reported that ADCK4 knockdown reduced podocyte migration 297 (Ashraf et al., 2013) , in the present study, we examined the cytoskeleton of ADCK4 KO 298 cells. Actin phalloidin staining revealed shrunk cellular area in ADCK4 KO podocytes 299 (Fig 4M) , whereas ADCK4 KO HK-2 showed surface area similar to that of control HK-2 300 cells (Appendix Fig S8K and L) . Interestingly, shrunk cellular area in ADCK4 KO 301 podocytes was not restored by 2,4-diHB supplementation, suggesting that this cellular 302 phenotype is not related to decreased CoQ 10 level and that ADCK4 might have other 303 cellular functions in addition to its role in the CoQ biosynthesis pathway. AA treatment 304 significantly reduced cellular area in both the control and ADCK4 KO podocytes (Fig   305   4M ). Shrunk cellular area by AA treatment was not rescued by 2,4-diHB, further 306 confirming that this phenotype is not related to CoQ 10 deficiency (Fig 4M and N) . 307 308 ADCK4 interacted with and stabilized COQ proteins 309 We performed proteomic analysis to understand the function of ADCK4 via the 310 identification of its interactome. We generated HEK293 cells that stably overexpressed 311 C-terminal FLAG-tagged bacterial alkaline phosphatase (BAP; BAP-3xFLAG) and 312 ADCK4 (ADCK4-3xFLAG) (Appendix Fig S9A) . We confirmed that ADCK4-3xFLAG 313 mostly localized to the mitochondria (Appendix Fig S9B) . Following affinity purification 314 using anti-FLAG beads (Appendix Fig S9C and D) , protein eluates were analyzed 315 using a liquid chromatograph coupled to a high-resolution mass spectrometer (LC- 

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