BAP31 stimulates translocation of NDUFS4 from cytosol to mitochondria

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Fig. 4 BAP31 stimulates translocation of NDUFS4 from cytosol to mitochondria. BAP31 stimulates translocation of NDUFS4 from cytosol to mitochondria. (A) Decreased NDUFS4 localization at MAM and mitochondria with BAP31 knockout. U2OS cells with sgBAP31 (+) or sgNegative (−) were fractionated into three parts (MAM, mitochondria, and ER) and blotted using antibodies to NDUFS4, Tom40, BAP31, FACL-4 (MAM marker), cytochrome c (mitochondria marker), and calnexin (ER and MAM markers). The protein concentration was equalized between each sample fraction. (B) Mitochondrial complex I activity is suppressed by BAP31 knockdown. U2OS cells transfected with siControl or siBAP31 for 24 hours were subsequently assessed for mitochondrial respiratory complex I activity. Data are presented as mean ± SD of three simultaneously performed experiments. mOD, millioptical density. (C) BAP31 expression level does not affect NDUFS4 mRNA expression. Total RNA of U2OS cells transfected with siControl or siBAP31 for 24 hours was extracted and subjected to real-time quantitative polymerase chain reaction. Data are presented as means ± SD (three different datasets). (D) BAP31 expression level does not affect total pre-NDUFS4 expression. U2OS sgControl or sgBAP31-2 cells were subjected to immunoblotting with the indicated antibodies. The clear band intensity was determined and expressed relative to the sgControl (n = 3). (E) BAP31 interacts with pre-NDUFS4. U2OS cells were harvested, and a co-immunoprecipitation assay was performed with cell lysates for pre-NDUFS4 detection using the indicated antibodies, followed by Western blotting. (F) Loss of BAP31 inhibits pre-NDUFS4/NDUFS4 translocation to mitochondria and stimulates proteolysis by the proteasome. U2OS sgControl (−) or sgBAP31-2 (+) cells were treated with the proteasome inhibitor MG132 for 1 hour, and cells were subsequently subjected to fractionation and immunoblotting with the indicated antibodies. DMSO, dimethyl sulfoxide. Cytosol fraction was concentrated by using amicon ultra spin. (G) BAP31 interacts with VDAC1 and NDUFB11. The co-immunoprecipitation assay is described in Fig. 3A. (H) BAP31 regulates NDUFS4 and NDUFB11 localization to the mitochondria. U2OS sgControl (−) or sgBAP31-2 (+) cells were subjected to fractionation and immunoblotting with the indicated antibodies. (I) The clear band intensity was determined [gels are shown in (H)] and expressed relative to the sgControl whole-cell fraction (n = 3). P value was calculated using two-way ANOVA; *P < 0.05; **P < 0.01 (B, D, and I). FACL-4, fatty acid-CoA ligase 4; VDAC1, voltage-dependent anion-selective channel 1; NDUFB11, NADH:ubiquinone oxidoreductase subunit B11; HSP60, heat shock protein 60. Takushi Namba Sci Adv 2019;5:eaaw1386 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).