Figure 3. CipB localizes to mitochondria... | Rockefeller University Press

Figure 3.

$CipB localizes to mitochondria through TUFM. (A) Co-IP analysis of CipB with mitophagy-related proteins in 293T cells. Cells were co-transfected with HA-CipB and Flag-tagged mitophagy proteins for 14 h. Cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel. The immunoprecipitate was eluted with SDS loading buffer and analyzed by immunoblotting. (B) Co-IP analysis of RFP-CipB with mitochondrial protein (HSP60, ATP5A, TUFM, Tim23, and Smac) in 293T cells treated as in A. (C) Co-IP analysis of endogenous TUFM with 3×Flag-GFP, 3×Flag-CipB, 3×Flag-SipB, or 3×Flag-IpaB in 293T cells treated as in A. (D)In vitro pull-down of purified GST-CipB and TUFM-His proteins. (E) 293T cells transfected with RFP or RFP-CipB were subjected to cytosolic-mitochondrial fractionation, and RFP signals were analyzed by immunoblotting. (F) Co-IP of CipB and endogenous TUFM after subcellular fractionation. 293T cells transfected with Flag-BFP or Flag-CipB were subjected to subcellular fractionation. Mitochondrial and cytosolic fractions were analyzed by Co-IP. (G) Western blot analysis of Tom20 and Tim23 in 293T cells transfected with Flag-BFP, Flag-CipB, or Flag-CipB-HA for 21 h. (H and I) Submitochondrial localization of CipB. 293T cells transfected with Flag-BFP (H), Flag-CipB (H), or Flag-CipB-HA (I) were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated concentration of proteinase K for 30 min on ice and analyzed by immunoblotting with anti-Tom20 (OMM), anti-Tim23 (IMS), anti-HSP60 (matrix), anti-Flag, and anti-HA. (J) Structural model of CipB within mitochondria. CipB is a mitochondrial transmembrane protein with its N terminus and C terminus exposed to the cytoplasm. The transmembrane domain spans the outer membranes that directly interact with TUFM. (K) Co-localization of TUFM and MitoTracker in HeLa cells transfected with GFP or GFP-CipB (yellow). Cells were incubated with 100 nM MitoTracker (magenta) for 45 min. Cells were then subjected to immunofluorescence by anti-TUFM (cyan). Nuclei (gray) were stained with DAPI. Scale bar, 10 μm. (L) qRT-PCR analysis of TUFM transcripts in TUFM+/−293T cells transfected with si-Neg or si-TUFM (75 nM, 24 h). GAPDH transcripts were used for normalization. Data are mean ± SEM (n = 3). Unpaired two-sided Student’s t tests were used to measure significance. (M) TUFM is required for the mitochondrial localization of CipB. 293T cells were transfected with 75 nM si-Neg or si-TUFM for 30 h, followed by transfection with RFP or RFP-CipB for 15 h. Cytosolic and mitochondrial fractions were immunoblotted. (N and O) WCL, whole cell lysate; (N-O) TUFM is required for CipB-induced mitophagy in 293T cells. Cells were treated as in M. Whole-cell lysates were immunoblotted with the indicated antibodies. (O) shows quantification of Tom20 and TUFM levels. Tom20 and TUFM levels were normalized to tubulin and set to 1.00 in corresponding control cells. Data are represented as mean ± SEM (n = 3, three independent experiments, two-way ANOVA). (P) Sanger sequencing confirmed the TUFM+/− genotype in 293T cells. (Q) WT or TUFM+/−293T cells transfected with RFP or RFP-CipB for 21 h were immunoblotted for LC3B-II and TUFM. The data are representative of three independent experiments. ns, not significant; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F3.$

CipB localizes to mitochondria through TUFM. (A) Co-IP analysis of CipB with mitophagy-related proteins in 293T cells. Cells were co-transfected with HA-CipB and Flag-tagged mitophagy proteins for 14 h. Cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel. The immunoprecipitate was eluted with SDS loading buffer and analyzed by immunoblotting. (B) Co-IP analysis of RFP-CipB with mitochondrial protein (HSP60, ATP5A, TUFM, Tim23, and Smac) in 293T cells treated as in A. (C) Co-IP analysis of endogenous TUFM with 3×Flag-GFP, 3×Flag-CipB, 3×Flag-SipB, or 3×Flag-IpaB in 293T cells treated as in A. (D)In vitro pull-down of purified GST-CipB and TUFM-His proteins. (E) 293T cells transfected with RFP or RFP-CipB were subjected to cytosolic-mitochondrial fractionation, and RFP signals were analyzed by immunoblotting. (F) Co-IP of CipB and endogenous TUFM after subcellular fractionation. 293T cells transfected with Flag-BFP or Flag-CipB were subjected to subcellular fractionation. Mitochondrial and cytosolic fractions were analyzed by Co-IP. (G) Western blot analysis of Tom20 and Tim23 in 293T cells transfected with Flag-BFP, Flag-CipB, or Flag-CipB-HA for 21 h. (H and I) Submitochondrial localization of CipB. 293T cells transfected with Flag-BFP (H), Flag-CipB (H), or Flag-CipB-HA (I) were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated concentration of proteinase K for 30 min on ice and analyzed by immunoblotting with anti-Tom20 (OMM), anti-Tim23 (IMS), anti-HSP60 (matrix), anti-Flag, and anti-HA. (J) Structural model of CipB within mitochondria. CipB is a mitochondrial transmembrane protein with its N terminus and C terminus exposed to the cytoplasm. The transmembrane domain spans the outer membranes that directly interact with TUFM. (K) Co-localization of TUFM and MitoTracker in HeLa cells transfected with GFP or GFP-CipB (yellow). Cells were incubated with 100 nM MitoTracker (magenta) for 45 min. Cells were then subjected to immunofluorescence by anti-TUFM (cyan). Nuclei (gray) were stained with DAPI. Scale bar, 10 μm. (L) qRT-PCR analysis of TUFM transcripts in TUFM^+/−293T cells transfected with si-Neg or si-TUFM (75 nM, 24 h). GAPDH transcripts were used for normalization. Data are mean ± SEM (n = 3). Unpaired two-sided Student’s t tests were used to measure significance. (M) TUFM is required for the mitochondrial localization of CipB. 293T cells were transfected with 75 nM si-Neg or si-TUFM for 30 h, followed by transfection with RFP or RFP-CipB for 15 h. Cytosolic and mitochondrial fractions were immunoblotted. (N and O) WCL, whole cell lysate; (N-O) TUFM is required for CipB-induced mitophagy in 293T cells. Cells were treated as in M. Whole-cell lysates were immunoblotted with the indicated antibodies. (O) shows quantification of Tom20 and TUFM levels. Tom20 and TUFM levels were normalized to tubulin and set to 1.00 in corresponding control cells. Data are represented as mean ± SEM (n = 3, three independent experiments, two-way ANOVA). (P) Sanger sequencing confirmed the TUFM^+/− genotype in 293T cells. (Q) WT or TUFM^+/−293T cells transfected with RFP or RFP-CipB for 21 h were immunoblotted for LC3B-II and TUFM. The data are representative of three independent experiments. ns, not significant; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F3.