1. Volume 67, Issue 3, Pages 471-483.e7 (August 2017)
Acylglycerol Kinase Mutated in Sengers Syndrome Is a Subunit of the TIM22 Protein Translocase in Mitochondria 
Milena Vukotic, Hendrik Nolte, Tim König, Shotaro Saita, Maria Ananjew, Marcus Krüger, Takashi Tatsuta, Thomas Langer 
Molecular Cell 
Volume 67, Issue 3, Pages e7 (August 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 67, 471-483.e7DOI: (10.1016/j.molcel.2017.06.013)
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3. Figure 1 AGK Interacts with TIM22 Complex Subunits
(A) Volcano plot representation of AGK interacting partners. Mitochondria isolated from AGK−/− HEK293 cells and AGK−/− cells expressing AGKFLAG were lysed in digitonin-containing buffer and subjected to immunoprecipitation. Co-purified proteins were analyzed by LC-MS/MS (n = 3). AGK and significantly enriched components of the TIM22 complex are highlighted (fold change > 2 and p value < 0.05).
(B) Immunoblot analysis of proteins co-purified with AGKFLAG. Total (20%), unbound (Unb., 20%), and eluate (100%) fractions are shown. #, unspecific cross-reaction.
(C) Complexome analysis of mitochondria isolated from mouse embryonic fibroblasts (n = 3). Representative heatmaps and migration profiles of AGK, TIMM22, and TIMM29 are shown.
(D) Subcellular fractionation of HEK293 cells.
(E) Submitochondrial fractionation. Mitochondria (Mito) isolated from HEK293 cells were subjected to osmotic swelling (Swell) or treated with Triton X-100 (0.5% [v/v]). Where indicated, samples were treated with proteinase K (PK, 50 μg/ml).
(F) Sodium carbonate extraction of mitochondrial membranes at different pH. T, total; S, supernatant; P, pellet.
(G) Salt (1 M NaCl) extraction of mitochondrial proteins. T, total; S, supernatant; P, pellet.
See also Figures S1 and S2 and Table S1.
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4. Figure 2 Assembly of Catalytically Inactive AGK into the TIM22 Complex
(A) AGK+/+ cells, AGK−/− cells, and AGK−/− cells expressing either AGK or AGKG126E under the control of a tetracycline-inducible promoter were lysed and analyzed by SDS-PAGE and immunoblotting.
(B) Quantification of (A). Protein amounts in control cells were set to 100%. Data are presented as the mean ± SD (n = 3). ∗∗p < 0.01, ∗∗∗p <
(C) BN-PAGE analysis of the assembly of the TIM22 complex. Mitochondria were isolated from the indicated cell lines, lysed in digitonin-containing buffer (6 g/g digitonin), and analyzed by BN-PAGE and immunoblotting using antibodies directed against TIMM2, TIMM29, and, for control, ATP5A.
(D) Proteome-wide identification of AGK-dependent substrates of the TIM22 complex. Heatmap of the relative abundance of proteins after hierarchical cluster analysis (Z scores). p < Membrane fractions of AGK+/+ cells, AGK−/− cells, and AGK−/− cells complemented with either AGK or AGKG126E were analyzed by LC-MS/MS (n = 4). Shown are proteins whose steady-state levels were decreased in AGK−/− cells but restored upon expression of AGK. Not all replicates co-cluster, indicating that the experimental variability exceeds the (small) biological variability (e.g., upon expression of AGK or AGKG126E).
(E) Boxplots of label-free quantification (LFQ) intensities for selected proteins obtained by LC-MS/MS analysis (n = 4).
See also Table S2.
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5. Figure 3 AGK-Dependent Mitochondrial Import of ANT1 and SLC25A24
(A–D) [35S]-Labeled (A) ANT1, (B) SLC25A24, (C) TIMM23, and (D) EMRE were imported into mitochondria isolated from AGK+/+ cells, AGK−/− cells, and AGK−/− cells expressing AGK or AGKG126E for indicated times in the presence or absence of a membrane potential (Δψ) across the IM. After treatment of mitochondria with proteinase K (PK; 50 μg/mL), samples were analyzed by SDS-PAGE followed by autoradiography or immunoblotting using AGK and VDAC1-specific antibodies. Quantifications are shown in the bottom panels. Proteins imported into AGK+/+ mitochondria at the longest time point were set to 1. Data are presented as the mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01.
See also Figure S3.
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6. Figure 4 Cell Proliferation and OCRs in AGK−/− Cells
(A) Cell proliferation of AGK+/+ and AGK−/− cells in galactose-containing medium. Data are presented as the mean ± SD (n = 3).
(B) Continuous measurements of OCR in AGK+/+ cells, AGK−/− cells, and AGK−/− cells expressing either AGK or AGKG126E cultured in galactose. 1 μM oligomycin (OLG), 1.5 μM CCCP, and 0.5 μM antimycin A (AA)/0.5 μM rotenone (Rot) were add at indicated time points. OCR was normalized to protein amount. Data are presented as the mean ± SD (n = 3). ∗p < Basal, ATP-linked, and maximal OCR, proton leak, and spare respiratory capacity were quantified from the original trace shown above. Data are presented as the mean ± SD (n = 3). ∗p < 0.05.
See also Figure S1.
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7. Figure 5 AGK Preserves Mitochondrial Morphology and Cristae Formation
(A) Immunofluorescence analysis of AGK+/+, AGK−/−, and AGK−/− HeLa cells expressing either AGKFLAG or AGKG126E-FLAG using ATP5β-specific antibodies. Higher magnifications of boxed areas are shown in the lower panel. Scale bars represent 10 μm or 3 μm (magnified images).
(B) Quantification of mitochondrial morphologies in three independent experiments (≥100 cells were analyzed in each experiment). Data are presented as the mean ± SD (n = 3).
(C) Transmission electron microscopic analysis of AGK+/+, AGK−/−, and AGK−/− HEK293 cells expressing either AGK or AGKG126E. Scale bar, 0.5 μm.
(D) SDS-PAGE analysis of indicated cell lines and immunoblotting using antibodies directed against OPA1, AGK, VDAC1, and SDHA. L, long, uncleaved forms of OPA1; S, short, proteolytically cleaved forms of OPA1.
(E) Quantification of (D). The sum of L and S was set to 100% for each cell type, and individual L and S were calculated as the percentage of that value. Data are presented as the mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01.
See also Figures S4 and S5.
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8. Figure 6
The Apoptotic Resistance of Cells Depends on Catalytically Active AGK
(A–D) Apoptotic sensitivity of HeLa AGK+/+ and AGK−/− HeLa cells transfected with either a control plasmid (ev) or plasmids expressing AGKFLAG or AGKG126E-FLAG was assessed by monitoring PARP cleavage. To induce apoptosis, cells were treated with (A) actinomycin D (ActD; 200 nM) for 6 hr or (C) TNF-α (20 ng/mL) and CHX (10 μg/mL) for 8 hr. Cells were harvested, lysed, and analyzed by SDS-PAGE and immunoblotting with indicated antibodies. cPARP, cleaved PARP. Quantifications of three independent experiments of (A) are shown in (B) and of (C) in (D). Data are presented as the mean ± SD (n = 3). ∗∗∗p < # unspecific cross-reaction. See also Figure S6.
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9. Figure 7
Increased Release of Pro-apoptotic Proteins from Mitochondria Lacking Catalytically Active AGK
(A) To induce apoptosis, AGK+/+ and AGK−/− HeLa cells expressing AGKFLAG or AGKG126E-FLAG were incubated with actinomycin D (ActD; 200 nM) for 6 hr in the presence of the caspase-inhibitor Z-VAD-FMK (50 μM). Cells were treated with MitoTracker DeepRed (magenta) and subjected to immunofluorescence analysis with antibodies directed against Smac/DIABLO (green) and cytochrome c (red). Higher magnifications of the boxed areas are shown in the bottom panel. Scale bars represent 75 μm or 25 μm (magnified images).
(B) Quantification of cells harboring cytosolic Smac/DIABLO or cytochrome c in three independent experiments (≥100 cells were analyzed in each experiment). Data are presented as the mean ± SD (n = 3). ∗∗p < 0.01, ∗∗∗p <
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