Volume 67, Issue 3, Pages 457-470.e5 (August 2017) Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex  Yilin Kang, David A. Stroud, Michael J. Baker, David P. De Souza, Ann E. Frazier, Michael Liem, Dedreia Tull, Suresh Mathivanan, Malcolm J. McConville, David R. Thorburn, Michael T. Ryan, Diana Stojanovski  Molecular Cell  Volume 67, Issue 3, Pages 457-470.e5 (August 2017) DOI: 10.1016/j.molcel.2017.06.014 Copyright © 2017 Terms and Conditions

Molecular Cell 2017 67, 457-470.e5DOI: (10.1016/j.molcel.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 1 AGK Is a Subunit of the TIM22 Complex (A) Schematic of acylglycerol kinase (AGK) protein, depicting the predicted position of the transmembrane domain (TMD) and diacylglycerol kinase (DGK) domain. Bottom: immunofluorescence images of AGK3XFLAG transiently expressed in HeLa cells. Cells were immunostained with antibodies against the FLAG epitope to visualize the FLAG-tagged AGK, NDUFAF2 (mitochondrial marker protein) and Hoechst 33258 dye (nuclear stain). Scale bars, 10 μm. (B) Isolated mitochondria from cells stably expressing AGK3XFLAG were subjected to submitochondrial fractionation. Untreated mitochondria (lanes 1 and 2); hypotonic swelled mitochondria (lanes 3 and 4), and TX-100 solubilized mitochondria (lanes 5 and 6) were treated with (+) or without (−) Proteinase K (PK). (C) Mitochondria isolated from AGK3XFLAG expressing cells were resuspended in sodium carbonate (Na2CO3; pH 9.5–12.5). Following incubation on ice, samples were centrifuged to separate integral membrane proteins (P) from soluble/peripherally associated membrane proteins (S). T indicates total mitochondrial function. (D) Mitochondria isolated from AGK3XFLAG expressing cells were resuspended in NaCl containing buffer (pH 7.4) (0−1.5 M NaCl) and subsequently centrifuged to separate integral membrane proteins (P) from soluble/peripherally associated membrane proteins (S). T indicates total mitochondrial fraction. (E) Control mitochondria and mitochondria expressing either AGK3XFLAG or hTim10b3XFLAG were solubilized in digitonin-containing buffer before immunoprecipitation with FLAG affinity resin. Samples were analyzed using SDS-PAGE and immunoblotting with the indicated antibodies. (F) [35S]-hTim22 protein was imported into isolated mitochondria (control, AGK3XFLAG, Tim293XFLAG, or hTim10b3XFLAG). Following import, mitochondria were isolated, solubilized in digitonin-containing buffer, and incubated with or without FLAG antibodies. Samples were subjected to BN-PAGE analysis and autoradiography. TIM22SHIFT indicates antibody-bound TIM22 complex. Molecular Cell 2017 67, 457-470.e5DOI: (10.1016/j.molcel.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 2 AGK Is Required for the Stability of the TIM22 Complex and Biogenesis of Carrier Proteins (A and B) Mitochondria isolated from wild-type (control) or AGK KO (AGKKO) HEK293T cells were subjected to (A) SDS-PAGE or (B) BN-PAGE and western-blotted using the indicated antibodies. Protein levels on SDS-PAGE were quantified and normalized to the loading control (Mfn2). The amount of protein in control mitochondria was set to 100%, and data represent mean ±± SD (n = 3). (C) Mitochondria were isolated from control, AGKKO, and AGKKO cells stably expressing wild-type AGK (AGKKO+WT) or a kinase-dead mutant (AGKKO+G126E 3XFLAG) and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Protein levels were quantified and normalized to the loading control, with the amount of protein in control mitochondria set to 100%. Data represent mean ± SD (n = 3). (D and E) Mitochondrial samples as described in (C) were solubilized in digitonin-containing buffer and analyzed by BN-PAGE and immunoblotting using the indicated antibodies. See also Figures S1 and S2. # indicates a cross-reactive band observed with AGK antibodies; ˆ indicates aTim29 subcomplex/monomer; ∗ indicates migration of AGKG126E 3XFLAG; ANTII and GC1II refer to ANT and GC1 dimers, respectively. Molecular Cell 2017 67, 457-470.e5DOI: (10.1016/j.molcel.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 3 AGK Functions in the Import and Assembly of Mitochondrial Carrier Proteins (A–F) The [35S]-labeled proteins (A) hTim22, (B) hTim23, (C) adenine nucleotide transporter (ANT1), (D) glutamate carrier (GC1), (E) phosphate carrier (PiC), and (F) hTom22 were imported into mitochondria isolated from control cells, AGKKO cells, AGKKO cells re-expressing AGKWT, or AGKG126E for the indicated times with (+) or without (−) membrane potential (ΔΨ). Mitochondria were re-isolated and solubilized in digitonin-containing buffer before separation on BN-PAGE and analysis using autoradiography. Equal protein loading was assessed by western blotting (bottom). See also Figures S3–S5. ANTII, GC1II, and PiCII refer to assembled dimers of ANT1, GC1, and PiC, respectively. Molecular Cell 2017 67, 457-470.e5DOI: (10.1016/j.molcel.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 4 Loss of AGK Affects Maximal Respiration Rates and Supercomplex Stability (A) Control and AGKKO cells were seeded at equal density and grown in glucose- or galactose-containing cell culture medium for 24 hr. Cell proliferation was then assessed by growing cells in the presence of BrdU for 24 hr and measuring absorbance at 450–550 nm. Data represent mean ± SD (n = 3). (B) Mitochondrial basal and maximal respiration rates in control and AGKKO cells. Data are mean ± SD (n = 3). (C) Control and AGKKO mitochondria were analyzed on SDS-PAGE, followed by immunoblotting using the indicated antibodies. (D) Mitochondria isolated from control, AGKKO, and AGKKO+G126E cells were solubilized in digitonin buffer, and complexes were resolved using BN-PAGE and immunoblotting using the indicated antibodies. SC indicates CI+III+IV respiratory supercomplexes, CIV refers to complex IV, and CII to complex II. High and Low refer to high and low exposure time, respectively. # indicates cross-reactive band observed with AGK antibody. Molecular Cell 2017 67, 457-470.e5DOI: (10.1016/j.molcel.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 5 Cells Lacking AGK Display Defects in Central Carbon Metabolism (A) Left: relative abundance of intracellular metabolites extracted from control or AGKKO cells. The data were normalized to the control and represented as mean ± SEM. Unpaired Student’s t test was performed to examine the differences in metabolite abundance between control and AGKKO cells, and the p values generated were further adjusted to control for false positives using Benjamini-Hochberg (BH) adjustment to produce the BH-adjusted p values, which are represented as follows: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005; n = 6 for control and n = 5 for AGKKO. Right: table showing the numerical mean values and BH-adjusted p values of the tabulated metabolites. (B) Graphic representation of central carbon metabolism through glycolysis and the TCA cycle upon glucose uptake. (C and D) Percentage of 13C-label in glycolytic (C) and TCA cycle intermediates (D) in 13C-labeled control cells, AGKKO cells, and AGKKO cells re-expressing AGKWT. Data are represented as mean ± SD (n = 2). See also Tables S1 and S2. Glc, glucose; 3PGA, 3-phosphoglyceric acid; Lac, lactate; Ala, alanine; Cit, citrate; Iso, isocitrate; Fum, fumarate; Mal, malate; Asp, aspartate; Glu, glutamate. Molecular Cell 2017 67, 457-470.e5DOI: (10.1016/j.molcel.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 6 Sengers Syndrome Patient Fibroblasts Display Defects in the TIM22 Complex and Import Pathway (A) Mitochondria isolated from control or patient (P1 and P2) fibroblasts were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Protein levels were quantified and normalized to the loading control (SDHA). The amount of protein in control mitochondria was set to 100%, and data represent mean ± SD (n = 3). (B) Mitochondria isolated from control and patient fibroblasts were solubilized in digitonin buffer and analyzed by BN-PAGE and immunoblotting using the indicated antibodies. (C–E) [35S]-hTim22 (C), [35S]-ANT1 (D), and [35S]-hTim23 (E) were imported into mitochondria isolated from control and patient (P1 and P2) fibroblasts in the presence (+) or absence (−) of membrane potential (ΔΨ) for 60 min, followed by PK treatment. Mitochondria were re-isolated and solubilized in digitonin-containing buffer prior to BN-PAGE and autoradiography (top) and western blot analyses (bottom). See also Figure S6. ANTII and GC1II refer to ANT and GC1 dimers, respectively. Molecular Cell 2017 67, 457-470.e5DOI: (10.1016/j.molcel.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 7 Mitochondria Isolated from Sengers Syndrome Patient Tissue Display Destabilization of TIM22 and Reduced Steady-State Levels of Carrier Proteins (A–C) Mitochondria isolated from control or P2 liver tissue were subjected to SDS-PAGE (A) and BN-PAGE (B and C) and immunoblotting using the indicated antibodies. (D) Control and P2 muscle-derived mitochondria were lysed in digitonin buffer and resolved by BN-PAGE, followed by western blotting using the indicated antibodies. (E and F) Mitochondria isolated from P2 fibroblasts and P2 fibroblasts stably expressing wild-type or kinase-dead AGK were analyzed using SDS-PAGE (E) or BN-PAGE (F), followed by western blotting using the indicated antibodies. ANTII and GC1II refer to the assembled dimers of ANT and GC1, respectively. SC refers to the respiratory supercomplex CI+III+IV, and CII and CIV represent complex II and complex IV of the mitochondrial respiratory chain, respectively. Molecular Cell 2017 67, 457-470.e5DOI: (10.1016/j.molcel.2017.06.014) Copyright © 2017 Terms and Conditions