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Volume 64, Issue 1, Pages (October 2016)

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Presentation on theme: "Volume 64, Issue 1, Pages (October 2016)"— Presentation transcript:

1 Volume 64, Issue 1, Pages 148-162 (October 2016)
The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria  Tim König, Simon E. Tröder, Kavya Bakka, Anne Korwitz, Ricarda Richter-Dennerlein, Philipp A. Lampe, Maria Patron, Mareike Mühlmeister, Sergio Guerrero-Castillo, Ulrich Brandt, Thorsten Decker, Ines Lauria, Angela Paggio, Rosario Rizzuto, Elena I. Rugarli, Diego De Stefani, Thomas Langer  Molecular Cell  Volume 64, Issue 1, Pages (October 2016) DOI: /j.molcel Copyright © 2016 Elsevier Inc. Terms and Conditions

2 Molecular Cell 2016 64, 148-162DOI: (10.1016/j.molcel.2016.08.020)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3 Figure 1 The Neuronal Interactome of AFG3L2
(A) Expression of hexa-histidine-tagged AFG3L2trap in brain mitochondria of 6-week-old mice. (B) Kaplan-Meier survival curves of control, Afg3l2NTE, and Afg3l2NKO mice (n = 32 each). (C) Forebrain atrophy in 9-week-old Afg3l2NKO and Afg3l2NTE mice (the scale bar represents 2 mm). Nissl staining of coronal hippocampal sections in Afg3l2NKO and Afg3l2NTE mice at 8 weeks of age (the scale bar represents 0.2 mm) is shown. (D) Progressive decrease of brain weight in Afg3l2NTE mice (SD; n = 3). (E) Neuronal interactome of AFG3L2trap determined by quantitative label-free LC-MS/MS analysis after affinity purification from forebrain mitochondria of 5-week-old mice. Only proteins identified in three independent experiments are shown (Table S1). Ethe1 (fold change 2.12, p value 0.41) is not shown. (F) Immunoblot analysis of proteins co-purifying with AFG3L2trap (input, IN [10%]; flow through, FT [10%]; elution, E [100%]; cross-reaction, ∗; cortex, CO; hippocampus, HC; striatum, ST; cerebellum, CB; CaMKIIα-Cre recombinase, Cre; ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001). See also Figure S1. Molecular Cell  , DOI: ( /j.molcel ) Copyright © 2016 Elsevier Inc. Terms and Conditions

4 Figure 2 MAIP1 Is a Novel m-AAA Protease Binding Protein in the Matrix
(A) Immunoblot analysis of proteins co-purifying with hexa-histidine-tagged AFG3L2wt expressed in HEK293 cells (input, IN [20%]; flow through, FT [20%]; and elution, E [100%]). (B) BN-PAGE analysis reveals loss of MAIP1-containing ∼2.3 MDa complexes upon Cre-mediated deletion of Afg3l2 in Afg3l1Δ/ΔAfg3l2fl/fl MEFs. (C) Complexome analysis of MEF mitochondria (n = 3) (Table S2). The heatmaps and migration profiles of MAIP1, m-AAA protease subunits, and prohibitins are shown. (D) Submitochondrial localization of MAIP1 in HEK293 mitochondria (proteinase K, PK). (E) Membrane association of MAIP1 after sonication of mitochondria and treatment with HEPES/KOH (pH 7.6) and 1 M NaCl or Na2CO3 as indicated (total, T; pellet, P; and supernatant, S). (F) Proteins containing a TIMM44-like domain in human (h) and yeast (y) (grey, mitochondrial targeting sequence [M]; green, import machinery interaction site; and orange, TIMM44-like domain). See also Figure S2. Molecular Cell  , DOI: ( /j.molcel ) Copyright © 2016 Elsevier Inc. Terms and Conditions

5 Figure 3 MAIP1 Assists Sorting of EMRE in Mitochondria
(A) Steady-state level of MCU complex subunits in HeLa mitochondria analyzed by immunoblotting. (quantification of precursor [p] and mature [m] EMRE) (SD; n = 7). (B) Topology of EMRE in the IM (red, mitochondrial targeting sequence, green, transmembrane domain, IMS, and matrix, M). (C) Import of 35S-EMRE into isolated mouse liver mitochondria (proteinase K, PK and degradation product generated by PK, ∗). (D) Import of 35S-EMRE and variants into HEK293 mitochondria. (E) Localization of p-EMRE and m-EMRE in HeLa mitochondria. (F) Steady-state level of EMRE in HeLa mitochondria depleted of proteins indicated. (G) Chemical crosslinking of newly imported 35S-EMRE to MAIP1 with disuccinimidyl suberate (DSS, 100 μM) in HEK293 mitochondria. After immunoprecipitation with MYC-specific antibodies, samples were analyzed by SDS-PAGE and autoradiography (input 5% and elution 100%). (H and I) Interaction of purified HIS-MAIP1 synthesized in a cell-free system with 35S-EMRE (amino acid [aa] 1–107 or aa 1–63) in vitro (input 4% and elution 100%) (H). Binding was assessed by affinity purification and quantified in (I) (SD; n = 4) (scrambled, SCR; ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001). See also Figure S3. Molecular Cell  , DOI: ( /j.molcel ) Copyright © 2016 Elsevier Inc. Terms and Conditions

6 Figure 4 The m-AAA Protease Limits the Availability of EMRE for MCU Assembly (A) Assembly of newly imported 35S-MCU into MCU complexes was analyzed by BN-PAGE. MCU was imported into HEK293 mitochondria, which were depleted of EMRE or contained overexpressed EMRE, and assembled into ∼300 kDa (black arrow) and ∼400 kDa (red arrow) complexes, respectively. (B) BN-PAGE analysis using mitochondria isolated from liver, cerebellum, or forebrain demonstrating the existence of ∼300 kDa and ∼400 kDa MCU complexes at different ratios. (C) Assembly of imported 35S-EMRE or MCU into ∼300 kDa and ∼400 kDa complexes in mouse liver mitochondria analyzed by BN-PAGE. (D and E) Facilitated assembly of imported 35S-MCU into ∼400 kDa complexes in HEK293 mitochondria depleted of SPG7/AFG3L2 (D). The ratio of 400 kDa to 300 kDa complexes was quantified (E) (SD; n = 3). (F) Affinity purification of mature EMRE with AFG3L2trap by metal chelating chromatography using mitochondria isolated from HEK293 cells stably expressing hexa-histidine-tagged AFG3L2trap (input 5% and elution 100%). (G) Stability of assembled and non-assembled EMRE in HEK293 cells stably expressing EMRE-MYC was assessed by SDS-PAGE and (H) BN-PAGE using immunoblotting (cycloheximide, CHX; mature, m; scrambled control, SCR; ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001). See also Figure S4. Molecular Cell  , DOI: ( /j.molcel ) Copyright © 2016 Elsevier Inc. Terms and Conditions

7 Figure 5 Loss of AFG3L2 Disturbs MCU Assembly via EMRE
(A) MCU complexes in cortical mitochondria of 8-week-old Afg3l2fl/fl (−Cre) and Afg3l2NKO (+Cre) mice, monitored by BN-PAGE (CaMKIIα-Cre recombinase, Cre). (B) Quantification of MCU- (SD; n = 6) and MICU3-containing complexes (SD; n = 5) related to (A). (C) Transient assembly of imported 35S-EMRE into ∼100 kDa intermediates in HEK293 mitochondria analyzed by BN-PAGE. (D) Quantification of (C) (SD; n = 4). (E) BN-PAGE analysis of HEK293 mitochondria ectopically expressing EMRE demonstrating co-migration of EMRE, MICU1, and MICU2 in a ∼100 kDa assembly intermediate (cross-reaction of the MICU1 antibody, ∗). (F) BN-PAGE analysis using HeLa mitochondria depleted of MICU1, AFG3L2/SPG7, or both (scrambled control, SCR; ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001). See also Figure S5. Molecular Cell  , DOI: ( /j.molcel ) Copyright © 2016 Elsevier Inc. Terms and Conditions

8 Figure 6 Increased Ca2+ Influx into Mitochondrial Lacking the m-AAA Protease (A) Model for the assembly of the Ca2+ uniporter MCU. (B) EMRE accumulating upon loss of the m-AAA protease competes with the ∼100 kDa EMRE-MICU1-MICU2 assembly intermediates for MCU binding, resulting in the formation of both constitutive active ∼400 kDa MCU-EMRE and regulated ∼1.1 MDa MCU channels containing gatekeepers. (C–F) Mitochondrial and cytosolic (E) Ca2+ influx after CPA (20 μM) induced Ca2+ leak from the ER in intact HeLa cells measured with aequorin probes and quantifications of the peak values (D and F) (SD; n = 4). For control, the gatekeeper MICU1 was depleted (D and F) (scrambled control, SCR; ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001). See also Figure S6. Molecular Cell  , DOI: ( /j.molcel ) Copyright © 2016 Elsevier Inc. Terms and Conditions

9 Figure 7 Loss of m-AAA Proteases Accelerates MPTP Opening
(A) CRC of HEK293 mitochondria depleted of m-AAA protease subunits. The buffers were supplemented with ATP (0.4 mM), NADH (0.5 mM), sodium succinate (5 mM), and an ATP-regenerating system. The representative traces are shown. (B) Quantification of the CRC in (A) (SD; n = 5). (C and D) CRC of HeLa mitochondria depleted of the indicated proteins. The representative traces and quantification are shown in (C) and (D) (SD; n = 3). (E–H) Representative traces and quantification of the CRC of (E and F) cortical and (G and H) hippocampal mitochondria of 8-week-old Afg3l2fl/fl (black) and Afg3l2NKO mice (red) (SD; n = 5). The arrows indicate Ca2+ addition (15 μM); cyclosporine A, CsA (2 μM); rotenone, Rot (2 μM); ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < See also Figure S7. Molecular Cell  , DOI: ( /j.molcel ) Copyright © 2016 Elsevier Inc. Terms and Conditions


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