ARMC1 partitions between distinct complexes and assembles MIRO with MTFR to control mitochondrial distribution

Abstract

Maintaining an optimal mitochondrial distribution is critical to ensure an adequate supply of energy and metabolites to support important cellular functions. How cells balance dynamic mitochondrial processes to achieve homeostasis is incompletely understood. Here, we show that ARMC1 partitioning between distinct mitochondrial protein complexes is a key determinant of mitochondrial distribution. In one complex, the mitochondrial trafficking adaptor MIRO recruits ARMC1, which mediates the assembly of a mitochondrial fission regulator (MTFR). MTFR stability depends on ARMC1, and MIRO-MTFR complexes specifically antagonize retrograde mitochondrial movement. In another complex, DNAJC11 facilitates ARMC1 release from mitochondria. Disrupting MIRO-MTFR assembly fails to rescue aberrant mitochondrial distributions clustered in the perinuclear area observed with ARMC1 deletion, while disrupting ARMC1 interaction with DNAJC11 leads to excessive mitochondrially localized ARMC1 and distinct mitochondrial defects. Thus, the abundance and trafficking impact of MIRO-MTFR complexes require ARMC1, whose mito-cytoplasmic shuttling balanced by DNAJC11 tunes steady-state mitochondrial distributions.

Fig. 1.. Mitochondrial ARMC1 stabilizes MTFRs.

(A) ARMC1 CTD is required for mitochondrial localization. Fluorescence microscopy of fixed ARMC1 KO COS7 cells stably re-expressing near-endogenous levels of WT mNG-ARMC1 or mNG-ARMC1 lacking 15 C-terminal residues (ΔCTD), showing mNG-ARMC1 (green), MitoTracker CMXRos (magenta), and nuclei [4′,6-diamidino-2-phenylindole (DAPI), blue]. Scale bar, 15 μm. (B) Manders’ colocalization coefficients (MCCs; mean + SD and measurements for n = 82 WT and n = 100 ΔCTD cells) of the mNG-ARMC1 variants with MitoTracker CMXRos as in (A). ****P < 0.0001. (C) Immunoblotting of ARMC1 KO Flp-In 293 T-REx cells re-expressing WT or ΔCTD F-ARMC1 before (t, total) or after fractionation into cytosolic (c) and membrane (m) fractions for F-ARMC1, the cytosolic protein SGTA, and the mitochondrial protein TOM20. (D) Radiolabeled ARMC1 variants synthesized in an in vitro mammalian translation system were incubated without or with organelles (memb.) isolated from Expi293F cells and analyzed before (total) or after centrifugation to pellet the membrane fraction by SDS-PAGE and autoradiography (top). The ratio of the F-ARMC1 variant recovered in the pellet fraction relative to WT F-ARMC1 was quantified for four to five independent replicates (bottom). Shown are mean + SD and individual measurements. ****P < 0.0001. (E) ARMC1 stabilizes MTFRs. Volcano plot of multiplexed proteomics data showing fold change (FC) of protein levels in ARMC1 KO Flp-In 293 T-REx cells with or without F-ARMC1 rescue. (F) SDS-PAGE and immunoblotting of WT or ARMC1 KO Flp-In 293 T-REx cells without or with re-expression of WT or ΔCTD F-ARMC1.

Fig. 2.. ARMC1 partitions into distinct mitochondrial complexes.

(A) SDS-PAGE and Coomassie staining of pulldowns (PDs) of F-ARMC1 stably expressed in ARMC1 KO Flp-In 293 T-REx cells, with proteins identified by mass spectrometry indicated. Orange dots, MTFRs. Gray dots, mitochondrial intermembrane bridging (MIB) complex proteins. (B) Immunoblotting of the solubilized membrane fraction (input), flow-through, and elution of F-ARMC1 affinity purification as in (A). (C) SDS-PAGE and immunoblotting (top) or Coomassie staining (bottom) of F-ARMC1 PD as in (A) size fractionated over a 10 to 30% sucrose gradient. Orange, teal, and gray lines indicate distinct complexes. (D) TMEM11 and MTCH2 bind the ARMC1 CTD. In vitro translation reactions of radiolabeled F-ARMC1 with the UV-activated Bpa probe at position 275 (F275Bpa) in the CTD incubated with organelles isolated from Expi293F cells were UV irradiated, immunoprecipitated (IP) for crosslinks to MTCH2 and TMEM11, and assayed by SDS-PAGE and autoradiography.

Fig. 3.. Uncoupling ARMC1 association with distinct mitochondrial complexes.

(A) MTCH2 is required for ARMC1 association with DNAJC11. Chemical crosslinking reactions of ΔCTD or WT-radiolabeled F-ARMC1 incubated with WT, MTCH2 KO, MIRO1 and MIRO2 double KO (DKO), or TMEM11 KO semi-permeabilized (SP) cells, analyzed by SDS-PAGE and phosphor imaging. Crosslinks to MIRO (orange dot), DNAJC11, and MTX1 (teal dots) are indicated. (B) Alphafold models of the indicated ARMC1 complexes with inferred placement of the OMM and protein topologies. IMS, intermembrane space. Circled numbers note interaction interfaces between two proteins as indicated by colored dots (ARMC1 helical domain, dark red; ARMC1 HMAL domain, salmon; ARMC1 CTD, light blue; MTFR, light orange; MIRO, gray; TMEM11, brown; DNAJC11, dark teal; MTCH2, tan; MTX1, light teal). (C) Structure-guided mutations uncouple ARMC1 complex association. Immunoblotting of membrane fractions (input, left) and pulldowns (right) of the indicated F-ARMC1 variants expressed in ARMC1 KO Flp-In 293 T-REx cells. 4K: D239K, E243K, D244K, and E245K.

Fig. 4.. ARMC1 partitioning impacts mitochondrial distribution.

(A) ARMC1 levels and variants change mitochondrial distribution. Immunofluorescence of WT and ARMC1 KO COS7 cells complemented without or with the indicated mNG-tagged ARMC1 variants, showing the mitochondrial protein HSP60 (magenta), the Golgi marker GOLGA2 (yellow), and nuclei (DAPI, blue). Scale bar, 15 μm. O/E, overexpressed. (B) Proportion of HSP60 signal within a dilated nuclear mask of cells as in (A). Shown are the mean, 95% confidence interval, the number of fields of view, and P values. (C) Distances between the COMs of HSP60 (mito.) to DAPI (left) or GOLGA2 (right) signals in WT (n = 329), ARMC1 KO (n = 457), or mNG-ARMC1 rescue (n = 141) cells as in (A). Shown are median, interquartile range, individual values, and P values. (D) ARMC1 is important for mitochondrial distribution in iNeurons. Live-cell images of WT or ARMC1 KO iNeurons after 12 days of NGN2-induced differentiation showing polarized mitochondria (TMRE, white and top), total mitochondria (MitoTrackerGreen, magenta), nuclei (Hoechst, blue), and microtubules (SiR-tubulin, green). Scale bar, 10 μm. (E) Percentage of MitoTrackerGreen (MT) signal outside of a soma mask to total MT signal in a SiR-tubulin mask of WT and three clonal lines of ARMC1 KO iNeurons after 20 days of differentiation from eight different fields of view each. (F) Ratio of TMRE to MT signal of WT and ARMC1 KO iNeurons after 20 days of differentiation from 26 different fields of view each.

Fig. 5.. ARMC1-mediated MIRO-MTFR assembly competes with TRAK2 activity.

(A) MTFR and TRAK engage a common MIRO binding site. Alphafold models of MTFR1L or TRAK2 with MIRO2 predict a common interaction interface with the N-GTPase (NG) and first EF-hand (EF1) domain of MIRO. (B) ARMC1 impacts TRAK mitochondrial recruitment. Immunoblotting of membrane fractions of WT and ARMC1 KO COS7 cells without or with overexpression of mNG-tagged ARMC1 shows increased TRAK2 recovery in the absence of ARMC1. (C) TRAK2 depletion specifically rescues the ARMC1 KO phenotype. Immunofluorescence showing the mitochondrial protein TOM20 (top or magenta), the Golgi marker GOLGA2 (yellow), and nuclei (DAPI, blue) of ARMC1 KO cells without or with siRNA-mediated knockdown (KD) of TRAK1 or TRAK2. Scale bar, 15 μm. (D) Proportion of TOM20 signal within a dilated nuclear mask of WT or ARMC1 KO cells treated with control (cont.) siRNAs or siRNAs against TRAK1 or TRAK2 and imaged as in (C). Shown are the mean, 95% confidence interval, the number of fields of view analyzed, and P values. (E) Distance between the COMs of TOM20 (mito.) to DAPI (nucleus, left) or GOLGA2 (Golgi, right) signals in cells imaged as in (C). Shown are median, interquartile range, and individual values (WT, n = 393, 201, and 208; ARMC1 KO, n = 430, 274, and 280 for control, TRAK1, and TRAK2 KD cells, respectively).

Fig. 6.. MIRO and DNAJC11 have opposing effects on ARMC1 localization.

(A) ARMC1 membrane association decreases with MIRO deletion. Immunoblotting of F-ARMC1 Flp-In 293 T-REx rescue cells without or with MIRO1 and MIRO2 double KO (DKO) directly (total, t) or after separation of cytosolic (c) and membrane (m) fractions. (B) Volcano plot of multiplexed proteomics data showing the fold-change (FC) of proteins associated with cellular membranes in MIRO1 and MIRO2 DKO relative to WT Flp-In 293 T-REx cells. Individual, OMM, and other mitochondrial proteins are indicated. (C) ARMC1 and MTFR membrane association increases with DNAJC11 depletion. Immunoblotting of the cytosolic and membrane fractions of F-ARMC1 rescue cells without or with siRNA-mediated KD of DNAJC11. (D) As in (A) but using 4K F-ARMC1 rescue cells without or with MIRO DKO. (E) DNAJC11 promotes ARMC1 release from mitochondria. Scheme (left) and immunoblots (right) of F-ARMC1 mitochondrial release assays, in which the membrane fraction (memb.) of F-ARMC1 rescue cells without or with DNAJC11 KD were incubated without or with cytosol and then separated into supernatant (S) and membrane pellet (P) fractions. (F) Ratios of radiolabeled F-ARMC1 signal in the supernatant (S) and pellet (P) fractions of release assays in which in vitro synthesized radiolabeled F-ARMC1 was first pre-incubated with membrane fractions (memb.) from cells without or with DNAJC11 KD, which were then re-isolated for release assays in the presence of buffer (buff.) or cytosol as in (E). Shown are means + SD, individual values, and P values for three to four independent replicates.

Fig. 7.. ARMC1 partitioning is a major determinant of mitochondrial distribution.

Model (center) of ARMC1 partitioning between the cytosol and distinct mitochondrial complexes, which requires the CTD of ARMC1. ARMC1 mediates the assembly of MIRO with MTFR and TMEM11. This complex is required for MTFR stability and competes with TRAK2 recruitment to mitochondria. ARMC1 can also engage a complex containing MTX1, MTCH2, and DNAJC11, which promotes ARMC1 release from mitochondria. Imbalances in ARMC1 partitioning between these interactors result in aberrant mitochondrial distributions. If ARMC1 fails to assemble with MIRO and MTFR, then too much mitochondrial recruitment of TRAK2 drives aberrantly excessive mitochondrial movement to produce a clustered mitochondrial distribution in the perinuclear region of cells (left). An excess of ARMC1 results in an aberrant enrichment of mitochondria at the cellular periphery (right).