Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Oct 21;378(6617):317-322.
doi: 10.1126/science.add1856. Epub 2022 Oct 20.

MTCH2 is a mitochondrial outer membrane protein insertase

Alina Guna #  1 , Taylor A Stevens #  2 , Alison J Inglis #  2 , Joseph M Replogle  1   3   4   5 , Theodore K Esantsi  1   5 , Gayathri Muthukumar  1   6 , Kelly C L Shaffer  2 , Maxine L Wang  1   2   6 , Angela N Pogson  1   5 , Jeff J Jones  2 , Brett Lomenick  2 , Tsui-Fen Chou  2 , Jonathan S Weissman  1   5   6   7 , Rebecca M Voorhees  2
Affiliations

MTCH2 is a mitochondrial outer membrane protein insertase

Alina Guna et al. Science. .

Abstract

In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane β-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction.

PubMed Disclaimer

Conflict of interest statement

Competing interests: JMR consults for Maze Therapeutics and is a consultant for and equity holder in Waypoint Bio. JSW declares outside interest in 5 AM Venture, Amgen, Chroma Medicine, KSQ Therapeutics, Maze Therapeutics, Tenaya Therapeutics, Tessera Therapeutics and Third Rock Ventures. RMV is a consultant and equity holder in Gate Bioscience.

Figures

Fig. 1.
Fig. 1.. Systematic characterization of human mitochondrial TA biogenesis.
(A) An 35S-methionine labelled TOM substrate (made from a fusion of the canonical TOM targeting sequence Su9 and the globular protein MBP) or OMP25 (a mitochondrial TA protein) were translated in rabbit reticulocyte lysate and released from the ribosome using puromycin. Competition assays were performed by incubation with purified mitochondria (see fig. S1) in the presence of increasing concentrations of a recombinant TOM competitor (Su9-DHFR). Mitochondrial insertion was assessed by protease protection and analyzed by SDS-PAGE and autoradiography. See also fig. S2. (B) Schematic of the split GFP reporter system used to specifically query integration of substrates into the outer mitochondrial membrane. A mitochondrial membrane protein fused to GFP11 is expressed in a cell constitutively expressing GFP1–10 in the intermembrane space (IMS) along with a translation normalization marker (RFP) Successful integration into the outer membrane results in complementation and GFP fluorescence. (C) Volcano plot of GFP:RFP stabilization phenotype for the three strongest sgRNAs versus Mann-Whitney p values from two independent replicates of a genome-wide CRISPRi screen using OMP25-GFP11. Individual genes are displayed in grey, and specific factors that increase or decrease OMP25 mitochondrial integration are highlighted and labelled. (D) Integration into mitochondria of the OMP25-GFP11 reporter described in (B) was assessed in K562 cells expressing a non-targeting (control) or MTCH2 knock down sgRNA. GFP fluorescence relative to the normalization marker RFP was determined by flow cytometry and displayed as a histogram. Individual channels are also shown. (E) Biogenesis of USP30-GFP11, an outer membrane resident signal anchored protein, and TIM9A-GFP11, an IMS localized protein, were assessed as in (D).
Fig. 2.
Fig. 2.. MTCH2 is required for mitochondrial outer membrane protein biogenesis.
(A) Label-free mass spectrometry analysis of purified mitochondria isolated from K562 cells using a percoll gradient (fig. S1B) expressing a MTCH2 targeting sgRNA (kd) compared to a non-targeting control (nt). Displayed are proteins that across four biological replicates were statistically altered in MTCH2 depleted versus non-targeting guide expressing cells colored according to the indicated key (signal anchored: SA). (B) Immunoblotting of endogenous proteins in mitochondria isolated from MTCH2 depleted (kd) and control cells in (generated as in A; left), and wild type (wt) and MTCH2 knock out (ko) cells (right). Substrates are colored by topology based on the key shown in (A). Quantification of fold-change in depleted vs control cells is displayed as determined using a dilution series for each antibody. (C) Flow cytometry analysis of integration of outer membrane protein reporters using the split GFP system described in Fig. 1B. GFP fluorescence relative to an RFP expression control are displayed as histograms in MTCH2 knockdown versus non-targeting K562 CRISPRi cells. Displayed are representative examples of a TA, signal anchored (SA), and multipass membrane protein that have a MTCH2 dependent biogenesis defect. (D) Summary of dependence on MTCH2 for the indicated outer membrane substrates determined using the fluorescent reporter system shown in (C) and colored by topology based on the key in (A).
Fig. 3.
Fig. 3.. MTCH2 inserts diverse mitochondrial TAs into the outer membrane.
(A) Schematic of the fusion between an inert N-terminal globular protein (VHP) and the TMDs of a panel of mitochondrial TAs (see also fig. S9) generated to probe TMD dependent insertion by MTCH2. (B) The indicated 35S-methionine labelled TA proteins were analyzed for in vitro insertion over time into mitochondria isolated from wild type (wt) or MTCH2 knockout (ko) K562 cells. Displayed are the samples prior to addition of protease (−PK; top right) and the protease protected fragment that has been affinity purified via a 6xHIS tag on the C-terminus of each substrate (+PK+IP; top left), ensuring insertion in the correct topology. (Bottom) Quantification of three biological replicates are plotted with error bars indicating one standard deviation at each time point. (C) As in (B) comparing insertion of the indicated TA proteins into wild type, MTCH2 ko, and MTCH2 ko + MTCH2 rescue mitochondria. (D) (Top) Schematic showing the photocrosslinking strategy. OMP25 containing the photoactivatable amino acid BpA within its TMD was expressed and purified from E. coli as a complex with calmodulin. OMP25BpA was released from calmodulin by addition of EGTA in the presence of mitochondria purified from K562 cells using a percoll gradient (fig. S1B). Crosslinking was activated by UV-irradiation, and the resulting crosslinked species were affinity purified via the Alfa-tag on the N-terminus of OMP25BpA for identification by mass spectrometry. (Bottom) All proteins identified by mass spectrometry were ranked by iBAQ abundance, and those specifically enriched in the UV compared to the -UV control are highlighted. Though TOM40 and CISD1 were identified, they were not significant hits in our screen (fig. S12), while TOM40 was not required for biogenesis both in vitro (Fig. 1A) and in cells (fig. S12B) (E) As in (D) with the resulting elution analyzed by immunoblotting to assess levels of crosslinked OMP25 BpA-MTCH2. (F) MTCH2 was expressed and purified from human cells and analyzed by SDS-PAGE and Sypro-Ruby staining. (G) Following reconstitution (see fig. S13 for optimization of conditions), the recovered proteoliposomes were analyzed by immunoblotting for incorporation of MTCH2. Using a protease protection assay, the indicated MTCH2 dependent (OMP25, CYB5B) and independent (MFF, USP30) 35S methionine labelled substrates synthesized in rabbit reticulocyte lysate were tested for insertion into liposomes reconstituted with increasing amounts of purified MTCH2 compared to an empty control. The resulting protease protected fragments were immunoprecipitated, imaged by autoradiography (autorad). (H) Mitochondria from wt K562 cells were treated with trypsin and their ability to insert TOM (Su9-DHFR) or TA substrates (OMP25) was assayed by protease protection as in (A). The indicated outer membrane proteins were confirmed to be degraded in a trypsin-dependent manner by immunoblot, while MTCH2 remained largely intact.
Fig. 4.
Fig. 4.. MTCH2 is a master regulator of outer membrane function.
(A) (Top) SLC25 transporters are composed of three sets of two TMDs (six total). The location of the characteristic Px[D/E]xx[K/R] motif within a single SLC25 repeat is indicated. (Bottom) Sequence alignment of helices 1, 3, and 5 (with starting residues indicated) from two canonical inner membrane SLC25 transporters (ADT1, UCP1) and two diverged outer membrane SLC25 transporters (MTCH1, MTCH2), with residues from the Px[D/E]xx[K/R] motif highlighted. (B) Flow cytometry analysis of OMP25-GFP11 integration into the outer membrane using the reporter assay described in Fig. 1B. MTCH1 was depleted by transient knockout in either wild type (wt) or MTCH2 knock out (ko) cell lines. (C) (Top) AlphaFold2 predicted model of MTCH2 highlighting conserved polar and charged residues within the bilayer colored based on their effects on OMP25 shown below. (Bottom) using the reporter strategy shown in Fig. 1B, the indicated MTCH2 mutants, which alter the electrostatic potential of its TMDs, were tested for their effect on the indicated reporters (fig. S20). Depicted is a heat map summarizing the stimulation of each mutant relative to wild type MTCH2 on biogenesis of MTCH2 independent (MICU1, LACTB1, TIM9A, USP30) and dependent (MAVS, OMP25, FUNDC1) substrates. (D) Cell lines expressing GFP1–10 in the ER lumen were used to monitor mislocalization to the ER of mitochondrial TAs fused to a C-terminal GFP11. Table summarizing the analysis when either MTCH2 is depleted or overexpressed (data in fig. S20A, fig. S21, and fig. S22). (E) K562 cells expressing varying levels of MTCH2 or inactive (D189R) or hyperactive MTCH2 mutants (E127R or K25E; Fig. 4C) were treated with the chemotherapeutic imatinib mesylate (IB; 1 μM) or carrier (DMSO) for 72 hours. Apoptosis was assessed by staining with Annexin-V-FITC and analyzed by flow cytometry. Shown are representative dot plots displaying the fraction of apoptotic cells upon IB treatment expressing wt MTCH2 compared to in inactive (D189R) or hyperactive mutant (K25E) (Top) as well as a summary table for all MTCH2 constructs in IB vs carrier treated control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Friedman JR, Nunnari J, Mitochondrial form and function. Nature 505, 335 (2014). - PMC - PubMed
    1. Wang W, Zhao F, Ma X, Perry G, Zhu X, Mitochondria dysfunction in the pathogenesis of Alzheimer’s disease: recent advances. Mol Neurodegener 15, 30 (2020). - PMC - PubMed
    1. Bose A, Beal MF, Mitochondrial dysfunction in Parkinson’s disease. J Neurochem 139 Suppl 1, 216 (2016). - PubMed
    1. Vyas S, Zaganjor E, Haigis MC, Mitochondria and Cancer. Cell 166, 555 (2016). - PMC - PubMed
    1. N W, N P, Mitochondrial Machineries for Protein Import and Assembly The Annual Review of Biochemistry (2017). - PubMed

Publication types

MeSH terms

Substances