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Review
. 2016 Feb;73(4):775-95.
doi: 10.1007/s00018-015-2087-8. Epub 2015 Nov 26.

Mitophagy programs: mechanisms and physiological implications of mitochondrial targeting by autophagy

Anne Hamacher-Brady  1   2 , Nathan Ryan Brady  3   4   5
Affiliations
Review

Mitophagy programs: mechanisms and physiological implications of mitochondrial targeting by autophagy

Anne Hamacher-Brady et al. Cell Mol Life Sci. 2016 Feb.

Abstract

Mitochondria are an essential source of ATP for cellular function, but when damaged, mitochondria generate a plethora of stress signals, which lead to cellular dysfunction and eventually programmed cell death. Thus, a major component of maintaining cellular homeostasis is the recognition and removal of dysfunctional mitochondria through autophagy-mediated degradation, i.e., mitophagy. Mitophagy further constitutes a developmental program, and undergoes a high degree of crosstalk with apoptosis. Reduced mitochondrial quality control is linked to disease pathogenesis, suggesting the importance of process elucidation as a clinical target. Recent work has revealed multiple mitophagy programs that operate independently or undergo crosstalk, and require modulated autophagy receptor activities at outer membranes of mitochondria. Here, we review these mitophagy programs, focusing on pathway mechanisms which recognize and target mitochondria for sequestration by autophagosomes, as well as mechanisms controlling pathway activities. Furthermore, we provide an introduction to the currently available methods for detecting mitophagy.

Keywords: Bnip3; FUNDC1; LC3-interacting region (LIR); Macroautophagy; Mitophagy; Nix; Parkin E3 ligase; Ubiquitin.

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Figures

Fig. 1
Fig. 1
Mitochondrial function and dysfunction. a A mitochondrion is enclosed by two membranes, the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). The mitochondrial compartment between the OMM and IMM is referred to as the intermembrane space (IMS). The respiratory chain (electron transport chain, ETC), which is composed of complexes I–V and localized in the IMM, drives ATP synthesis in the mitochondrial matrix. b ROS are implicated in both physiological and pathophysiological signaling. c Electrons from the tricarboxylic acid (TCA) cycle substrates are transferred through the respiratory chain complexes (along the yellow arrows), driving the extrusion of protons (+) from the matrix, thereby generating the proton motive force. Proton flow through the ATP synthase (complex V) drives ATP production. Oxygen (O2) serves as the terminal electron acceptor at complex IV, forming H2O. At complexes I and/or III electron leak can produce the reactive oxygen species (ROS) superoxide anion (O2 −˙)
Fig. 2
Fig. 2
Mitochondrial dynamics are linked to mitophagy. The GTPases Mitofusin 1 (Mfn1) and Mitofusin 2 (Mfn2) mediate OMM fusion, and the GTPase OPA1 (Optic atrophy 1) mediates IMM fusion. Mitochondrial fragmentation requires the translocation of the GTPase Drp1. Normally cytosolic, Drp1 is recruited to mitochondria via OMM-bound receptor proteins Fis1, Mff, MID49, and MID51. Protein kinase A (PKA)-mediated phosphorylation of Drp1 at serine 656 inhibits its activity, resulting in hyperfused mitochondrial networks, while dephosphorylation by the phosphatase calcineurin activates Drp1. Active Drp1 constricts and fragments mitochondria. Drp1-driven mitochondrial fragmentation is a critical quality control event upstream of mitophagy
Fig. 3
Fig. 3
Autophagosome formation and degradation. a Autophagy involves phagophore-nucleated autophagosome formation, fusion with endolysosomes to form the autolysosome, and subsequent degradation of the autophagosome and its contents by lysosomal hydrolases. Mammalian Atg8 proteins include proteins of the LC3 subfamily, which participate in phagophore elongation, and of the GABARAP subfamily, which coordinate closure of autophagosome. Proper LC3 function is positively and negatively regulated through phosphorylation. Fusion between autophagosome and endolysosomes is coordinated by Pleckhm1, which at the endolysosome binds Rab7 and the HOPS complex, and at the autophagosome binds LC3 and the HOPS complex. The SNARE member Syntaxin17 (STX17) binds autophagosomes through interaction with the endolysosomal SNARE VAMP8, thereby mediating autophagosomal-lysosomal membrane fusion. Autophagosomes and content are then degraded by lysosomal hydrolases. b Cytosolic microtubule-associated protein light chain 3 (LC3)-I is conjugated to phosphatidylethanolamine (PE) to form lipidated LC3-II (LC3-PE), an integral membrane component of the autophagosome and binding partner for autophagy receptors. Two ubiquitin-like conjugation reactions coordinate LC3 lipidation. (1) E1-like and E2-like Atg7 and Atg10 conjugate Atg12 to Atg5, which then complex with Atg16. (2) Pro-LC3 is cleaved at the C-terminus by Atg4, forming LC3-I, which is activated by E1-like Atg7. Through the E2-like Atg3 and E3-like Atg12-Atg5-Atg16 complex, LC3 is lipidated with PE
Fig. 4
Fig. 4
The LC3-interacting region (LIR) motif as a mechanistic basis for mitophagy. a LC3B structure (PDB 1UGM) [179], analyzed using Chimera [180]. Upper panel ribbon structure diagram indicating α-helices and β-sheets. LC3 proteins contain two N-terminal α-helices and a ubiquitin-like core formed from β strands. Hydrophobic pockets between β2 and α2 form the W-site, and between β2 and α3 form the L-site. Middle panel surface representation indicates topology of W- and L-sites (adapted from [49]), phosphorylated residues at S12 and T50, and putative binding sites for cardiolipin (R10 and R11) and ceramide (I35 and F52). Lower panel, N-terminal sequence alignments of LC3 homologues. Tcoffee alignment [181] of human LC3 member N-terminal regions using Jalview [182]. Blue shading indicates percentage identity, positively charged R, K and H residues, aligning to LC3B R10 and R11, are indicated in red. b The LIR motif, also known as Atg8 family-interacting motif (AIM), is a short linear peptide motif found in autophagy receptors, which binds LC3-II and thereby underlies selective autophagy. The LIR has a [W/Y/F]xx[L/I/V] core motif, and receptor-ligand interaction occurs through formation of an intermolecular β-sheet via hydrophobic interactions between the LIR motif and the conserved W and L sites on Atg8 proteins. c Sequence alignment of reported mitophagy receptor core LIR motifs and neighboring upstream and downstream regions. Phosphorylation can positively or negatively regulate LIR activity. d Two main groups of autophagy receptors target mitochondria. E3 ligase-mediated ubiquitylation of OMM proteins recruits ubiquitin-binding, LIR motif-containing receptors, which leads to the binding of receptors and engagement of sequestration by autophagosomes. Alternatively, a group of autophagy receptors contain a transmembrane domain and are constitutively targeted to the OMM. LIR activity of these mitophagy receptors is regulated by phosphorylation events
Fig. 5
Fig. 5
Mitophagy program pathways: triggers and post-translational regulation. a Upon mitochondrial depolarization PINK1 is stabilized at the OMM, resulting in phosphorylation of ubiquitin and Mfn2 and consequent recruitment of the E3 ligase Parkin. In the cytosol, Parkin de-ubiquitylation by USP8 is required for mitochondrial translocation. At the mitochondria, Parkin ubiquitylates OMM proteins, resulting in recruitment of ubiquitin-binding autophagy receptors such as p62, OPTN, and NBR1 which then can attach to autophagosomes via their LIR motifs. In addition, PINK1 directly recruits autophagy receptors OPTN and NDP52 through generation of phospho-ubiquitin. AMBRA1 can increase Parkin-mediated mitophagy by direct binding with LC3 and local autophagy stimulation. Sequestration is coordinated by the Rab7 GTPase-activating protein TBC1D15, which localizes to mitochondria by binding Fis1 and binds forming autophagosomes via a LIR. b Bnip3 is induced by hypoxia, and localizes to the OMM via one transmembrane domain. Bnip3 LIR activity requires serine 17 phosphorylation, and is enhanced by serine 24 phosphorylation. Responsible kinase(s) and phosphatase(s) have not yet been identified. Pro-survival Bcl-xL activity functions to enhance Bnip3 binding to LC3. Nix contains an identical SWxxL LIR motif and is expected to undergo similar regulation by phosphorylation. c FUNDC1 contains three transmembrane domains which accomplish its OMM localization. Under normal conditions, Src kinase phosphorylates tyrosine 18 and thereby inactivates the FUNDC1 LIR. Hypoxia inactives Src kinase, permitting FUNDC1 LIR-mediated binding to autophagosomes. As a second negative regulatory mechanism, CK2 phosphorylates FUNDC1 at serine 13, inhibiting its LIR activity, and PGAM5 phosphatase antagonizes this reaction. The pro-survival Bcl-xL can bind PGAM5, inhibiting its pro-mitophagy activity. Under hypoxia and in response to depolarization serine 17 phosphorylation by ULK1 enhances LIR activity. d Bcl2L13 induces Drp1-independent mitochondrial fragmentation and is a LIR-containing mitophagy receptor and mammalian homologue of yeast Atg32 mitophagy protein. e, f Mitophagy receptor systems undergo crosstalk with mitochondrial apoptosis. e Pro-survival Bcl-2 signaling suppresses Parkin translocation to mitochondria, and pro-apoptotic BH3-only proteins can activate Parkin-mediated mitophagy. At longer timescales, Parkin ubiquitylates pro-survival Mcl-1, resulting in its degradation and consequent activation of mitochondrial apoptosis. f Bnip3 LIR and BH3 domains confer dual functionality during apoptosis. Under conditions of LIR inactivation, the BH3-only protein Bnip3 increases TNF-mediated caspase activation. Under conditions of engaged LIR activity, the increased degradation of mitochondria can suppress the mitochondrial participation in apoptosis activation. Green arrows indicate positive regulation; red arrows indicate negative regulation
Fig. 6
Fig. 6
Transcriptional regulation of mitophagy. a Under conditions of mitochondrial and endoplasmic reticulum (ER) stress, Parkin expression can be induced by the PERK/ATF4 pathway, and suppressed by the JNK/c-Jun pathway. Further, p53 and Parkin interactions form complex crosstalk. p53 can induce Parkin expression, and Parkin can inhibit p53 expression, forming a negative feedback circuit. In addition, cytosolic p53 can inhibit Parkin translocation to mitochondria. b Nix expression is induced by G(q) signaling via SP1, by hypoxia via HIF1 and p53, and FOXO3. Nix expression can be suppressed via FOXO3 activation of CITED2. Both Nix and FUNDC1 are negatively co-regulated by microRNAs. miR-351 and hsa-miR-125a-5p target Nix, and miR-137 co-targets Nix and FUNDC1. c Bnip3 expression is increased by Ras activity and induced during hypoxia by HIF1. E2F and FOXO3 activate, and NFkB suppresses Bnip3 expression. Black arrows indicate post-translational regulation; green arrows indicate transcriptional activation; red arrows indicate transcriptional repression
Fig. 7
Fig. 7
Alternative, autophagy-independent modes of mitochondrial processing by endolysosomes. a Under sub-threshold conditions of mitochondrial stress, and prior to depolarization, PINK1 and Parkin mediate a pathway delivering mitochondria-derived vesicles (MDVs), carrying damaged mitochondrial components, directly to endolysosomes. b Under conditions of oxidative stress, Nix and Bnip3 can bind with MIEAP (mitochondrial-eating protein), to induce autophagy-independent interactions of mitochondria with lysosome-like organelles. c Activity of canonical BH3-only proteins, or mitochondrial depolarization, triggers mitochondrial ubiquitylation by the endogenous caspase inhibitor and E3 ligase XIAP and Bax/Bak-mediated entering of XIAP into mitochondria. Mitochondrial activity of XIAP recruits endolysosomal trafficking machinery into mitochondria, leading to the degradation of its inhibitor Smac, thereby reducing the apoptotic potential of mitochondria
Fig. 8
Fig. 8
Methodologies to measure mitophagy. Biochemical and imaging-based assays can measure different aspects of mitophagy program activities. a Mitochondria-containing autophagosomes can be detected via imaging of fluorescent protein (FP)-LC3-labeled autophagosome colocalization with immunofluorescence or FP-labeled mitochondria. Western blot and immunofluorescence detection of IMM and matrix proteins is most specific for detecting mitophagic degradation events. Mito-autolysosomes can be detected using FP-Rab7. b MitoTimer is a tetramer which matures as a green-to-red fluorescent protein. MitoTimer can be used under inducible- and constitutive-expression to analyze mitochondrial quality control dynamics. c Mitochondrial entry into the autolysosome and subsequent degradation can be measured using FP sensors targeted to mitochondria, that are sensitive to low pH and resistant to degradation by lysosomal hydrolases. Mito-Keima fluoresces green at neutral pH in the cytosol, and red upon entry into acidic autolysosomes. Mito-Tandem is a mitochondria-targeted RFP–GFP fusion. GFP fluorescence is acid-quenched while RFP fluorescence remains stable also at low pH, permitting live cell analysis of mitochondrial presence in autolysosomes. Note, lysosomal hydrolases degrade GFP more efficiently than RFP, permitting detection of mitophagy in fixed cells. d Example co-immunoprecipitation demonstrating phospho-regulated Bnip3 mitophagy receptor engagement, i.e., Bnip3 LIR binding with LC3B. MCF7 cells stably expressing GFP-LC3B were transfected with RFP-Bnip3 WT, or LIR-inactive 2SA or LIR-activated 2SE mutants. At 48 h of expression, co-immunoprecipitations (IP) were performed with anti-GFP antibody-coupled magnetic beads. Whole cell lysates (input) and IP samples were analyzed by western blotting (WB). This research was originally published in Zhu et al. [52]. © The American Society for Biochemistry and Molecular Biology
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