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In collaboration with Dr Ramona Lupi, Metabolism in Brain Diseases, European Brain Research Institute, Rita Levi-Montalcini Foundation and Michelangelo Campanella, University of London Consortium for Mitochondrial Research (CfMR), University College London.
Mitochondrial membrane proteins are encoded by both the nuclear and mitochondrial genomes. Nuclear-encoded proteins must pass the outer membrane via the central entry gate, the translocase of the outer membrane (TOM complex). The outer membrane (OM) additionally contains the sorting and assembly machinery (SAM complex) required for the biogenesis of OM proteins.
Precursor proteins follow different sorting pathways. The insertion of proteins with internal signal sequences to the inner membrane (IM) is mediated by the carrier translocase of the IM (TIM22 complex). Matrix-targeted and inner membrane-sorted pre-proteins with cleavable N-terminal presequences are directed to the translocase of the IM (TIM23 complex). Translocation of pre-protein domains across the IM requires ATP-driven pre-sequence translocase-associated motor (PAM).
A small number of IM proteins are encoded by mitochondrial DNA. Oxa1 is the main insertase and, together with Mdm38 and Mba1, binds ribosomes and inserts proteins into the IM. Intermembrane space proteins (IMS) with cysteine motifs require the machinery for import and assembly (MIA) in the mitochondrial IMS.
Mitochondria exist in a dynamic network within living cells, undergoing fusion and fission events that facilitate IM and OM fusion and the exchange of organelle contents.
Mitochondrial fusion depends on the action of three large GTPases: mitofusions (Mfn1 and Mfn2) mediating membrane fusion on the OM level, and Opa1 which is essential for inner mitochondrial membrane fusion.
Mitochondrial fission requires local organization of Fis1 and recruitment of GTPase DRP1 for assembly the fission machinery that subsequently leads to membrane scission.
Mitophagy is the selective removal of damaged mitochondria by autophagosomes and their subsequent catabolism by lysosomes.
The first event in quality control by mitophagy is the distinction between damaged mitochondria (with depolarized mitochondrial membrane potential) and healthy mitochondria. These are distinguished by the accumulation of PTEN induced putative kinase 1 (PINK1) following mitochondrial membrane depolarization of damaged mitochondria.
In healthy mitochondria, PARL, the rhomboid-like protein localized in the mitochondrial IM, processes PINK1. Newly synthesized PINK1 in the cytosol is imported and inserted into the mitochondrial IM, and is cleaved in its putative transmembrane domain by PARL to generate the 52 kDa form of PINK1. This processed form of PINK1 is rapidly removed by a proteasome-dependent pathway, likely after its release into the cytosol from the mitochondrial IMS.
Upon depolarization of the mitochondrial membrane potential, the IM insertion and subsequent processing of PINK1 by PARL may be inhibited, leading to full-length PINK1 accumulating in the mitochondrial OM, probably facing the cytosol. The accumulation of PINK1 with kinase activity is sufficient for Parkin recruitment to the mitochondrial surface.
Following Parkin recruitment, mitophagy induction involves the Parkin-mediated ubiquitination of mitochondrial substrates that prominently display Lys63-linked polyubiquitin chains, usually associated with signaling. Different Parkin substrates have been identified in mitochondria: the mitofusin mitochondrial assembly regulatory factor (MARF), mitofusin 1, mitofusin 2 and voltage dependent anion-selective channel protein 1 (VDAC1), all of which are embedded in the OM.
Parkin promotes the recruitment of p62, an ubiquitin-binding adaptor also known as sequestosome 1. The p62 protein can both aggregate ubiquitinated proteins by polymerizing with other p62 molecules and recruit ubiquitinated cargo into autophagosomes by binding to LC3. p62 accumulates on mitochondria, binds to Parkin-ubiquitinated mitochondrial substrates, mediates clumping of mitochondria and links ubiquitinated substrates to LC3 to facilitate the autophagic degradation of ubiquitinated proteins. The histone deacetylase HDAC6, also binds ubiquitinated substrates, accumulates on mitochondria following Parkin translocation and is required for Parkin-mediated mitophagy.
LC3 is synthesized as proLC3 in the cytosol. Soon after its translation, proLC3 is cleaved by Atg4B to expose the carboxyterminal Gly of LC3 (LC3-l). LC3-l is activated by the same E1-like enzyme, Atg7, transferred to Atg3 (a second E2-like enzyme), and conjugated to PE (phosphatidyl-ethanolamine). The LC3-PE conjugate is known as LC3-II, an autophagosome membrane bound form of LC3. The Atg12-Atg5 conjugate functions as an E3-like ligase for LC3 lipidation.
Following the formation of the isolation membrane, it elongates to engulf mitochondria. During elongation of the isolation membrane, the Atg5-Atg12=Atg16L complex localizes to the membrane to form a cup-shaped structure. LC3-II localizes to the isolation membrane, while the Atg5-Atg12=Atg16L complex dissociates from it. Soon after autophosome formation, its outer membrane fuses with lysosomes to form autolyosomes. Following autolysosome formation, the lysosomal hydrolases, including cathespins and lipases, degrade the intra-autophoagosomal contents, whereas cathepsins degrade LC3-II on the intra-autophagosomal surface.