Mitophagy pathway

Mitophagy is the selective removal of damaged mitochondria by autophagosomes and their subsequent catabolism by lyosomes. Learn the role of PINK1, Parkin, and the autophagy machinery in the mitophagy pathway with our interactive pathways and identify key reagents to further your research by reading the pop ups and product tables that include Abcam's full product offering.  

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Contents

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Mitophagy pathway: an overview

Mitophagy is an important mitochondrial quality control mechanism that eliminates damaged mitochondria. In mammals, the mitophagy pathway involves PTEN-induced putative protein kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin.

The mitophagy pathway needs to distinguish between damaged (with depolarized mitochondrial membrane potential) and healthy mitochondria. These are distinguished by the accumulation of PINK1 following mitochondrial membrane depolarization of damaged mitochondria.

In healthy mitochondria, newly synthesized PINK1 in the cytosol is imported and inserted into the mitochondrial inner membrane (IM). Then PINK1 is cleaved in its putative transmembrane domain by PARL (rhomboid-like protein) 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 intermembrane space proteins (IMS).

In unhealthy mitochondria, the inner mitochondrial membrane becomes depolarized. 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.

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The mitophagy pathway includes the following main steps:

  1. PINK1 stabilization: Under mitochondrial stress/depolarisation, full-length PINK1 is stabilized at the outer mitochondrial membrane as PINK1 import is blocked (via TOM and TIM complexes). PINK1 is subsequently cleaved by the inner membrane protease PARL.
  2. PINK1 activation: When PINK1 stabilizes at the surface of the mitochondria, it is activated by auto-phosphorylation at Ser228.
  3. Ubiquitin phosphorylation: Activated PINK1 phosphorylates ubiquitin at Ser65 on outer mitochondrial membrane substrates.
  4. Parkin recruitment: Following phosphorylation at Ser65 by PINK1, ubiquitin binds Parkin with high affinity (cytosolic E3 ubiquitin ligase) from the cytosol to the mitochondria.
  5. Parkin phosphorylation: Once at the mitochondria, Parkin is activated by both its phosphorylation at Ser65 by PINK1 and by binding to phosphorylated ubiquitin (also phosphorylated at Ser65 by PINK1)
  6. Parkin-dependent substrate ubiquitination: Once active, Parkin ubiquitinates numerous substrates at the outer mitochondrial membrane. More ubiquitin chains mean more substrate for PINK1, resulting in more phosho-ubiquitin and increased Parkin recruitment in a feed-forward amplification loop.
  7. Recruitment of autophagy receptors: Ubiquitin molecules at the surface of the mitochondria act as “eat me signals”, leading to the recruitment of both the ubiquitin-proteasome system (UPS) machinery and the autophagy machinery. Autophagy receptors then bridge the ubiquitinated mitochondria to the LC3-positive autophagosome. Phosphorylation of autophagy receptors by TBK1 increases interaction between LC3 and ubiquitin.
  8. Autophagosome: The ubiquitinated mitochondria is engulfed by the autophagosome.
  9. Fusion with lysosome: Autophagosome fuses with the lysosome allowing for the degradation of the autophagosome contents by the lysosome hydrolases.


Role of Parkin in the mitophagy pathway

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, a 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.

​Mitochondrial import pathway

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.

Mitochondrial fission and fusion

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: mitofusins (Mfn1 and Mfn2), mediating membrane fusion on the OM level, and Opa1, essential for the inner mitochondrial membrane fusion.

Mitochondrial fission requires a local organization of Fis1 and recruitment of GTPase DRP1 for assembly of the fission machinery that subsequently leads to membrane scission.

References

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