Atg12-Atg5-Atg16 and LC3 complexes localize to isolation membranes. In nutrient deprivation (starvation), isolation membranes target individual mitochondria by unknown signals in a process inhibited by the PI3K inhibitors, 3-methyladenine (3MA) and wortmannin. Isolation membranes completely envelop individual mitochondria to form double membrane vesicles (autophagosomes). After this sequestration, mitochondria depolarize in a CsA and NIM811 sensitive fashion, and Atg12-Atg5/Atg16 complexes are released from the autophagosomal surface. Autophagosomes then acidify and fuse with lysosomal vesicles to form autolysosomes. Lysosomal hydrolases digest the inner autophagosomal membrane and degrade LC3 trapped inside autophagosomes. Remaining LC3 on the surface of autophagosomes is released. After mitochondrial damage, mitochondria first depolarize and then are recognized and sequestered by isolation membranes recognizing unknown markers on the damaged mitochondria. 3MA and wortmannin do not inhibit this process but actually seem to augment it. In both pathways, sequestered mitochondria are completely digested and their molecular components recycled to the cytoplasm.

In one model (A), the PT pore is composed of ANT from the inner membrane (IM), CypD from the matrix, VDAC from the outer membrane (OM) and other proteins, including hexokinase (HK), creatine kinase (CK) and Bax, a proapoptotic Bcl2 family member. Ca²⁺, inorganic phosphate (Pi), ROS and oxidized pyridine nucleotides NAD(P)⁺ and glutathione (GSSG) promote PT pore opening, whereas CsA, Mg²⁺ and pH less than 7 inhibit opening. In an alternative model (B), PT pores form from misfolding and aggregation of damaged mitochondrial membrane proteins at hydrophilic surfaces facing the hydrophobic membrane bilayer. CypD and other chaperones bind to the nascent PT pores and block conductance of solutes through the aqueous channels formed by the protein clusters. High Ca²⁺ opens these regulated channels acting through CypD, an effect blocked by CsA. As misfolded protein clusters exceeds the number of chaperones available to regulate them, constitutively open unregulated channels form that are not inhibited by CsA.

Cultured rat hepatocytes were loaded with MitoTracker Green and TMRM and imaged by confocal microscopy before (Baseline) and 60 min after (Nutrient Deprivation) changing from complete growth medium to a modified Krebs-Ringer buffer containing glucagon. Green-fluorescing structures (circles) in the overlay images are newly depolarized mitochondria.

Hepatocytes were loaded with TMRM to monitor mitochondrial membrane potential and subjected to nutrient deprivation in modified Krebs-Ringer buffer containing glucagon. In the time-lapse confocal images, note green-fluorescing GFP-LC3 enveloping and then sequestering a red-fluorescing mitochondrion (arrows). After sequestration, the mitochondrion loses its red fluorescence, indicating depolarization.

GFP-LC3 transgenic hepatocytes were loaded with TMRM (Baseline), and 5 to 10 mitochondria were exposed to a pulse of 488-nm laser light at full power (circles). Mitochondria lost red TMRM within a minute, indicating mitochondrial depolarization. After 30 min, green GFP-LC3 fluorescence localized to the region where mitochondria were damaged. After 55 min, the photodamaged mitochondria were sequestered into GFP-LC3-labeled autophagosomes, which indicated that the damaged mitochondria were selectively targeted for mitophagy.

Inducers of the MPT include NAD(P)⁺, GSSG, ROS, Ca²⁺, and mutations of mtDNA (mtDNA_X). As the MPT involves an increasing proportion of mitochondria, cellular responses progress from mitophagy, a repair mechanism, to apoptosis driven by release of cytochrome c and other proapoptotic factors from mitochondria, and finally to necrosis due to ATP depletion. As ATP becomes depleted, mitophagy and apoptosis become inhibited.