Bulk protein degradation and organelle clearance occurs via two major systems in eukaryotic cells. The ubiquitin-proteasome pathway predominantly degrades short-lived nuclear and cytosolic proteins. The lysosomal-vacuolar pathway degrades larger substrates such as protein complexes and organelles.
The processing and degradation of cytoplasmic components and organelles by the lysosome/vacuole is known as autophagy (“self-eating”). This catabolic process is enhanced during the stress response such as with nutrient deprivation and starvation. It thus maintains the balance between the biogenesis and production of cell organelle structures, and also destroys bacteria or aggregated proteins. Autophagy plays a role in development, aging, immunity and cell death and in human has been associated with cancer, neurodegeneration and certain muscular diseases.
There are three primary forms of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy. During macroautophagy (the most common), the concerted actions of several different proteins lead to the formation of double-membrane sequestering vesicles (autophagosomes), followed by fusion with the lysosome or vacuole, and subsequent delivery of the inner vesicle (autophagic body) into the lumen of the degradative compartment. Hydrolytic enzymes in the lumen degrade the protein components which are recycled.
Autophagosome formation involves two inter-related pathways that are analogous to ubiquitin-like protein (UBL) conjugation. These pathways utilize two proteins which are the ubiquitin-like modifier proteins Apg8 and Apg12, which are conjugated to their respective substrates in conjunction with similar E1 activating and E2 conjugating enzymes. Both Apg8 and Apg12 are highly conserved in eukaryotes and in humans the Apg8 consists of a multigene family. Structurally, all Apg8 proteins have an N-terminal helical subdomain, a C-terminal domain with a conserved ubiquitin fold, and a conserved C-terminal glycine. Mammalian Apg8 homologues (including GABARAP, GATE-16 and MAPLC3a) are all implicated in membrane processes but have diverse functions. However, they all function with a single set of conserved activating, conjugating and deconjugating enzymes.
Both Apg8 and Apg12 proteins are activated by Apg7, an ATP-dependent homodimeric E1 activating enzyme. This E1 enzyme charges the Apg proteins by forming a high-energy thiolester intermediate which is transferred to the active site cysteine of the E2-like conjugating enzymes Apg3 (in the case of Apg8) or Apg10 (in the case of Apg12). Apg8 proteins are covalently but transiently attached to membrane lipids. Activated Apg8 proteins eventually form conjugates with phosphatidylethanolamine (PE), which becomes a component of the complete autophagosome vesicle. Apg8 precursor processing and deconjugation is catalyzed by a novel cysteine protease Apg4b (also known as autophagin-1). This protease removes the residue following the C-terminal glycine, which is exposed in the mature Apg8 form that is active in subsequent conjugation reactions. Apg4b also removes Apg8 proteins from PE and releases the modifier from the vesicle membrane to regenerate the soluble form.
Apg12 is synthesized in its mature form and becomes conjugated to a single substrate, Apg5 via an isopeptide linkage to a conserved lysine residue. The Apg12-Apg5 complex binds Apg16 non-covalently, and this trimer forms a large multimeric complex through self-association. The exact function of this complex is unknown, but these proteins localize at sites of autophagosome formation.