Macro-autophagy, hereafter autophagy, is an intracellular degradative process conserved among eukaryotes.
The hallmark of autophagy is the sequestration of the cargo destined to destruction inside double-membrane vesicles called autophagosomes.
Cargoes include aberrant or dysfunctional protein and protein complexes, superfluous or damaged organelles and invading pathogens.
Autophagy is defined as bulk or non-selective when the cargo destined to turnover is heterogeneous in composition and appears to be captured randomly; while they are considered selective when a distinct cargo, for example, a mitochondrion, is exclusively targeted.
Around twenty autophagy-related or ATG proteins compose the highly conserved core machinery that controls autophagosome biogenesis in all eukaryotes.
The proteins encoded by these genes have been divided into six functional modules: the ULK kinase complex, the autophagy-specific phosphatidylinositol 3-kinase complex, the ATG9A-positive vesicles, the ATG2-WIPI complex, the ATG12 and the LC3 conjugation systems.
However, there are forms of macro-autophagy that do not require the function of all ATG modules, and those have been defined as unconventional. Conversely, a single or group of ATG proteins operate in other cellular processes.
Autophagosome biogenesis and consumption can be divided into five discrete and consecutive steps, each one involving specific sets of core ATG proteins, but also other factors: Initiation, Expansion, Maturation, Tethering & Fusion and Breakdown & Recycling.
Although both bulk and selective types of autophagy involve autophagosomes, their induction mechanism differs.
The Initiation step of bulk autophagy is characterized by the activation of the ULK kinase complex. Multiple signaling cascades regulating autophagy directly act on this complex.
The supramolecular assembly and activation of multiple ULK kinase complexes generate a scaffold for the formation of the phagophore, the precursor structure of the autophagosome, which is initiated by heterotypic fusion of ATG9A-positive vesicles with vesicles that are probably derived from multiple membrane sources, including recycling endosomes and ER.
This event takes place adjacently to the ER. The autophagy-specific phosphatidylinositol 3-kinase complex also participates in phagophore nucleation by catalyzing the synthesis of phosphatidylinositol-3-phosphate on nascent autophagosomal membranes. This is important for the recruitment of the components of the ATG machinery involved in the phagophore expansion.
The Expansion step relies on the association to the phagophore of the ATG machinery components such as the ATG2-WIPI complex, and the ATG12 and LC3 conjugation systems that eventually conjugate the members of the ubiquitin-like LC3 protein family, to the phosphatidylethanolamine present in the membrane of the growing phagophore.
ATG2 proteins are key in supplying part of the lipids required for the phagophore expansion through their direct transfer from the endoplasmic reticulum, in conjunction with the ATG9A lipid scramblase activity.
The ATG12 and LC3 conjugation systems marginally participate in this process and are more crucial for the closure of the phagophore into an autophagosome and its subsequent transport and fusion. Phagophore closure appears to also require the ESCRT-III complex.
The Maturation step is characterized by the dissociation of autophagosomes from the endoplasmic reticulum and the release in the cytoplasm for re-utilization, of the components intervening during autophagosome biogenesis.
Two important aspects of this step are the hydrolysis of phosphatidylinositol-3-phosphate into phosphatidylinositol by phosphatases from the myotubularin family, and the deconjugation of the LC3 proteins from their lipid anchor by ATG4 cysteine proteases.
For the Tethering & Fusion steps, motor proteins and microtubule tracks ensure the encounter of autophagosomes first with late endosomes, to form amphisomes, and then lysosomes, or directly with lysosomes, to generate autolysosomes.
Tethering of autophagosomes with these compartments of the endolysosomal system is coordinated by the small GTPase RAB7, its guanosine exchange factor, its downstream effector HOPS complex and additional tethering factors. The subsequent fusion is mediated by SNARE proteins.
The Breakdown & recycling steps consists in the lysis of the inner autophagosomal membrane and the turnover of the cargo by lysosomal hydrolytic enzymes. This event generates the basic metabolites that are subsequently transported into the cytoplasm, for their use as either building blocks for the synthesis of new macromolecules or energy sources.
Selective autophagy is involved in the regulated turnover of portions of organelles including mitochondria, peroxisomes, and the endoplasmic reticulum, but also large protein/RNA complexes such as protein aggregates, and pathogens.