This article reviews the main processes and stages involved in autophagy pathway – a complex process that is closely regulated at the molecular level and has a crucial housekeeping role.
In autophagy, cells remove harmful or damaged components via catabolism and recycle them to sustain energy and nutrient homeostasis. In addition, autophagy is known to be an important protective mechanism which enables cell survival in response to different stress conditions such as:
- growth factor or nutrient deprivation
- DNA damage
- reactive oxygen species (ROS)
- intracellular pathogens (Levine & Kroemer, 2008)
Figure 1. Overview of autophagy process. An expanding membrane structure (phagophore) enwraps portions of the cytoplasm, followed by the formation of a double-membrane sequestering vesicle (autophagosome). The autophagosome fuses with the lysosome and releases its inner compartment into the lysosomal lumen. The inner membrane part of the autophagosome is degraded together with the enclosed cargo. The resulting macromolecules are released into the cytosol for recycling through lysosomal membrane permeases (Mizushima, 2007).
Induction and formation of phagophore
A number of conserved Atg (autophagy-related) proteins are involved in autophagy’s molecular mechanism. Different stimuli, including nutrient starvation, promote the formation of phagophore – a step in which two protein complexes are involved:
- A complex that includes the class III PI3K/Vps34, Atg14, Atg6/Beclin1, and Vps15/p150.73
- A complex that contains the serine/threonine kinase Atg1/ULK1 – a key positive regulator of autophagosome formation
Elongation of phagophore and formation of autophagosome
Phagophore elongation promotes the formation of the typical double-membrane autophagosome. Two Atg7-catalyzed and ubiquitin-like conjugation pathways are required in this step.
- Atg5-Atg12 conjugation is caused by the first ubiquitin-like system that subsequently forms a multimeric complex with Atg16L. Next, the Atg5-Atg12-Atg16L complex binds to the external membrane of the extending phagophore (Glick et al., 2010; Kaur & Debnath, 2015).
- The second ubiquitin-like system results in LC3 processing, which is encoded by the mammalian homologue of the yeast Atg8.
- When LC3B is induced by autophagy, Atg4 proteolytically cleaves it to create LC3B-I. Atg7 activates this LC3B, which is later conjugated to phosphatidylethanolamine (PE) in the membrane to produce processed LC3B-II.
The processed LC3B-II is recruited onto the developing phagophore and its integration is dependent on Atg5-Atg12. LC3B-II, unlike Atg5-Atg12-Atg16L, is present on the outer and inner surfaces of the autophagosome, where it is needed to expand and complete the autophagic membrane.
Once the autophagosomal membrane is closed, the Atg16-Atg5-Atg12 complex detaches the vesicle, while a part of LC3B-II remains covalently bound to the autophagosomal membrane. As a result, LC3B-II can be utilized as a marker to track the extent of autophagy in cells.
In 2010, He & Klionsky hypothesized that many organelles, such as the endoplasmic reticulum (ER), the Golgi complex, and mitochondria, could be the origin of the autophagosomal membrane. New studies have shown that membrane tethering and/or fusion may result from self-multimerization of Atg9 (He et al., 2008).
Fusion, degradation, and recycling
After the autophagosome formation is complete, the LC3B-II bound to the outer autophagosomal membrane is cleaved from PE by Atg4 and transported back to the cytosol. The small GTPase Rab7 and the lysosomal membrane protein LAMP-2 may be required for the fusion between the lysosome and the autophagosome. The machinery in yeast includes:
- the NSF homolog Sec18
- the Ypt7 (Rab7 homolog)
- the SNARE proteins Vti1, Vam3, Vam7
- the class C Vps/HOPS complex proteins
Following fusion, the degradation of the sequestered cytoplasmic cargo commences, recruiting a chain of acid hydrolases. The degradation produces tiny molecules which are sent back to the cytosol (most of all, amino acids) for maintenance of cellular functions and protein synthesis under starvation conditions.
During yeast autophagy, the detection of Atg22 along with other vacuolar permeases (such as Avt4 and Avt3) as vacuolar amino acid effluxers has provided a deeper insight into the mechanisms of nutrient recycling. The final stage in the degradation and recycling process is represented by these vacuolar permeases (He & Klionsky, 2010).
- Glick D, Barth S, Macleod KF (2010). Autophagy: cellular and molecular mechanisms. J. Pathol. 221(1), 3-12
- He C, Baba M, Cao Y, Klionsky DJ (2008). Self-interaction is critical for Atg9 transport and function at the phagophore assembly site duting autophagy. Mol. Biol. Cell., 19(12), 5506-5516
- He C & Klionsky DJ (2010). Analyzing autophagy in zebrafish. Autophagy, 6(5), 642-644
- Kaur J & Debnath J (2015). Autophagy at the crossroads of catabolism and anabolism. Nat. Rev. Mol. Cell Biol., 16(8), 461-472
- Levine B & Kroemer G (2008). Autophagy in the pathogenesis of disease. Cell, 11(132), 27-42
- Mizushima N (2007). Autophagy: process and function. Genes Dev., 21(22), 2861-2873.
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