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Molecular mechanisms of autophagy

Autophagy is a complex process that is tightly regulated at the molecular level. Here, we review the key stages and processes involved in this pathway.

Autophagy is a tightly regulated pathway with an important housekeeping role, allowing cells to eliminate damaged or harmful components through catabolism and to recycle them to maintain nutrient and energy homeostasis. Autophagy is also a major protective mechanism which allows cell survival in response to multiple stress conditions such as nutrient or growth factor deprivation, hypoxia, reactive oxygen species (ROS), DNA damage or 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 phagophore formation

The molecular mechanism of autophagy involves several conserved Atg (autophagy-related) proteins. Various stimuli, such as nutrient starvation, lead to the formation of the phagophore, a step that involves two protein complexes:

  • A complex that contains the class III PI3K/Vps34, Atg6/Beclin1, Atg14 and Vps15/p150.73.
  • A complex that includes the serine/threonine kinase Atg1/ULK1, an essential positive regulator of autophagosome formation.

Phagophore elongation and autophagosome formation

The elongation of the phagophore results in the formation of the characteristic double-membrane autophagosome. This step requires two ubiquitin-like conjugation pathways, both catalyzed by Atg7.

  • The first ubiquitin-like system leads to the conjugation of Atg5-Atg12, which then form a multimeric complex with Atg16L. The Atg5-Atg12-Atg16L complex associates with the outer membrane of the extending phagophore (Glick et al., 2010; Kaur & Debnath, 2015).
  • The second system results in the processing of LC3, encoded by the mammalian homologue of the yeast Atg8. Upon autophagy induction LC3B is proteolytically cleaved by Atg4 to generate LC3B-I. LC3B-I is activated by Atg7 and then conjugated to phosphatidylethanolamine (PE) in the membrane to generate processed LC3B-II.

Processed LC3B-II is recruited onto the growing phagophore and its integration is dependent on Atg5-Atg12. Unlike Atg5-Atg12-Atg16L, LC3B-II is found on both the inner and outer surfaces of the autophagosome, where it is required for the expansion and completion of the autophagic membrane. After closure of the autophagosomal membrane, the Atg16-Atg5-Atg12 complex dissociates from the vesicle, whereas a portion of LC3B-II remains covalently bound to the membrane. Therefore, LC3B-II can be used as a marker to monitor the level of autophagy in cells.

It has been postulated that various organelles, including mitochondria, the Golgi complex and the endoplasmic reticulum (ER), can be the origin of the autophagosomal membrane (He & Klionsky, 2010). Recent studies demonstrate that self-multimerization of Atg9 may facilitate membrane tethering and/or fusion (He et al., 2008).


Fusion, degradation and recycling

When the autophagosome formation is completed, LC3B-II attached to the outer membrane is cleaved from PE by Atg4 and released back to the cytosol. The fusion between the autophagosome and the lysosome in thought to require the lysosomal membrane protein LAMP-2 and the small GTPase Rab7. In yeast, the machinery consists of the Ypt7 (Rab7 homolog), the NSF homolog Sec18, the SNARE proteins Vam3, Vam7, Vti1, and the class C Vps/HOPS complex proteins.

After fusion, a series of acid hydrolases are involved in the degradation of the sequestered cytoplasmic cargo. The small molecules resulting from the degradation, particularly amino acids, are transported back to the cytosol for protein synthesis and maintenance of cellular functions under starvation conditions. The identification of Atg22 together with other vacuolar permeases (such as Avt3 and Avt4) as vacuolar amino acid effluxers during yeast autophagy has helped in the understanding of the mechanisms of nutrient recycling. These permeases represent the last step in the degradation and recycling process (He & Klionsky, 2010).

References

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