Autophagosome Proteins

 Creative BioMart Autophagosome Proteins Product List
 Autophagosome Proteins Background

Mechanisms of autophagosome biogenesis

Autophagosome biogenesis requires a cascade of different autophagy genes (Atgs) to initiate the formation, elongation and closure of the double-membrane autophagosome around its cargo. In non-selective autophagy, the protein complexes and the recruitment order of these different autophagy genes have been studied in great detail. However, it is important to note that these steps may be different during selective autophagy, such as in the degradation of ubiquitinated Salmonella or mitochondria in which the omegasome/isolation membrane must selectively form around and engulf the ubiquitinated cargo, and thus the recruitment order of autophagy genes to selective autophagic cargo still remains to be examined.

Autophagosome biogenesis begins with the initiation, nucleation, and expansion of the autophagosome. During non-selective autophagy in mammalian cells, autophagosome biogenesis is initiated upon activation of the kinase ULK1 (Atg1 in yeast) and an ULK1/Atg13/FIP200 complex is recruited to the isolation membrane, the autophagosome precursor, and serves as a scaffold for the recruitment of downstream Atg proteins. Under nutrient rich conditions, ULK1 is negatively regulated by binding to the mammalian target of rapamycin complex 1 (mTORC1) complex, and is positively regulated by AMP-activated protein kinase (AMPK). However, upon amino acid starvation, mTORC1 is inactivated and dissociates from the ULK1 complex, allowing ULK1 to increase its kinase activity and facilitate autophagy initiation.

In the subsequent nucleation step, the ULK1/Atg13/FIP200 complex recruits a class III PI3K (phosphatidylinositol 3-kinase) complex consisting of Beclin1, Atg14L, p150 and VPS34, which is responsible for generating the lipid PI3P (phosphatidylinositol 3-phosphate) by phosphorylating PI (phosphatidylinositol). ULK1 phosphorylates Beclin-1 on Ser14 to help activate this class III PI3K complex during autophagy.

PI3P generation is crucial for autophagosome formation and is localized to the membranes of autophagosomes. PI3P is able to recruit PI3P-binding proteins WIPI1 and WIPI2 (WD repeat domain phosphoinositide-interacting proteins) and DFCP1 (double FYVE-domain containing protein 1) to the forming isolation membrane. While it is still unclear what role DFCP1 plays, WIPIs are considered to be essential PI3P effectors during autophagy which bridge PI3P generation with LC3 recruitment, as WIPI1 is essential for LC3 lipidation and WIPI2 can bind Atg16L.

Following the nucleation step, expansion of the autophagosome occurs via two ubiquitinlike conjugation steps. In the first step, Atg7 and Atg 10 act as E1-like and E2-like enzymes respectively, conjugating Atg12 with Atg 5. The Atg12/Atg5 complex then binds Atg16L via a direct interaction between Atg5 and Atg 16L. The Atg12/Atg5/Atg16L complex is then recruited to the isolation membrane, potentially via Atg16L’s interactions with FIP200 or WIPI2, but leaves the membrane upon completion of autophagosome formation.

In the second step, Atg7 and Atg 3 act as E1-like and E2-like enzymes respectively, while Atg12/Atg5/Atg16L acts as an E3-like enzyme, to conjugate LC3 to the lipid PE (phosphatidylethanolamine) to form lipidated LC3 (also called LC3-II). This conjugation step occurs on the isolation membrane, generating lipidated LC3 on both the inner and outer surface of the autophagosome membrane during autophagy. As lipidated LC3 stably associates with the autophagosome membrane, it has been used as a well-established marker for autophagosomes, and lipidated LC3 on the inner membrane is later degraded along with the internalized autophagic cargo. LC3 is thought to be crucial for isolation membrane expansion and closure of the autophagosome. The LC3 family consists of LC3A, LC3B, LC3C, and its homologs GABARAP, GABARAPL1 and GABARAPL2. Of these, LC3B is the most prevalent and has been used most widely as an autophagosome marker.

The only transmembrane protein involved in autophagy is Atg9 which partially localizes to the trans-Golgi network and endosomes, and transiently colocalizes with LC3 in starvation-induced autophagy. As Atg9 also localizes to small vesicles, these Atg9-positive vesicles have been proposed to supply the initial membrane source which later becomes the isolation membrane.

Several other membrane sources have also been proposed to contribute to the formation of the autophagosome membrane. These include the ER (endoplasmic reticulum) which forms DFCP1-positive omega-shaped cradles termed omegasomes as isolation membrane precursors of the autophagosome. Mitochondria have also been suggested to contribute to the autophagosome membrane, supporting evidence showing that autophagosomes form at ER-mitochondria contact sites. In addition, other compartments such as ER exit sites where COPII vesicle formation occurs, the ER-Golgi intermediate compartment, the plasma membrane and recycling endosomes have also been found to contribute to autophagosome biogenesis. Thus, the exact autophagosome membrane source still remains to be understood, as well as whether this membrane source might vary depending on the cell type, the method in which autophagy is induced, and the autophagic cargo being degraded. In neurons, autophagosomes forming at the axon terminal during basal constitutive autophagy were found to incorporate an ER membrane source rather than plasma membrane or mitochondria.

Autophagosomes subsequently fuse with multiple lysosomes to acquire the necessary degradative machinery including hydrolytic enzymes such as proteases, lipases and nucleases, as well as the vacuolar ATPase (vATPase) proton pump which is needed to acidify the autolysosomal lumen for activation of the hydrolytic enzymes. Following degradation of the internal autophagic contents, mTOR signaling is reactivated during prolonged starvation and is responsible for attenuating autophagy and generating protolysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes. Thus, both the autophagic cargo and the lysosomal machinery are recycled by the cell to maintain cellular homeostasis in autophagy.