LC3 conjugation system in mammalian autophagy

Abstract

Autophagy is the bulk degradation of proteins and organelles, a process essential for cellular maintenance, cell viability, differentiation and development in mammals. Autophagy has significant associations with neurodegenerative diseases, cardiomyopathies, cancer, programmed cell death, and bacterial and viral infections. During autophagy, a cup-shaped structure, the preautophagosome, engulfs cytosolic components, including organelles, and closes, forming an autophagosome, which subsequently fuses with a lysosome, leading to the proteolytic degradation of internal components of the autophagosome by lysosomal lytic enzymes. During the formation of mammalian autophagosomes, two ubiquitylation-like modifications are required, Atg12-conjugation and LC3-modification. LC3 is an autophagosomal ortholog of yeast Atg8. A lipidated form of LC3, LC3-II, has been shown to be an autophagosomal marker in mammals, and has been used to study autophagy in neurodegenerative and neuromuscular diseases, tumorigenesis, and bacterial and viral infections. The other Atg8 homologues, GABARAP and GATE-16, are also modified by the same mechanism. In non-starved rats, the tissue distribution of LC3-II differs from those of the lipidated forms of GABARAP and GATE-16, GABARAP-II and GATE-16-II, suggesting that there is a functional divergence among these three modified proteins. Delipidation of LC3-II and GABARAP-II is mediated by hAtg4B. We review the molecular mechanism of LC3-modification, the crosstalk between LC3-modification and mammalian Atg12-conjugation, and the cycle of LC3-lipidation and delipidation mediated by hAtg4B, as well as recent findings concerning the other two Atg8 homologues, GABARAP and GATE-16. We also highlight recent findings regarding the pathobiology of LC3-modification, including its role in microbial infection, cancer and neuromuscular diseases.

Fig. 1

Schematic representation of Crosstalk between two ubiquitylation-like modifications essential for mammalian autophagy. Atg12 is a first modifier essential for the formation of autophagosomal precursors and isolation membranes. Atg12 is activated by Atg7, transferred to Atg10, and conjugated to Atg5, which subsequently forms a complex with Atg16L. LC3 is a second modifier essential for the later formation of autophagosomes. After translation, most proLC3 is processed to LC3-I, which is localized in the cytosol. LC3-I is activated by Atg7, transferred to Atg3, and conjugated to phospholipid. Overexpression of Atg10 facilitates the modification of LC3-I to LC3-II (Facilitation, Red color). Overexpression of Atg3 facilitates the conjugation of Atg12 to Atg5 (Facilitation, Blue color). Excess amount of the Atg12-Atg5 conjugate inhibits LC3-modification (Negative Feedback).

Fig. 2

Tissue distribution of the three Atg8 homologues as analyzed by immunoblotting. Wistar male rats were maintained for at least two weeks in an environmentally controlled room (lights on 06:00 to 20:00) and fed a standard laboratory chow and tap water ad libitum. Under these conditions, the feeding-starvation cycle of the animals occurs earnestly dependent on circadian rhythm. Namely, immediately after lights are gone off, they begin to eat chow for ∼3 h and then stop eating. They have been fasting during daytime. Hence, at 11:00 it is assumed that the animals are starved for 12 h. Brain, kidney, liver, spleen, heart, and hind leg muscle were freshly isolated from a Wistar male rat (300 g body weight) at 11:00. Each tissue, suspended in a buffer containing 5 mM Tes-NaOH (pH 7.5), 0.3 M sucrose, 5 μg/ml leupeptin, and 5 μg/ml pepstatin, was homogenized with a glass/Teflon homogenizer. The homogenate was centrifuged at 700 × g for 5 min, and the resulting supernatant was centrifuged at 100,000 × g for one hour. Forty μg protein of the pellet (P) and supernatant (S) was separated on 12.5% SDS-polyacrylamide gels and subjected to immunoblotting analysis for LC3, GABARAP, and GATE-16.

Fig. 3

Lipidation and delipidation cycle of mammalian Atg8 homologues mediated by mammalian Atg4 homologues. The carboxyl termini of the pro-forms of LC3, GABARAP, and GATE-16 are cleaved by hAtg4B (or hAtg4A for proGATE-16) to expose Gly, and the resultant LC3-I, GABARAP-I, and GATE-16-I are modified to LC3-II, GABARAP-II, and GATE-16-II, respectively. While intra-autophagosomal LC3-II is degraded by lysosomal proteolytic enzyme after fusion of autophagosomes to lysosomes, LC3-II on the cytosolic surface of autophagosomes is delipidated by hAtg4B. Membrane-localized GABARAP-II on the cytosolic surface is also delipidated by hAtg4B. Little endogenous GATE-16-II (i.e. lipidated form) is observed in rat and mouse tissues and cell lines. The physiological function of GATE-16-II is not yet known, while GATE-16 itself is an essential component of intra-Golgi transport and post-mitotic Golgi reassembly. Apg8L has been reported to interact with hAtg4B in mammals, but cleavage of the carboxyl termini and ubiquitylation-like modification have not been observed.