Autophagy is an evolutionarily conserved intracellular self-degradative process that plays a crucial housekeeping role in removing misfolded proteins. It facilitates the survival of cells in severe environmental conditions such as nutrient limitation, by recycling cellular building blocks including nucleic acids and amino acids. Cytoplasmic materials destined for degradation during autophagy are compartmentalized in double membrane–bound organelles called autophagosomes. These autophagosomes go on to fuse with the vacuole and then their inner membrane–bound structures are released into the vacuolar lumen to become autophagic bodies, which are finally degraded by vacuolar hydrolases. Atg15 is a lipase required for the disintegration of autophagic body membranes and it has a lipase domain at the C-terminus and a transmembrane domain at the N-terminus. However, the roles of these two domains in vivo are poorly understood.
In a new study published in the Journal Molecular Biology of the Cell, Dr. Eri Hirata, Kyo Shirai, Dr. Tatsuya Kawaoka, Kosuke Sato, Fumito Kodama, and Associate Professor Kuninori Suzuki from the University of Tokyo investigated the roles of the N- and C-terminal domains of Atg15 by analyzing them separately. Their findings showed that the degradation of autophagic bodies required targeting of the C-terminal lipase domain to the vacuole while the N-terminal domain alone had the ability to travel to the vacuole via the multivesicular body pathway.
The research team found that during autophagy, the expansion of autophagic membranes in Atg15 deficient cells was normal. No significant differences were observed in the length of autophagic membranes between Atg15 deficient cells and wild-type cells cultured under nitrogen starvation conditions for 6 hours. The delivery of Atg15 to the vacuole was identified to be via the multivesicular body pathway. In cells deficient of Vps4, a known requirement for the multivesicular body pathway, Atg15 tagged with green fluorescent protein (Atg15-GFP) accumulated in an abnormal multivesicular body compartment termed the class E compartment.
In all strains, full-length Atg15-GFP was discovered at almost the same level. Contrastingly, among the strains, the amount of GFP moiety derived from Atg15-GFP varied. Atg15-GFP had the ability to disintegrate autophagic bodies efficiently. It was detected that the functions of Atg15 in degrading autophagic bodies occurred inside the vacuole after it was transported into the organelle, not outside the vacuole during transport.
The authors discovered that the N-terminal transmembrane domain of Atg15 served as a signal sequence for the multivesicular body pathway. In addition, they also established that the N-terminal region of Atg15 was sufficient for the delivery of Atg15 to the vacuole via the multivesicular body pathway. The N-terminal residues 50–466 in Atg15 were also found to be essential for the disintegration of autophagic bodies inside the vacuole.
Moreover, the C-terminal domain of Atg15 was identified as a critical component in the activity of Atg15 and transport of the C-terminal domain to the vacuolar lumen was also deemed important. The C-terminal lipase domain proved to be sufficient for the enzymatic activity of Atg15. In mutant cells of residue W466, a highly conserved residue at the extreme C terminal region among Atg15 orthologs, defective autophagic bodies were observed.
In conclusion, the authors have demonstrated through their findings in elegant experimental studies that Atg15 has two distinct functional domains. The C-terminal lipase domain, found to be responsible for the enzymatic activity of Atg15 and the N-terminal transmembrane domain, identified as a targeting sequence to the vacuole via the multivesicular body pathway. They therefore suggest that in order to clarify the mechanisms through which autophagic bodies are disintegrated, future studies need to focus on developing in an vitro monitoring system for the activity of Atg15.
Hirata E, Shirai K, Kawaoka T, Sato K, Kodama F, Suzuki K. Atg15 in Saccharomyces cerevisiae consists of two functionally distinct domains. Mol Biol Cell. 2021;32(8):645-663.Go To Mol Biol Cell