The mechanism of Atg15-mediated membrane disruption in autophagy

Abstract

Autophagy is a lysosomal/vacuolar delivery system that degrades cytoplasmic material. During autophagy, autophagosomes deliver cellular components to the vacuole, resulting in the release of a cargo-containing autophagic body (AB) into the vacuole. AB membranes must be disrupted for degradation of cargo to occur. The lipase Atg15 and vacuolar proteases Pep4 and Prb1 are known to be necessary for this disruption and cargo degradation, but the mechanistic underpinnings remain unclear. In this study, we establish a system to detect lipase activity in the vacuole and show that Atg15 is the sole vacuolar phospholipase. Pep4 and Prb1 are required for the activation of Atg15 lipase function, which occurs following delivery of Atg15 to the vacuole by the MVB pathway. In vitro experiments reveal that Atg15 is a phospholipase B of broad substrate specificity that is likely implicated in the disruption of a range of membranes. Further, we use isolated ABs to demonstrate that Atg15 alone is able to disrupt AB membranes.

Identification and Potential Participation of Lipases in Autophagic Body Degradation in Embryonic Axes of Lupin (Lupinus spp.) Germinating Seeds

“…The amino acid sequence of the Atg15 (QHB07198.1_SC) yeast with 38 amino acid sequences of lupin lipases with predicted vacuolar localization was compared ( Table 1 ). As reported previously, the Atg15 yeast possesses lipolytic activity and is involved in autophagic body degradation [ 11 , 12 , 13 , 14 , 23 , 24 , 25 ]. Our analysis revealed that three lupin lipases (XP_019430451.1, XP_019425758.1, and XP_019420123.1) had high amino acid sequence similarity to the Atg15 yeast ( Figure 1 ).…”

“…The Atg15 yeast possesses lipolytic activity and is one of the enzymes whose involvement in the degradation of the autophagic body has been confirmed [ 11 , 12 , 13 , 14 , 23 , 24 , 25 ]. Saccharomyces cerevisiae yeast lacking Atg15 accumulated autophagic bodies in vacuoles, demonstrating its essential role in autophagic degradation [ 11 , 12 ].…”

“…However, the degradation of cargo located inside an autophagic body requires earlier disintegration of its phospholipid-built membrane [ 10 ]. Therefore, enzymes with lipase or phospholipase activity must initiate their breakdown [ 13 , 14 , 26 ]. It has been reported that the phospholipase A2 (LPLA-2) is required for the successful processing of autophagic degradation in Caenorhabditis elegans [ 27 ].…”

“…Two of the important uncertainties are the existence of a functional plant analog of the yeast vacuolar putative lipase Atg15/Aut5/Cvt17 and its involvement in the final steps of autophagy, in particular, the degradation of autophagic bodies [ 9 , 10 ]. Atg15 is the best-known and best-described protein involved in autophagic body breakdown in yeast [ 11 , 12 , 13 , 14 ]. Determining this unknown area of the involvement of particular proteins and their roles in the degradation of autophagic bodies in plants would be a major achievement on the road to a complete understanding of the course of autophagy in these organisms.…”

Atg45 is an autophagy receptor for glycogen, a non-preferred cargo of bulk autophagy in yeast

“…The outer membrane of the autophagosome fuses with the vacuole, which results in the release of the inner membrane-bound structure, the autophagic body, into the vacuolar lumen, allowing degradation by the vacuolar lipase Atg15 and a range of proteases. 1 , 2 , 3 , 4 , 5 , 6 Degradation products arising from autophagy, such as amino acids, are subsequently transported back to the cytoplasm for reuse. 7 , 8 , 9 …”