The activation mechanism of Atg15, a vacuolar phospholipase required for the disintegration of organelle membranes

ABSTRACT

The disintegration of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies in vacuoles is essential to the Cvt pathway and macroautophagy in yeast. Atg15 is a vacuolar lipase required for the degradation of both Cvt and autophagic bodies. However, the molecular mechanism of their degradation by Atg15 remains poorly understood. In a recent study, we showed that recombinant Chaetomium thermophilum Atg15 (CtAtg15) possesses phospholipase activity, and that this activity is significantly elevated by proteolytic cleavage at a site away from the active center. The proteolytic cleavage of CtAtg15 causes a conformational change around the active center, resulting in the active open state. Interestingly, activated CtAtg15 can degrade not only Cvt and autophagic bodies but also organelle membranes. On the basis of these results, we propose an activation mechanism by which Atg15, as an “organellase,” functions only in vacuoles.

In Saccharomyces cerevisiae, as part of the cytoplasm-to-vacuole targeting (Cvt) pathway and the process of macroautophagy (hereinafter referred to simply as “autophagy”), the outer membranes of Cvt vesicles or autophagosomes fuse with vacuolar membranes, and then Cvt bodies or autophagic bodies are released into the vacuole lumen. Atg15 is the only vacuolar lipase in S. cerevisiae, and it is essential for the disintegration of Cvt and autophagic bodies. However, it remains unclear if these bodies are directly disrupted by Atg15. In a recent study [1], we characterized the Atg15 lipase activity involved in membrane disintegration by performing biochemical analyses using recombinant purified proteins. We examined the activity of the recombinant Atg15 proteins using nitrobenzoxadiazole (NBD)-labeled phospholipid as a substrate, and found that the recombinant Chaetomium thermophilum Atg15 (CtAtg15) lipase domain catalyzes the hydrolysis reaction that produces NBD-lysophospholipids and NBD-free fatty acids. This result suggests that CtAtg15 possesses phospholipase B activity, which generates lysophospholipids and free fatty acids by cleaving the acyl ester bond at the sn-1 and sn-2 positions. We also showed that CtAtg15 can degrade NBD-phosphatidylethanolamine (NBD-PE), NBD-phosphatidylcholine, and NBD-phosphatidylserine, and that CtAtg15 can degrade NBD-PE in liposomes. Moreover, CtAtg15 exhibits significant activity under acidic conditions but minimal activity under neutral conditions. These results suggest that Atg15 is active only inside vacuoles and can recognize and degrade membrane phospholipids.

The disintegration of Cvt bodies and autophagic bodies in vacuoles requires not only Atg15 but also Pep4 and Prb1, two vacuolar proteases of S. cerevisiae. Because Pep4 and Prb1 are unlikely to have lipase activity, we speculated that these vacuolar proteases are involved in the activation of the lipase activity of Atg15, and that activated Atg15 directly disintegrates Cvt and autophagic bodies. Indeed, the phospholipase activity of CtAtg15 is significantly elevated by treatment with proteinase K, a broad-spectrum serine protease. Interestingly, although S. cerevisiae Atg15 (ScAtg15) shows minimal phospholipase activity without proteinase K treatment, this activity, like that of CtAg15, is significantly increased by treatment with proteinase K. We also showed that cell lysates prepared from pep4Δ atg15Δ S. cerevisiae cells that overexpressed ScAtg15 exhibit phospholipase activity whose magnitude depends on treatment with proteinase K. These results support our hypothesis that Atg15 is activated upon cleavage by vacuolar proteases (Figure 1). SDS-PAGE analysis indicated that the CtAtg15 lipase domain is cleaved into two relatively stable peptides by treatment with proteinase K. Furthermore, gel filtration analysis and circular dichroism spectra showed that treatment with proteinase K does not affect overall folding of the CtAtg15 lipase domain, suggesting that Atg15 is activated by nicking the lipase domain, after which each fragment remains part of the active enzyme.

Figure 1.

A model for the activation of Atg15 in a vacuole. Atg15 is transported to the vacuole in a precursor form, and then Atg15 is activated through limited proteolysis by vacuolar proteases. This limited proteolysis causes conformational changes that expose the active site. Activated Atg15 disintegrates autophagic bodies, and then proteins and organelle membranes inside autophagic bodies are degraded by vacuolar proteases and Atg15, respectively.

Edman sequencing together with SDS-PAGE analysis indicated that the CtAtg15 lipase domain is cleaved between S159 and V160 by treatment with proteinase K. S159 and V160 are located on opposite sides of the active center in the AlphaFold2-predicted CtAtg15 structure. To investigate whether cleavage between S159 and V160 is involved in the activation of CtAtg15, the human rhinovirus (HRV) 3C protease recognition sequence was inserted between S159 and V160 of the CtAtg15 lipase domain. The lipase activity of CtAtg15 containing this sequence is elevated by cleavage between S159 and V160 by HRV 3C protease treatment. These results suggest that CtAtg15 cleavage between S159 and V160 increases phospholipase activity even though these two residues are not located near the active center.

In the AlphaFold2-predicted structure of CtAtg15, the active center S330 is buried inside the molecule and is covered by several loop regions, suggesting that a conformational change is required for CtAtg15 activity. To verify whether intramolecular cleavage between S159 and V160 of CtAtg15 causes this conformational change, we next performed molecular dynamics simulations of cleaved and non-cleaved CtAtg15. These simulations show a large conformational change in the loop region covering the active center, which exposes the active center in the structure of the cleaved CtAtg15. By contrast, a conformational change exposing the active center is not observed in the non-cleaved CtAtg15. Together, these results suggest that the conformational change upon cleavage between S159 and V160 contributes to the activation of CtAtg15.

We next investigated whether Atg15 can disintegrate organelle membranes. Vacuoles containing Cvt or autophagic bodies were collected from pep4Δ atg15Δ S. cerevisiae cells and subjected to a proteinase K protection assay. Precursor aminopeptidase I (prApe1), which is a cargo protein of both the Cvt pathway and autophagy and is enclosed in Cvt or autophagic bodies, is degraded only when the vacuoles are simultaneously treated with recombinant CtAtg15 and proteinase K. This suggests that proteinase K can access prApe1 enclosed in Cvt or autophagic bodies that are located in vacuoles. Using isolated mitochondria, we further showed that CtAtg15 treated with proteinase K degrades the mitochondrial outer and inner membranes. These results indicated that activated CtAtg15 can disintegrate Cvt bodies, autophagic bodies, and organelle membranes enclosed within autophagic bodies.

We revealed that Atg15 possesses phospholipase activity and disintegrates organelle membranes. Because Atg15 degrades membranes, its activity must be limited to vacuoles. Our study showed that the activity of Atg15 is dependent on pH and that Atg15 is activated by limited proteolysis. These safety mechanisms probably restrict the activity of Atg15, which functions as an “organellase” to vacuoles. However, it remains unclear why Atg15 inside vacuoles does not degrade the vacuolar membrane. The proteinase K protection assay using isolated vacuoles showed that externally added CtAtg15 can disintegrate the vacuolar membranes. Thus, Atg15 potentially disintegrates membranes with a positive curvature. Future studies on the mechanism by which Atg15 recognizes membranes will provide further mechanistic insights into organelle membrane degradation during autophagy.

Funding Statement

This work was supported by a grant from the Naito Foundation (to K.S.), by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (22K06096 and 22H02569 to Y.W.; 20H05313, 21K19205, and 22H02569 to K.S.), and by CREST, the Japan Science and Technology Agency, Japan (JP201032912 to K.S.).

Disclosure statement

No potential conflict of interest was reported by the author(s).