A method for the isolation and characterization of autophagic bodies from yeast provides a key tool to investigate cargos of autophagy

Kawamata, Tomoko; Makino, Shiho; Kagohashi, Yoko; Sasaki, Michiko; Ohsumi, Yoshinori

doi:10.1016/j.jbc.2022.102641

Cited by 8 publications

(10 citation statements)

References 35 publications

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“…6 A ). To this end, we employed our recently established method for the isolation of ABs that retain autophagosome cargo ( Kawamata et al, 2022 ), which allowed us to use purified ABs as a native substrate of Atg15. We isolated ABs from atg15 Δ cells expressing GST-GFP, which localizes to the cytosol and is delivered to the vacuole via autophagy.…”

Section: Resultsmentioning

confidence: 99%

“…Vacuoles were obtained from cells as previously described with some modifications ( Kawamata et al, 2022 ; Ohsumi and Anraku, 1981 ; Takeshige et al, 1992 ). Cells grown in 10 L YPD to OD 600 = 1.0–2.0 were treated with 0.2 µM rapamycin for 1 h. Cells were then collected and washed once with water.…”

Section: Methodsmentioning

confidence: 99%

“…To separate liposomes from NBD-PE, the liposome solution was centrifuged at 125,000 g for 1 h at 4°C and the supernatant was removed carefully. Then, buffer E was added and liposomes were analyzed using a Zetasizer Nano S (Malvern Instruments) as described previously ( Kawamata et al, 2022 ).…”

Section: Methodsmentioning

confidence: 99%

“…ABs were isolated as described previously ( Kawamata et al, 2022 ). In brief, atg15 ∆ cells (SMY345) were grown in 2.5 L YPD cultures to a density of OD 600 = 1.0 and then treated with 0.2 µM rapamycin for 6 h. Vacuoles were isolated as described above and then passed through a 0.8-μm PC membrane filter (ATTP01300; Millipore) to liberate ABs.…”

Section: Methodsmentioning

confidence: 99%

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The mechanism of Atg15-mediated membrane disruption in autophagy

Kagohashi,

Sasaki,

May

et al. 2023

Journal of Cell Biology

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The mechanism of Atg15-mediated membrane disruption in autophagy

Kagohashi,

Sasaki,

May

et al. 2023

Journal of Cell Biology

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“…Such ultrastructures are unusual in sugar-starved axes (−S) as the process of autophagic body breakdown is rapid and vacuoles rather quickly become clear [ 16 ]. In addition, visualization of the autophagic body degradation in yeast cells was not easy to achieve, even with the use of live-cell microscopy [ 21 ]. This suggested that asparagine in plants has an inhibitory effect on this late step of autophagy.…”

Section: Introductionmentioning

confidence: 99%

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

Wleklik,

Stefaniak,

Nuc

et al. 2023

IJMS

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Autophagy is a fundamental process for plants that plays a crucial role in maintaining cellular homeostasis and promoting survival in response to various environmental stresses. One of the lesser-known stages of plant autophagy is the degradation of autophagic bodies in vacuoles. To this day, no plant vacuolar enzyme has been confirmed to be involved in this process. On the other hand, several enzymes have been described in yeast (Saccharomyces cerevisiae), including Atg15, that possess lipolytic activity. In this preliminary study, which was conducted on isolated embryonic axes of the white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet), the potential involvement of plant vacuolar lipases in the degradation of autophagic bodies was investigated. We identified in transcriptomes (using next-generation sequencing (NGS)) of white and Andean lupin embryonic axes 38 lipases with predicted vacuolar localization, and for three of them, similarities in amino acid sequences with yeast Atg15 were found. A comparative transcriptome analysis of lupin isolated embryonic axes cultured in vitro under different sucrose and asparagine nutrition, evaluating the relations in the levels of the transcripts of lipase genes, was also carried out. A clear decrease in lipase gene transcript levels caused by asparagine, a key amino acid in lupin seed metabolism which retards the degradation of autophagic bodies during sugar-starvation-induced autophagy in lupin embryonic axes, was detected. Although the question of whether lipases are involved in the degradation of autophagic bodies during plant autophagy is still open, our findings strongly support such a hypothesis.

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Receptor-mediated cargo hitchhiking on bulk autophagy

Takeda,

Isoda,

Hosokawa

et al. 2024

EMBO J

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While the molecular mechanism of autophagy is well studied, the cargoes delivered by autophagy remain incompletely characterized. To examine the selectivity of autophagy cargo, we conducted proteomics on isolated yeast autophagic bodies, which are intermediate structures in the autophagy process. We identify a protein, Hab1, that is highly preferentially delivered to vacuoles. The N-terminal 42 amino acid region of Hab1 contains an amphipathic helix and an Atg8-family interacting motif, both of which are necessary and sufficient for the preferential delivery of Hab1 by autophagy. We find that fusion of this region with a cytosolic protein results in preferential delivery of this protein to the vacuole. Furthermore, attachment of this region to an organelle allows for autophagic delivery in a manner independent of canonical autophagy receptor or scaffold proteins. We propose a novel mode of selective autophagy in which a receptor, in this case Hab1, binds directly to forming isolation membranes during bulk autophagy.

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A method for the isolation and characterization of autophagic bodies from yeast provides a key tool to investigate cargos of autophagy

Cited by 8 publications

References 35 publications

The mechanism of Atg15-mediated membrane disruption in autophagy

The mechanism of Atg15-mediated membrane disruption in autophagy

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

Receptor-mediated cargo hitchhiking on bulk autophagy

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