The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation

Agata Makar; Alina Boraman; Peter Mosen; Joanne Simpson; Jair Marques; Tim Michelberger; Stuart Aitken; Ann P Wheeler; Dominic Winter; Alexander von Kriegsheim; Noor Gammoh

doi:10.1038/s41467-024-52049-3

The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation

Agata Makar, Alina Boraman, Peter Mosen, Joanne Simpson, Jair Marques , Tim Michelberger, Stuart Aitken, Ann P Wheeler, Dominic Winter, Alexander von Kriegsheim, Noor Gammoh^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic
components. The final degradation step is essential for clearing autophagic cargo and recycling
macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved
transmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.

Original language	English
Journal	Nature Communications
Early online date	5 Sept 2024
DOIs	https://doi.org/10.1038/s41467-024-52049-3
Publication status	E-pub ahead of print - 5 Sept 2024

Keywords / Materials (for Non-textual outputs)

Autophagy
autophagosome
lisosome
lipase
V-ATPase

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10.1038/s41467-024-52049-3

The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation_finalFinal published version, 4.19 MB

Advanced Imaging Resource
Wheeler, A. (Manager), Murphy, L. (Other), Lee, M. (Other), Caldwell, H. (Other), Pearson, M. (Other) & Iremonger, J. (Other)
Deanery of Molecular, Genetic and Population Health Sciences
Facility/equipment: Facility
Institute for Genetics and Cancer Mass Spectrometry Facility
Von Kriegsheim, A. (Manager)
Deanery of Molecular, Genetic and Population Health Sciences
Facility/equipment: Facility

Cite this

@article{5c265a086f984be7a9df5b19d56ec02f,

title = "The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation",

abstract = "Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosoliccomponents. The final degradation step is essential for clearing autophagic cargo and recyclingmacromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conservedtransmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.",

keywords = "Autophagy, autophagosome, lisosome, lipase, V-ATPase",

author = "Agata Makar and Alina Boraman and Peter Mosen and Joanne Simpson and Jair Marques and Tim Michelberger and Stuart Aitken and Wheeler, {Ann P} and Dominic Winter and {von Kriegsheim}, Alexander and Noor Gammoh",

year = "2024",

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language = "English",

journal = "Nature Communications",

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T1 - The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation

AU - Makar, Agata

AU - Boraman, Alina

AU - Mosen, Peter

AU - Simpson, Joanne

AU - Marques , Jair

AU - Michelberger, Tim

AU - Aitken, Stuart

AU - Wheeler, Ann P

AU - Winter, Dominic

AU - von Kriegsheim, Alexander

AU - Gammoh, Noor

PY - 2024/9/5

Y1 - 2024/9/5

N2 - Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosoliccomponents. The final degradation step is essential for clearing autophagic cargo and recyclingmacromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conservedtransmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.

AB - Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosoliccomponents. The final degradation step is essential for clearing autophagic cargo and recyclingmacromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conservedtransmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.

KW - Autophagy

KW - autophagosome

KW - lisosome

KW - lipase

KW - V-ATPase

U2 - 10.1038/s41467-024-52049-3

DO - 10.1038/s41467-024-52049-3

M3 - Article

SN - 2041-1723

JO - Nature Communications

JF - Nature Communications

ER -

The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation

Abstract

Keywords / Materials (for Non-textual outputs)

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Institute for Genetics and Cancer Mass Spectrometry Facility

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