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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 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

DISEASE(S): Alzheimer's Disease

SUBMITTER: Peter Robert Mosen

LAB HEAD: Dr Dominic

PROVIDER: PXD042079 | Pride | 2024-08-02

REPOSITORIES: Pride

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			Action	DRS
	20210913_192114_PM_6_114-ELF_001.sne	Other
	PM_6_48-ELF-s1-1.raw	Raw
	PM_6_48-ELF-s1-2.raw	Raw
	PM_6_48-ELF-s1-3.raw	Raw
	PM_6_48-ELF-s2-1.raw	Raw

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A lysosomal delivery mechanism required for autophagosome degradation

Project description: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. We identified a highly conserved transmembrane protein named RNAseK as a novel regulator of autophagosome degradation. Analyses of RNAseK knockout cells revealed that, while autophagosome maturation was intact, cargo degradation was severely disrupted. Importantly, lysosomal protease activity and acidification remained intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions showed reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 led to a defect in autophagosome clearance. In addition, the lysosomal fraction of RNAseK-depleted cells exhibited an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, our findings identified a lysosomal hydrolase delivery pathway required to mediate efficient autolysosome degradation.

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A lysosomal delivery mechanism required for autophagosome degradation

Project description:A lysosomal delivery mechanism required for autophagosome degradation

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Project description:A lysosomal delivery mechanism required for autophagosome degradation

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Aneuploidy-induced cellular stresses limit autophagic degradation.

Project description:An unbalanced karyotype, a condition known as aneuploidy, has a profound impact on cellular physiology and is a hallmark of cancer. Determining how aneuploidy affects cells is thus critical to understanding tumorigenesis. Here we show that aneuploidy interferes with the degradation of autophagosomes within lysosomes. Mis-folded proteins that accumulate in aneuploid cells due to aneuploidy-induced proteomic changes overwhelm the lysosome with cargo, leading to the observed lysosomal degradation defects. Importantly, aneuploid cells respond to lysosomal saturation. They activate a lysosomal stress pathway that specifically increases the expression of genes needed for autophagy-mediated protein degradation. Our results reveal lysosomal saturation as a universal feature of the aneuploid state that must be overcome during tumorigenesis. RPE-1 cells either untreated or treated with one of Reversine, Bafilomycin A1 or MG132, each condition was done in triplicate. D14-*_Control: untreated control D14-*_Rev: cells treated with 0.5uM Reversine for 24hrs and harvested 48hrs later D14-*_Baf: cells treated with 0.1uM BafA1 for 6hrs D14-*_Mg: cells treated with 1uM MG132 for 24 hrs

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Project description:Toll-like receptor 7 (TLR7) plays a key role in the recognition of RNA viruses and the subsequent induction of an innate immune response. TLR7 contains two ligand binding pockets that recognize distinct RNA degradation products: Pocket 1 recognizes guanosine nucleotides and pocket 2 coordinates short, pyrimidine-rich RNA fragments, while the engagement of both pockets is required to activate TLR7. How these ligands are generated within the endolysosomal compartment is currently unknown. Using a pDC cell line, we here show that the endonuclease RNase T2 and the 5' exonucleases PLD3 and PLD4 act in concert to produce these ligands. On the one hand, RNase T2 generates 2',3'-cyclophosphate-guanosine terminated RNA fragments upon which PLD exonuclease activity acts to release the terminal 2',3'-cyclic GMP. This molecule serves as the physiological ligand for the first binding pocket. On the other hand, PLD activity is also required to generate short RNA fragments that occupy the second binding pocket of TLR7. While PLD3 and PLD4 exert comparable catalytic activities, the almost exclusive expression of PLD4 in pDCs suggests that PLD4 is the relevant exonuclease for pDC activation in the human system. Biochemical and structural studies of PLD3 and PLD4 show that these enzymes form homodimers with two ligand binding sites. Dimer formation is critical for catalytic activity, and previously identified disease-associated mutants fail to form stable dimers. In conclusion, our data provide a mechanistic basis for the recognition of RNA fragments by TLR7.

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TMEM41B is a novel regulator of autophagy and lipid mobilization

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2018-08-20 | PXD010207 | Pride

Spinster homolog 1 (spns1)-dependent endocardial autophagy-lysosomal pathway drives valve morphogenesis through the control of Notch1 signaling.

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Autophagy cargo receptors are secreted via extracellular vesicles and particles in response to endolysosomal inhibition or impaired autophagosome maturation

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Aneuploidy-induced cellular stresses limit autophagic degradation.

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Project description:Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyzed lysosomes and early endosomes. Based on the identification of 5,376 cross-links, we investigated protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and a heterodimeric structure of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identified >300 proteins presenting putative cargo, and confirmed several substrates for flotillin-dependent endocytosis, including the latrophilin family of adhesion G protein-coupled receptors.

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