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A Lysosome membrane regeneration pathway depends on TBC1D15 and autophagic lysosomal reformation proteins

ABSTRACT: Acute lysosomal membrane damage reduces the cellular population of functional lysosomes. However, these damaged lysosomes have a remarkable recovery potential independent of lysosomal biogenesis and remain unaffected in TFEB/TFE3-depleted cells. We combined proximity labelling based proteomics, biochemistry and high-resolution microscopy to unravel a new lysosomal membrane regeneration pathway which is dependent on ATG8, lysosomal membrane protein LIMP2, the Rab7 GAP TBC1D15, and proteins required for autophagic lysosomal reformation (ALR) including Dynamin2, Kinesin5B and Clathrin. Upon lysosomal damage, LIMP2 act as a lysophagy receptor to bind ATG8, which in turn recruits TBC1D15 to damaged membranes. TBC1D15 hydrolyses Rab7-GTP to segregate the damaged lysosomal mass and provides a scaffold to assemble and stabilize the ALR machinery, potentiating the formation of lysosomal tubulesand subsequent Dynamin2-dependent scission. TBC1D15-mediated lysosome regeneration was also observed in a cell culture model of oxalate nephropathy.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Anshu Bhattacharya

LAB HEAD: Ivan Dikic

PROVIDER: PXD040191 | Pride | 2023-03-07

REPOSITORIES: Pride

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			Action	DRS
	20181211_GT_AB_LC3PD_Ctrl_LC3_1.raw	Raw
	20181211_GT_AB_LC3PD_Ctrl_LC3_2.raw	Raw
	20181211_GT_AB_LC3PD_Ctrl_LC3_3.raw	Raw
	20181211_GT_AB_LC3PD_LLOMe_LC3_1.raw	Raw
	20181211_GT_AB_LC3PD_LLOMe_LC3_2.raw	Raw

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Project description:Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding Galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their inter-relationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and Galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosomal proteome remodeling during lysophagy. Among proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both Hela cells and induced neurons (iNeurons). While the related receptor OPTN can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.

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A Lysosome membrane regeneration pathway depends on TBC1D15 and autophagic lysosomal reformation proteins

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