Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

Abstract

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 lysosome-associated 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.

TBK1 is part of a galectin 8 dependent membrane damage recognition complex and drives autophagy upon Adenovirus endosomal escape

“…Tank binding kinase (TBK1) has recently been implicated in autophagy activation. It is recruited by autophagy receptors to damaged organelles such as mitochondria [ 24 ], aggregates [ 25 ], or lysosomes [ 26 ], leading to their elimination. TBK1 is also involved in the degradation of invasive bacteria by autophagy, following detection of cellular membrane penetration by galectins.…”

Early Endosome Capture Proteomics and its Application to Amyloid Precursor Protein Intramembrane Processing by β and γ-Secretases

“…We sought to develop an approach analogous to Lyso-IP for purification of early endosomes. First, we adopted a previously reported targeting approach 36 to create 293 cells wherein endogenous TMEM192 was tagged with a 3xHA epitope (referred to as 293 L for Lyso-IP cells) ( Extended Data Fig. 1a ; see METHODS ).…”

“…Gene editing was performed as described previously 79 . For endogenous tagging of TMEM192 with 3xHA, cells were co-transfected with pX459 containing a gRNA (5’-AGTAGAACGTGAGAGGCTCA) targeting adjacent to the translational termination sequence in TMEM192 and pSMART containing 5’ and 3’ homology arms for TMEM192 in which the termination codon is replaced by a 3xHA epitope sequence followed by a TAA stop codon, as described 36 , except that the puromycin resistance cassette was replaced by a neomycin resistance cassette. Homozygously targeted clones were identified by immunoblotting cell extracts with α-HA and α-TMEM192, as described 36 .…”

“…a, Scheme summarizing how 293 cells were gene edited to place a C-terminal 3xHA tag on the endogenous TMEM192 gene as described 36 to create 293 L cells. Cell extracts from 293 or 293 L cells were subjected to immunoblotting with α-TMEM192 and α-HA to demonstrate homozygous targeting of the TMEM192 gene to introduce the 3xHA tag, with α-HSP90 used as a loading control.…”

Maintenance and loss of endocytic organelle integrity: mechanisms and implications for antigen cross-presentation

“…Galectins initiate selective autophagy through recruitment of a number of cytosolic autophagy receptors, including p62, nuclear dot protein 52 (NDP52), optineurin and TAX1BP1 [70,[78][79][80], which direct galectin-decorated damaged endocytic organelles to the autophagy machinery. Autophagy receptors also recognize ubiquitin that has been added by E3 ubiquitin ligases onto proteins (or lipids as recently discovered in the case of Salmonella [81]) at sites of endocytic organelle damage.…”