VCP /p97 cooperates with YOD 1, UBXD 1 and PLAA to drive clearance of ruptured lysosomes by autophagy

Abstract

Rupture of endosomes and lysosomes is a major cellular stress condition leading to cell death and degeneration. Here, we identified an essential role for the ubiquitin-directed AAA-ATPase, p97, in the clearance of damaged lysosomes by autophagy. Upon damage, p97 translocates to lysosomes and there cooperates with a distinct set of cofactors including UBXD1, PLAA, and the deubiquitinating enzyme YOD1, which we term ELDR components for Endo-Lysosomal Damage Response. Together, they act downstream of K63-linked ubiquitination and p62 recruitment, and selectively remove K48-linked ubiquitin conjugates from a subpopulation of damaged lysosomes to promote autophagosome formation. Lysosomal clearance is also compromised in MEFs harboring a p97 mutation that causes inclusion body myopathy and neurodegeneration, and damaged lysosomes accumulate in affected patient tissue carrying the mutation. Moreover, we show that p97 helps clear late endosomes/lysosomes ruptured by endocytosed tau fibrils. Thus, our data reveal an important mechanism of how p97 maintains lysosomal homeostasis, and implicate the pathway as a modulator of degenerative diseases.

Ubiquitination of exposed glycoproteins by SCF FBXO27 directs damaged lysosomes for autophagy

“…Although previous reports, including Chauhan et al, demonstrate that K63-linked ubiquitin chains are important for selective autophagy including lysophagy (23,24), our data indicate that K48-linked ubiquitination is also important. Although the role in lysophagy at first seems inconsistent with reports implicating K48-linkages in proteasomal degradation, a recent study suggests that pathway choice between proteasomal degradation and selective autophagy is largely regulated by the oligomerization state of ubiquitin receptors, rather than specific linkage type (25).…”

Triggered recruitment of ESCRT machinery promotes endolysosomal repair

“…S11) and the ubiquitin-dependent disaggregase VCP/p97 (fig. S12), consistent with known roles for ubiquitination and associated proteins in lysophagy (2, 5, 30). Early ESCRT recruitment and delayed accumulation of GAL3 and ubiquitinated material were also apparent in macrophage-like THP-1 cells (fig.…”

VPS37A directs ESCRT recruitment for phagophore closure

“…It has recently been reported that the ESCRT machinery can be recruited to damaged lysosomes for the membrane repair that occurs independent of lysophagy induction (Radulovic et al, 2018; Skowyra et al, 2018). To determine if the phagophore localization of VPS37A is distinct from the ESCRT recruitment to damaged membranes, GFP-FL– and HT-LC3–expressing VPS37A KO cells were transfected with the damaged endomembrane marker mCherry-GAL3 (Papadopoulos et al, 2017) and treated with the lysosome-damaging agent l-leucyl-l-leucine O-methyl ester (LLOME) in the presence or absence of the phosphatidylinositol-3-kinase inhibitor wortmannin (WM) followed by labeling with MIL. We observed that nearly all damaged lysosomes labeled with mCherry-GAL3 were enwrapped with MIL + immature autophagosomal membranes in a WM-sensitive manner (Fig.…”

“…ON-TARGETplus SMART Pool nontargeting (D-001810-10) and CHMP2A (L-020247-01) siRNAs were purchased from GE Healthcare. The following plasmids were obtained from Hartmut Land (University of Rochester Medical Center, Rochester, NY), Jay Morgenstern (Imperial Cancer Research Fund, London, UK), Bob Weinberg (Whitehead Institute for Biomedical Research, Cambridge, MA), Feng Zhang (Massachusetts Institute of Technology, Cambridge, MA), Brett Stringer (QIMR Berghofer Medical Research Institute, Brisbane, Australia), Christopher Vakoc (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Noboru Mizushima (The University of Tokyo, Tokyo, Japan), Wesley Sundquist (University of Utah School of Medicine, Salt Lake City, UT), Konrad Buessow (Max Planck Institute for Molecular Genetics, Berlin, Germany), and Hemmo Meyer (University of Duisburg-Essen, Essen, Germany) through Addgene: pBabe-Hygro (#1765; Morgenstern and Land, 1990), lentiCas9-Blast (#52962; Sanjana et al, 2014), lentiCRISPRv2 (puro; #98290; Brett Stringer Lab), LRG (Lenti_sgRNA_EFS_GFP, #65656; Shi et al, 2015), pMXs-IP-EGFP-mATG5 (#38196; Hara et al, 2008), pMXs-IP-EGFP-ULK1 (#38193; Hara et al, 2008), pMXs-IP-EGFP-LC3 (#38195; Hara et al, 2008), pEGFP-VPS4-E228Q (#80351; Votteler et al, 2016), pQTEV-VPS28 (#34803; Büssow et al, 2005), and pmCherry-GAL3 (#85662; Papadopoulos et al, 2017). To generate pCDH1-CMV-MCS-SV40-Hygro, the puromycin resistance gene (BamHI–SalI [blunted] in pCDH1-CMV-MCS1-EF1-Puro; #CD510A-1; System Biosciences) was replaced to the hygromycin resistance gene (BanHI–ClaI [blunted]) from pBabe-Hygro.…”