A delicate decision between repair and degradation of damaged lysosomes

ABSTRACT

The destination of a damaged lysosome is either being repaired if the damage is small or degraded through a lysosome-specific macroautophagy/autophagy pathway named lysophagy when the damage is too extensive to repair. Even though previous studies report lumenal glycan exposure during lysosome damage as a signal to trigger lysophagy, it is possibly beneficial for cells to initiate lysophagy earlier than membrane rupture. In a recently published article, Gahlot et al. determined that SPART/SPG20 senses lipid-packing defects and recruits and activates the ubiquitin ligase ITCH, which labels damaged lysosomes with ubiquitin chains to initiate lysophagy.

KEYWORDS:

• ESCRT
• lipid-packing defect
• lysophagy
• lysosome damage
• ubiquitination

As an essential organelle for degradation – and hence the site of a vast array of hydrolases – and the terminal destination of many internalized cargos, lysosomes play critical physiological roles; thus, maintaining the integrity of the lysosome membrane is significant for cell homeostasis. However, at the same time, lysosomes are frequently subjected to damaging materials, such as incoming pathogens, sharp crystals and protein aggregates [1]. When lysosome membranes are damaged, a decision needs to be made to either repair the damage or initiate the lysosome degradation pathway, named lysophagy, if the damage is large and too extensive to repair [2]. Small perforations on the lysosome membrane trigger calcium influx and three major pathways were identified for lysosome damage repair. The first pathway depends on endosomal sorting complexes required for transport (ESCRT), which are recruited to damaged lysosomes and mediate membrane repair; this pathway is found to react to the damage prior to lysophagy [3,4]. The second pathway involves the calcium-activated scrambling of sphingomyelin toward the cytosolic side of the lysosome limiting membrane, which will be proceeded by sphingomyelinases and promote lysosome repair [5]. In addition, damaged lysosomes can also be repaired through increasing lysosome and endoplasmic reticulum (ER) contact sites and the ER-to-lysosome transfer of phosphatidylserine and cholesterol [6]. When the damage is too extensive/costly to repair, lysophagy will be initiated to start lysosome degradation. Currently, the best studied mechanism of lysophagy initiation is mediated by the damage-induced exposure of lumenal glycans to the cytosol, which leads to the ubiquitination of the lysosome through directly binding to ubiquitin ligases or via recognition by galectins, which recruit ubiquitin ligases. The ubiquitinated lysosome will be recognized by autophagy receptors and brought to the phagophore for degradation [2]. However, glycan exposure suggests the formation of huge pores on the lysosome that creates permeability for proteins, a situation that may already cause harm to the cell. Therefore, lysophagy may be initiated before glycan exposure, but the mechanism is not clear. In the article highlighted here, Gahlot et al. determined that SPART/SPG20 senses lipid-packing defects, binds to damaged lysosomes, and recruits the ubiquitin ligase ITCH, eventually leading to lysosome ubiquitination and lysophagy [7].

The authors first determined that the recruitment of SPART to damaged lysosomes triggered by L-leucyl-L-leucine methyl ester (LLOMe) treatment coincides with K63 ubiquitination and this happens after the recruitment of ESCRT component IST1. The fact that SPART needs a higher LLOMe concentration than IST1 to reach maximal recruitment indicates that SPART recruitment requires an additional trigger besides IST1. In addition, SPART localizes on damaged lysosomes that cannot be repaired and colocalizes with the autophagy receptor OPTN, suggesting that SPART initiates lysophagy rather than a repair pathway. In addition, the recruitment of SPART to damaged lysosomes happens before the cytosolic appearance of LGALS3 (galectin 3), a signal identified previously for lysophagy initiation [8], suggesting that SPART may initiate lysophagy independently from LGALS3. In addition, the fact that SPART and LGALS3 can be subsequently recruited to an ESCRT-positive lysosome indicates that different damage responses can successively occur on an individual damaged lysosome.

To identify how SPART mediates the ubiquitination of damaged lysosomes, the authors screened for the NEDD4-like family of ubiquitin ligases, which can interact with the PPAY motif in SPART, and found that ITCH is the only one that translocates to lysosomes after LLOMe treatment in a SPART-dependent manner. In addition, ITCH is activated by the LLOMe-induced damage and mediates the K63-linked ubiquitination of damaged lysosomes. Knocking out SPART or ITCH leads to compromised clearance of damaged lysosomes, and the deficiency can only be rescued by expressing SPART with an intact PPAY motif or by ITCH with normal catalytic activity. In addition, in the ITCH or SPART knockout cells, LC3 and the autophagy receptor SQSTM1 cannot be efficiently recruited to the damaged lysosome and, as a result, lysophagy flux is reduced and cells are more prone to lysosome damage compared to wild-type cells. In summary, SPART on damaged lysosomes recruits ITCH, which initiates the ubiquitination of lysosomes and lysophagy for clearance.

The researchers next examined the mechanism through which SPART is recruited to the damaged membrane. First, IST1 can interact with the MIT domain in SPART and help with its recruitment to damaged lysosomes; the removal of the MIT domain from SPART or knocking down IST1 partially impairs the recruitment of SPART to damaged lysosomes. Additionally, SPART contains an SC domain at the C terminus that mediates membrane binding; the SC domain is necessary and sufficient for the location of SPART on the lysosome after LLOMe-induced membrane damage. These results suggest that the SC domain is the major mediator, but the MIT domain also contributes to SPART recruitment to damaged lysosomes. Further investigation into how SPART senses membrane damage, revealed that SPART is colocalized with an amphipathic lipid-packing sensor, indicating that SPART senses lipid-packing defects associated with lysosome membrane damage. The SC domain is predicted to contain four amphipathic helices; helices 1 and 3 have bulky hydrophobic residues, which can sense the spaces between loosely packed lipids. Mutating eight of these amino acids into the small hydrophobic amino acid valine (the 8xV mutant) is sufficient to block SPART and ITCH translocation to damaged lysosomes and their subsequent clearance. Consistent with these results, bulky hydrophobic residue-dependent SPART recruitment is also observed in the lysosome damage caused by peroxidation. In vitro experiments using giant unilamellar vesicles confirmed the conclusion that SPART is recruited to peroxidation-damaged membranes and that this recruitment depends on an intact SC domain; the 8xV mutant impairs SPART binding to the membrane. These results demonstrate that the SC domain of SPART detects lipid-packing defects and binds to these sites.

In summary, this study identifies SPART-ITCH as new initiators of lysophagy. Unlike glycan exposure-induced lysophagy, which usually occurs when the lysosome membrane is permeable to proteins, SPART recognizes lipid-packing defects when lysosomes are not too severely damaged. This mechanism allows the detection and degradation of compromised lysosomes at an early stage and avoids further damage to the cells. In addition, this study also sheds light on how the decision is made between lysosome repair or degradation. Upon lysosome damage, ESCRT complexes that are sensitive to calcium influx are recruited to the damaged site for repair. If the damage continues, IST1 recruits SPART. At the same time, the SC domain of SPART senses the lipid-packing defects and facilitates SPART recruitment to damaged lysosomes. SPART will further recruit the ubiquitin ligase ITCH, which ubiquitinates lysosomes and initiates lysophagy. Given the important role of SPART in lysophagy, combined with the fact that mutations in SPART are identified in Troyer syndrome [9] and SPART is recognized as a biomarker for colon cancer detection [10], how SPART affects lysophagy in the context of these diseases is worth further investigation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the National Institute of General Medical Sciences [GM131919].