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A conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation
['Weichao Zhang', 'Department Of Molecular', 'Cellular', 'Developmental Biology', 'University Of Michigan', 'Ann Arbor', 'Michigan', 'United States Of America', 'Xi Yang', 'Liang Chen']
Date: None

The lysosome is an essential organelle to recycle cellular materials and maintain nutrient homeostasis, but the mechanism to down-regulate its membrane proteins is poorly understood. In this study, we performed a cycloheximide (CHX) chase assay to measure the half-lives of approximately 30 human lysosomal membrane proteins (LMPs) and identified RNF152 and LAPTM4A as short-lived membrane proteins. The degradation of both proteins is ubiquitin dependent. RNF152 is a transmembrane E3 ligase that ubiquitinates itself, whereas LAPTM4A uses its carboxyl-terminal PY motifs to recruit NEDD4-1 for ubiquitination. After ubiquitination, they are internalized into the lysosome lumen by the endosomal sorting complexes required for transport (ESCRT) machinery for degradation. Strikingly, when ectopically expressed in budding yeast, human RNF152 is still degraded by the vacuole (yeast lysosome) in an ESCRT-dependent manner. Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins.

Funding: This project has been funded by grants from National Institutes of Health, with R01GM133873 to ML, R35GM130331 to YW, and R01GM122434 to PH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we screened approximately 30 human LMPs using a cycloheximide (CHX) chase assay and identified a few candidates with short half-lives. Among those candidates, we focused on RNF152 (a lysosome membrane–anchored E3 ligase) and LAPTM4A (a 4-transmembrane LMP) as cargoes to examine the possible mechanisms of LMP turnover. We discovered that their degradation is both ubiquitination- and lysosome dependent. Further, we showed that the conserved ESCRT machinery plays a vital role in cargo internalization. Collectively, our work suggests that the ubiquitin- and ESCRT-dependent degradation pathway is a conserved and general mechanism to down-regulate LMPs.

At the protein level, selective removal of proteins from the lysosome surface is essential for adjusting membrane composition in response to environmental cues. However, very little is known about its underlying mechanism. A process like lysophagy, which engulfs whole lysosomes, could not accomplish selectivity. This leads to important questions as to how human lysosomes selectively down-regulate their membrane proteins and what machinery might be involved in the process.

Given the physiological importance and clinical implications of LMPs, we wonder how human LMPs are regulated and quality controlled. At the organelle level, if the lysosomal membrane is mildly damaged by insults like lysosomotropic compounds, such as L-leucyl-L-leucine methyl ester (LLOMe) or iron-dependent oxidative stress, the endosomal sorting complexes required for transport (ESCRT) machinery can be recruited to the lysosome surface to repair the membrane [ 16 – 18 ]. If the damage is too severe to be repaired, ruptured lysosomes will be sequestered and degraded by selective autophagy, a process termed “lysophagy” [ 19 , 20 ].

As an essential organelle, the lysosome is responsible for various cellular processes, including protein turnover and recycling, energy metabolism, intracellular signaling, and nutrient storage [ 1 – 3 ]. The lysosome membrane contains hundreds of transmembrane proteins, many of which are transporters and channels that shuttle metabolites (ions, amino acids, cholesterol, etc.) across the membrane [ 4 – 7 ]. Malfunction of these lysosomal membrane proteins (LMPs) can give rise to inherited genetic disorders called lysosomal storage diseases (LSDs). Many LSD patients will develop severe neurodegeneration symptoms [ 8 ]. Furthermore, growing evidence suggests that mutations in LMPs and other lysosome dysfunction are associated with age-related neurodegeneration such as Alzheimer disease, frontotemporal dementia, and Parkinson disease [ 9 – 11 ]. As we age, the lysosome membrane gradually accumulates damaged proteins and loses its integrity, which dampens the cell’s ability to remove pathogenic protein aggregates and damaged organelles, eventually leading to cell death and inflammation [ 12 – 15 ]. Strategies to maintain the lysosome membrane integrity during aging will likely delay the onset of neurodegenerative symptoms.

Results

Macroautophagy machinery and CMA pathway are not involved in the degradation of GFP-RNF152 In mammalian cells, there are 4 possible mechanisms to deliver intracellular materials into lysosomes for degradation: macroautophagy, microautophagy, ESCRT-dependent formation of intraluminal vesicles, and chaperone-mediated autophagy (CMA) [30]. How is ubiquitinated RNF152 internalized into the lysosome then? Although lysophagy can deliver an entire damaged lysosome into other healthy lysosomes for degradation [31], it cannot selectively turnover a particular membrane protein while leaving others intact. Recently, Lee and colleagues reported that glucose starvation and certain drug treatments could trigger a microautophagy process to selectively turnover some LMPs. Although the mechanism remains to be identified, it was shown that the LC3 lipidation machinery, such as ATG5, is critical to initiate microautophagy [32]. To test if this microautophagy is involved in RNF152 degradation, we knocked out either ATG5 or ATG7 using the CRISPR/Cas-9 method [33,34]. In wild-type (WT) cells, Atg5 forms a stable 55-kDa conjugate with Atg12 in an Atg7-dependent manner (S5A Fig, left 3 lanes) [35]. After knocking out Atg7, the conjugate no longer forms, and Atg5 appears as a 33-kDa band (S5A Fig, last 3 lanes). However, neither ATG5 nor ATG7 knockout cells exhibited any defect in GFP-RNF152 degradation (S5A and S5B Fig). Autophagy and the ubiquitin-proteasome system (UPS) are the 2 major pathways to degrade proteins in eukaryotic cells [36]. There is mounting evidence to show that the 2 pathways can crosstalk. It is possible that the UPS pathway is up-regulated to compensate for the loss of autophagy [37–40]. To rule out the possibility that GFP-RNF152 is redirected to the proteasome after knocking out macroautophagy, we treated the ATG7KO cells with BafA1 and MG132. As shown in S5C and S5D Fig, GFP-RNF152 degradation is still mainly dependent on the lysosome in autophagy-deficient cells. Thus, the macroautophagy machinery and likely the LC3 lipidation–triggered microautophagy is not involved in RNF152 degradation. We also examined whether CMA is involved. In the CMA pathway, the chaperone Hsc70 recognizes a KFERQ-like motif of its substrates and delivers them to the lysosome for degradation [41]. Using a web-based motif finder, we identified one putative KFERQ-like motif in the cytosolic domain of RNF152: 46QKDVR50 (S5E Fig) [42]. However, mutating 46QK47 to AA does not affect RNF152 degradation (S5F and S5G Fig), suggesting that the CMA pathway may not be involved.

[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001361

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