CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export

We report here two genome-wide CRISPR screens performed to identify genes that, when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also performed under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight coregulation of cholesterol and bis(monoacylglycero)phosphate in cells and reveal an unexpected role for the ER-localized SNX13 protein as a negative regulator of lysosomal cholesterol export and contributor to ER–lysosome membrane contact sites. In the absence of NPC1 function, SNX13 knockdown redistributes lysosomal cholesterol and is accompanied by triacylglycerol-rich lipid droplet accumulation and increased lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the regulation of lysosomal lipids and modification of these processes by novel gene products.

Gene-dependent yeast cell death pathway requires AP-3 vesicle trafficking leading to vacuole membrane permeabilization

Unicellular eukaryotes are suggested to undergo self-inflicted destruction. However, molecular details are sparse by comparison to the mechanisms of cell death known for human cells and animal models. Here we report a molecular pathway in Saccharomyces cerevisiae leading to vacuole/lysosome membrane permeabilization and cell death. Following exposure to heat-ramp conditions, a model of environmental stress, we observed that yeast cell death occurs over several hours, suggesting an ongoing molecular dying process. A genome-wide screen for death-promoting factors identified all subunits of the AP-3 adaptor complex. AP-3 promotes stress-induced cell death through its Arf1-GTPase-dependent vesicle trafficking function, which is required to transport and install proteins on the vacuole/lysosome membrane, including a death-promoting protein kinase Yck3. Time-lapse microscopy revealed a sequence of events where AP-3-dependent vacuole permeability occurs hours before the loss of plasma membrane integrity. An AP-3-dependent cell death pathway appears to be conserved in the human pathogen Cryptococcus neoformans.

A conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation

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.

Biomarkers in rock oysters (Saccostrea mordax) in response to organophosphate pesticides

Chlorpyrifos is a xenobiotics contaminants that threats the marine environment and the living organism within the habitat. Although several marine bivalve species have been used as the indicator of marine pollution, the used of Saccostrea mordax is remaining unknown. This study aimed at investigating the suitability of lysosome membrane integrity, carboxylesterase activity, 8-oxo-2′-deoxyguanosine and condition index as biomarkers in adult S. mordax following their exposure to 0.0, 5.0 and 500 μg.L-1 of Chlorpyrifos for 21 days under laboratory conditions. Results indicated that the lysosome membrane integrity showed a dose-dependent response with a significant statistical number of destabilized cells between all the treatment groups. Carboxylesterase activity was significantly inhibited in 500 μg.L-1 chlorpyrifos treated group, while the environmentally relevant concentration (5 μg.L-1) did not induce a significant inhibition with reference to the control. Similarly, the condition index showed a dose-dependent response with the oysters exposed to 500 μg.L-1 chlorpyrifos exhibiting a significantly reduced growth rate. There was no statistical significance in the means of both 8-oxo-2′-deoxyguanosine in all treatment groups. The reaction of S. mordax to chlorpyrifos contamination demonstrates that the species can potentially be used as sentinel organisms in environmental monitoring programs. Lysosome membrane integrity was a single out as a sensitive biomarker for exposure to chlorpyrifos and is therefore suitable for environmental monitoring for sublethal concentrations of chlorpyrifos contaminations. Additionally, the use of multiple biomarkers was found to be robust in this study and can be extrapolated to other ecotoxicological studies

Lysosomal agents inhibit store-operated Ca2+ entry

Pharmacological manipulation of lysosome membrane integrity or ionic movements is a key strategy for probing lysosomal involvement in cellular processes. However, we have found an unexpected inhibition of store-operated Ca2+ entry (SOCE) by these agents. Dipeptides (GPN and LLOMe) that are inducers of lysosomal membrane permeabilization (LMP) uncoupled ER Ca2+-store depletion from SOCE by interfering with Stim1 oligomerization and/or Stim1 activation of Orai. Similarly, the K+/H+ ionophore, nigericin, that rapidly elevates lysosomal pH, also inhibited SOCE in a Stim1-dependent manner. In contrast, other strategies for manipulating lysosomes (bafilomycin A1, lysosomal re-positioning) had no effect upon SOCE. Finally, the effects of GPN on SOCE and Stim1 was reversed by a dynamin inhibitor, dynasore. Our data show that lysosomal agents not only release Ca2+ from stores but also uncouple this release from the normal recruitment of Ca2+ influx.

A conserved ubiquitin- and ESCRT-dependent pathway to regulate human lysosomal membrane proteins

The lysosome is an essential organelle to recycle cellular materials and maintain nutrient homeostasis, but the mechanism to down-regulate lysosomal membrane proteins is poorly understood. In this study, we developed a cycloheximide chase assay to measure the half-lives of ~30 human lysosomal membrane proteins, and identified RNF152 as a short-lived protein. The degradation of RNF152 depends on ubiquitin and the endosomal sorting complexes required for transport (ESCRT) machinery. Ubiquitinated RNF152 is sorted and internalized by the ESCRT machinery into the lysosomal lumen for degradation. Strikingly, when expressed in budding yeast, human RNF152 is also degraded by the vacuole (yeast lysosome) in an ESCRT-dependent manner. Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins.

Mitochondria–lysosome membrane contacts are defective in GDAP1-related Charcot–Marie–Tooth disease

Mutations in the GDAP1 gene cause Charcot–Marie–Tooth (CMT) neuropathy. GDAP1 is an atypical glutathione S-transferase (GST) of the outer mitochondrial membrane and the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs). Here, we investigate the role of this GST in the autophagic flux and the membrane contact sites (MCSs) between mitochondria and lysosomes in the cellular pathophysiology of GDAP1 deficiency. We demonstrate that GDAP1 participates in basal autophagy and that its depletion affects LC3 and PI3P biology in autophagosome biogenesis and membrane trafficking from MAMs. GDAP1 also contributes to the maturation of lysosome by interacting with PYKfyve kinase, a pH-dependent master lysosomal regulator. GDAP1 deficiency causes giant lysosomes with hydrolytic activity, a delay in the autophagic lysosome reformation, and TFEB activation. Notably, we found that GDAP1 interacts with LAMP-1, which supports that GDAP1–LAMP-1 is a new tethering pair of mitochondria and lysosome membrane contacts. We observed mitochondria–lysosome MCSs in soma and axons of cultured mouse embryonic motor neurons and human neuroblastoma cells. GDAP1 deficiency reduces the MCSs between these organelles, causes mitochondrial network abnormalities, and decreases levels of cellular glutathione (GSH). The supply of GSH-MEE suffices to rescue the lysosome membranes and the defects of the mitochondrial network, but not the interorganelle MCSs nor early autophagic events. Overall, we show that GDAP1 enables the proper function of mitochondrial MCSs in both degradative and nondegradative pathways, which could explain primary insults in GDAP1-related CMT pathophysiology, and highlights new redox-sensitive targets in axonopathies where mitochondria and lysosomes are involved.

Lipids and membrane-associated proteins in autophagy

Autophagy is essential for the maintenance of cellular homeostasis and its dysfunction has been linked to various diseases. Autophagy is a membrane driven process and tightly regulated by membrane-associated proteins. Here, we summarized membrane lipid composition, and membrane-associated proteins relevant to autophagy from a spatiotemporal perspective. In particular, we focused on three important membrane remodeling processes in autophagy, lipid transfer for phagophore elongation, membrane scission for phagophore closure, and autophagosome-lysosome membrane fusion. We discussed the significance of the discoveries in this field and possible avenues to follow for future studies. Finally, we summarized the membrane-associated biochemical techniques and assays used to study membrane properties, with a discussion of their applications in autophagy.