See Table S1 for complete genome-wide display datasets. Immunofluorescence To visualize endolysosomes in suspension K562 cells (Fig. contributor to ERClysosome membrane contact sites. In the absence of Alogliptin NPC1 function, SNX13 knockdown redistributes lysosomal cholesterol and is accompanied by triacylglycerol-rich lipid droplet build up and improved lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the rules of lysosomal lipids and changes of these processes by novel gene products. Intro Cellular lipid homeostasis is definitely managed by complex and dynamic interorganelle communication processes that coordinate uptake, biosynthesis, and degradation of 1,000 lipid varieties. Among all the organelles involved in lipid rules, the lysosome takes on a central part. Lysosomes are the final station at which endocytosed lipoprotein particles and membranes derived from intralumenal budding and autophagy undergo a series of degradative reactions to yield unesterified cholesterol and additional lipid precursors (Gruenberg, 2020; Ballabio and Bonifacino, 2020). Free cholesterol is then exported out of the lysosome and either recycled for de novo synthesis of biological membranes and additional sterol products or esterified and stored in lipid droplets (LDs). Probably one of the most analyzed lipid storage disorders is definitely Niemann-Pick type C (NPC) disease, caused by genetic problems in the lysosomal cholesterol transport system, Niemann-Pick C1 and C2 proteins (NPC1 and NPC2). NPC mutant cells accumulate cholesterol and glycosphingolipids in lysosomes, leading to neurodegeneration and premature death (Pentchev, 2004). Despite recent advances in understanding of how transmembrane NPC1 and lumenal NPC2 export cholesterol from lysosomes (Pfeffer, 2019), the precise molecular events that regulate this process and function downstream of NPC1 are still unclear (Das et al., 2014; Infante and Radhakrishnan, 2017). Once cholesterol exits lysosomes, membrane contact sites between the lysosome surface and additional juxtaposed compartments deliver cholesterol to the ER, a process that involves transit via the plasma membrane (PM; Infante and Radhakrishnan, 2017). An important player in cholesterol rules is definitely bis(monoacylglycero)phosphate (BMP; also known as lysobisphosphatidic acid), which is found almost specifically in intralumenal vesicles of multivesicular endosomes (MVEs; Alogliptin Gruenberg, 2020). Elevation of BMP levels occurs in many lipid storage disorders, including NPC, and BMP takes on important tasks in lipid catabolism and lysosomal cholesterol egress (Chevallier et al., 2008; examined in McCauliff et al., 2019). In the absence of active NPC1, cells fed phosphatidylglycerol increase their BMP content material, which decreases their lysosomal cholesterol levels by a process that requires NPC2 (McCauliff et al., 2019). These findings support Alogliptin the living of NPC1-self-employed, relatively sluggish cholesterol export pathways. Because much remains to be learned regarding the mechanisms of cholesterol transport Alogliptin and its rules, we performed genome-wide CRISPR screens to identify regulators of lysosomal cholesterol and BMP homeostasis. We repeated these screens under conditions of NPC1 inhibition to identify cellular parts that may function in parallel with the NPC1 pathway to accomplish cholesterol export; such gene products might present pathways to benefit individuals with NPC disease. This strategy CALML3 allowed us to confirm known parts that regulate cholesterol transport and rate of metabolism and revealed additional previously unrecognized players. As one example, we display here that SNX13 is an ER-resident inter-organelle tether that regulates lysosomal cholesterol export. Amazingly, SNX13-depleted cells are able to redistribute cholesterol to the PM and additional compartments, despite the absence Alogliptin of NPC1 function. Results Genome-wide screens to identify regulators of endolysosomal cholesterol We founded two screening protocols to monitor changes in either cholesterol or BMP using fluorescently tagged perfringolysin O* (PFO*) to label accessible cholesterol (Das et al., 2013) or an mAb to detect BMP in K562 cells in conjunction with circulation cytometry (Fig. 1 A). Briefly, Cas9-expressing.