LAUSR

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase–mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder. Description LYSET helps load lysosomes Lysosomes are major degradative compartments within the cell, and their dysfunction results in both rare and common disorders. Certain viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), hijack lysosomes to gain entry into the cell and start their destructive infection cycle. Richards et al. identified a small protein named LYSET that is critical for proper lysosomal function. In cells lacking LYSET, the trafficking of enzymes to the lysosomes was severely disrupted resulting in the accumulation of undigested material in the lysosome. Independently, Pechincha et al. identified LYSET as being selectively essential when cells feed on extracellular proteins. Cancer cells commonly rely on extracellular proteins to provide amino acids. LYSET helped to anchor N-acetylglucosamine-1-phosphotransferase in Golgi membranes for tagging enzymes with the lysosomal trafficking signal mannose-6-phosphate. Without LYSET, lysosomes were depleted of catabolic enzymes, losing their ability to digest extracellular proteins. —SMH Genome-scale CRISPR screens identify a key protein in lysosome biogenesis relevant for inherited disease and viral infection. INTRODUCTION Lysosomes are key degradative compartments within the cell that are essential to maintain protein homeostasis. Their dysfunction causes over 70 rare genetic diseases collectively known as lysosomal storage disorders (LSDs). Intracellular sorting of most soluble lysosomal enzymes occurs by tagging with mannose 6-phosphate (M6P) residues in the Golgi apparatus, which are recognized by specific receptors that direct transport to the endosomal/lysosomal system. GlcNAc-1-phosphotransferase catalyzes the first step in M6P-tagging. Inherited loss of GlcNAc-1-phosphotransferase function causes the severe LSD mucolipidosis type II (MLII). The M6P signal-mediated lysosomal sorting pathway is well studied and thought to be completely understood. However, it remains unknown whether additional critical components exist. RATIONALE Certain viruses program successful entry into cells by co-opting lysosomal cathepsin proteases to cleave and activate viral structural proteins allowing delivery of their genome into the cytoplasm. This infection strategy is shared among different virus families including reovirus, Ebola virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, these viruses are sensitive probes for lysosomal function. To identify genes important for lysosomal homeostasis, we performed genome-scale CRISPR screens using susceptibility to reovirus infection as phenotypic selection. RESULTS The genetic screens identified TMEM251—a small, uncharacterized protein—as an essential component of lysosomal biogenesis. Cells with knockout (KO) mutations in TMEM251 were refractory to infection by reovirus, SARS-CoV-2, and vesicular stomatitis virus pseudotyped with the Ebola virus glycoprotein. This was due to strongly reduced activity of lysosomal cathepsin proteases. Moreover, quantitative proteomics revealed a severe global sorting defect in which intracellular enzymes destined for lysosomal delivery were instead secreted into the medium. Thus, we renamed TMEM251 to LYSET (for lysosomal enzyme trafficking factor). Mechanistically, we showed that LYSET binds to GlcNAc-1-phosphotransferase in the Golgi apparatus and controls its stability. LYSET KO caused aberrant localization of GlcNAc-1-phosphotransferase to lysosomes and subsequent degradation, resulting in M6P tagging failure. LYSET KO mice displayed typical diagnostic features of MLII including elevated levels of lysosomal enzymes in blood serum and enlarged lysosomes with accumulation of electron dense material in isolated cells. Recently, an MLII-like genetic disorder in patients carrying biallelic mutations in LYSET has been described. We showed that complementation of LYSET KO cells with these pathogenic mutants failed to rescue lysosomal sorting defects. CONCLUSION Our work identifies LYSET as an indispensable component of the biosynthetic pathway that directs transport of soluble enzymes to the lysosome. As such, LYSET is essential for entry of diverse, highly pathogenic viruses that rely on endo-lysosomal activation by cathepsins. We uncovered an unexpected molecular mechanism in which LYSET regulates GlcNAc-1-phosphotransferase function by binding to and retaining it in the Golgi apparatus. The key role of LYSET in lysosome biogenesis likely explains MLII-like symptoms observed in patients with LYSET mutations. Altogether, our findings provide insights in fundamental cell physiology with relevance for human inherited disease and viral infection. LYSET is an essential component of the M6P lysosomal transport pathway. By using genome-scale genetic screens for viral infection we identified LYSET as a protein required for lysosome biogenesis. LYSET controls GlcNAc-1-phosphotransferase (GlcNAc-1-PT) function by binding to and retaining it in the Golgi apparatus. In LYSET KO cells M6P tagging is severely disrupted resulting in strong resistance to infection by certain viruses, aberrant secretion of enzymes normally present in the lysosome, and abnormally large lysosomes filled with storage material as a result of drastically reduced levels of active hydrolytic enzymes.