In:
Science, American Association for the Advancement of Science (AAAS), Vol. 378, No. 6615 ( 2022-10-07)
Abstract:
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.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.abn5648
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2022
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
