The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

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.

RapiGest precipitation depends on peptide concentration.

The mass spectrometry-compatible surfactant RapiGest promotes the enzymatic digestion of proteins by facilitating their unfolding while retaining enzymatic activity. RapiGest consists of a hydrophilic head and a hydrophobic tail, which can be separated by acid hydrolysis. This allows for removal of RapiGest prior to mass spectrometric analysis via precipitation and solid phase extraction. During in-solution digestion experiments with RapiGest, we noticed a high variability in the formation of precipitates after acid hydrolysis, implying that RapiGest precipitation is sample-dependent. We show that RapiGest hydrolyses efficiently under acidic conditions and that differences in precipitation are solely due to protein/peptide concentration. Furthermore, we demonstrate that RapiGest precipitation can be triggered by the addition of intact proteins, providing a strategy for its efficient removal from highly diluted samples. Data are available via ProteomeXchange with identifier PXD025982.