Functional analysis of TMEM251, a novel component of the lysosomal enzyme trafficking pathway

• Applicant: Dr. Wilhelm Palm
• Subject Area: Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 504920634

Mammalian cells can meet their demands for exogenous amino acid supply either through uptake of monomeric amino acids or through macropinocytosis and lysosomal catabolism of extracellular proteins. Although cells preferentially import monomeric amino acids, most extracellular biomass in vivo is contained within proteins. By tapping into the copious nutrient stores of extracellular proteins, cells can sustain a supply of bioenergetic and biosynthetic precursors during periods of starvation. Macropinocytosis and lysosomal catabolism are commonly upregulated in cancer. By feeding on extracellular proteins as an alternative nutrient source, cancer cells gain the ability to grow in nutrient-poor tumor microenvironments. To systematically discover pathways underlying this process, we performed proliferation-based CRISPR screens with monomeric amino acids versus extracellular proteins as nutrients. One striking hit that is selectively required when cells feed on proteins was TMEM251, an uncharacterized transmembrane protein. Preliminary results suggest a critical function of TMEM251 for the generation of mannose-6-phosphate groups, lysosomal enzyme trafficking and catabolism of endocytic and autophagic cargoes. In this grant proposal, we aim to characterize the molecular function of TMEM251 and to address its relevance for cancer cell growth and hereditary disorders. Specifically, we propose three independent and complementary objectives:Objective 1: Characterize the lysosomal defects of TMEM251-deficient cellsWe will combine subcellular fractionation and quantitative proteomics to determine the lysosomal proteome and secretome of TMEM251-deficient cells, and measure their lysosomal enzyme activities.Objective 2: Identify the molecular function of TMEM251 in the mannose 6-phosphate pathwayWe will identify TMEM251 interaction partners through coimmunoprecipitation and mass spectrometry and mechanistically dissect the function of TMEM251 in the mannose-6-phosphate pathway.Objective 3: Investigate pathological roles of TMEM251We will determine the relevance of TMEM251 for cancer cell growth in vitro and in vivo, and characterize the molecular phenotypes of disease-associated mutations in TMEM251.The results will reveal the molecular function of TMEM251 as a novel core component of the lysosomal enzyme trafficking pathway and address its pathological relevance in cancer metabolism and genetic disorders.

• DFG Programme: Research Grants