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Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable because of a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer. 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 LYSET enables lysosomal nutrient generation through lysosomal enzyme trafficking that is particularly important in cancer cells. INTRODUCTION Mammalian cells are surrounded by a range of different nutrients, including amino acids and extracellular proteins. In nutrient-rich conditions, cells prefer to import free amino acids to meet their nutritional demands. However, most amino acids in circulation and in the extracellular space are contained within proteins. Cells can ingest proteins from the environment and deliver them to lysosomes—organelles with digestive enzymes that break proteins down into their constituent amino acids. By generating an intracellular nutrient source, lysosomes can sustain cellular functions during starvation. This process is commonly exploited by cancer cells, which can feed on extracellular proteins to thrive in poorly vascularized, nutrient-poor tumors. However, the molecular pathways that enable cells to use extracellular proteins as a nutrient source remain incompletely understood. RATIONALE We set out to identify genes that are essential for survival and growth when cells rely on extracellular proteins as nutrients. Genetic screens have been instrumental in functionally characterizing genes in mammalian cells and identifying genes that become essential in specific cancer contexts. However, such screens have commonly been performed in cell culture media that provide most amino acids at supraphysiological levels while being strongly depleted in extracellular proteins. Conceivably, such unphysiological nutrient mixtures enforce metabolic activities that differ from in vivo phenotypes. To address this, we developed screening conditions where cancer cells grow either by the import of free amino acids or by the uptake and lysosomal degradation of extracellular proteins. RESULTS Through CRISPR screens in defined nutrient environments, we identified LYSET, a transmembrane protein (TMEM251) selectively required for cell survival and growth when extracellular proteins were an obligatory amino acid source. Mechanistically, we characterized LYSET as a protein that anchors GlcNAc-1-phosphotransferase in Golgi membranes for tagging catabolic enzymes with the lysosomal trafficking signal, mannose-6-phosphate. GlcNAc-1-phosphotransferase stability depended on LYSET because of its transmembrane domain, which was found to contain multiple hydrophilic amino acid residues and to co-occur with LYSET in multicellular animals. Without LYSET, the GlcNAc-1-phosphotransferase transmembrane domain was unstable, which led to degradation of the protein. Consequently, catabolic enzymes did not reach the lysosome and were instead mistrafficked to the cell surface. LYSET-deficient cells were unable to generate nutrients through lysosomal breakdown of proteins and accumulated lysosomes that were filled with undigested cargo. Although LYSET-deficient cancer cells grew normally under nutrient-rich conditions, they failed to grow in amino acid–poor environments and displayed a severely reduced ability to form tumors in mice. CONCLUSION Our results identified LYSET as a core component of the mannose-6-phosphate pathway for lysosomal enzyme trafficking. A clue for the function of LYSET came from our discovery that GlcNAc-1-phosphotransferase contains an energetically unfavorable transmembrane domain, which was predicted to depend on LYSET for stable membrane integration. The co-occurrence of LYSET and the unstable GlcNAc-1-phosphotransferase transmembrane domain in the same organisms suggests that they became functionally linked during evolution of the mannose-6-phosphate pathway. Conceivably, controlling GlcNAc-1-phosphotransferase levels through LYSET constitutes a mechanism to regulate lysosomal enzyme trafficking. LYSET is relevant for several human pathologies: LYSET-deficient cells lack a functional mannose-6-phosphate pathway, which provides a mechanistic explanation for the association of LYSET mutations with hereditary syndromes that resemble the lysosomal storage disorders mucolipidosis II and III. Moreover, LYSET enables cancer cells to exploit extracellular proteins as a nutrient source, thereby gaining metabolic flexibility and resilience. Thus, inhibiting LYSET and the lysosomal enzyme trafficking pathway might be a promising strategy to suppress a key metabolic adaptation in cancer. LYSET enables lysosomal nutrient generation. LYSET anchors GlcNAc-1-phosphotransferase (GlcNAc-PT) in the Golgi for tagging catabolic enzymes with the lysosomal trafficking signal, mannose-6-phosphate (M6P). The generation of catabolically active lysosomes enables cells to acquire amino acids through the breakdown of extracellular proteins. Loss of LYSET abrogates the mannose-6-phosphate pathway, depletes lysosomes of catabolic enzymes, and blocks the survival and growth of cells that rely on extracellular proteins as nutrients.