PROJECT SUMMARY/ABSTRACT Lysosomes control a substantial part of cellular metabolism by acting as the main catabolic hub of the cell and serving as a platform for the integration of numerous signals that modulate cell death, growth and proliferation. Most lysosomal functions rely on a set of more than 50 acid hydrolases that degrade a wide variety of macromolecules. Lysosomal enzymes are trafficked to the lysosome in two stages: transport of the newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi complex, and their subsequent receptor-assisted transfer from the Golgi to endolysosomal compartments. How lysosomal enzymes are transported from the ER to the Golgi complex is unknown and, to our knowledge, the simple model of a bulk, unregulated transportation has never been questioned. We have identified two candidate ER receptors, CLN6 and CLN8, whose deficiency results in altered maturation of lysosomal enzymes and lysosomal storage disorder-like diseases. We propose to study how CLN6 and CLN8 function in the pathway of maturation of lysosomal enzymes. First, we will test the hypothesis that CLN6 and CLN8 directly interact with lysosomal enzymes and that such interaction is disrupted by disease-associated mutations on either CLN6/CLN8 or on the surface of lysosomal enzymes (Aim 1). Second, we will examine the trafficking and maturation of newly synthesized lysosomal enzymes to identify the exact step that is disrupted by CLN6 and CLN8 deficiency. We will also define CLN6 and CLN8 functions in vivo by carrying out detailed tissue-specific analyses of lysosomal composition in CLN6- and CLN8-deficient mouse lines by LC-MS/MS-based proteomics. To this aim, we have generated a knock-in Lamp1FLAG mouse line to efficiently isolate lysosomes from the desired tissues (Aim 2). Third, we will identify the protein domains and motifs that are involved in CLN6/CLN8 interaction and that direct their sorting across the compartments of the early secretory pathway via COP-coated vesicles (Aim 3). We will accomplish our goals with a multi-disciplinary approach that uses the tools of biochemistry, molecular biology, cell biology and mouse engineering and we will also develop a new method of in vivo lysosome isolation from mouse tissues. Our results are likely to have important consequences for our understanding of the mechanisms governing lysosomal biogenesis and of the molecular pathogenesis of numerous human diseases. Some of the regulatory mechanisms we uncover may serve in the future as targets for modulating lysosomal biogenesis in diseases resulting from impaired lysosomal function or in conditions, such as certain types of cancer, that are characterized by aberrant or unrestricted lysosomal activation.