Systematic Analysis of GARP- and EARP-Mediated Trafficking Pathways Using Lysosomal Proteomics

Eukaryotic cells utilize tethering complexes for sorting of proteins within the endo-lysosomal system. The Golgi-associated retrograde protein (GARP) complex and the endosome-associated recycling protein (EARP) are two tethering complexes involved in endosomal recycling pathways. Both complexes share three common subunits (VPS51, VPS52, and VPS53), with each relying on one specific subunit: VPS54 for GARP and VPS50 for EARP. Mutations in both complexes are linked to various neurodegenerative disorders, such as progressive cerebello-cerebral atrophy. While model cargoes have been described, the cargo spectrum of both complexes remains largely elusive. Here, we combined lysosomal proteomics with knockdowns of GARP and EARP to analyze their cargo spectra. We first generated a deep lysosomal reference proteome. Using the lysosome as a terminal reference point for mis-sorted proteins, we show that GARP depletion causes a loss in lysosomal hydrolases and accumulation of non-lysosomal cargo. EARP depletion results in lysosomal accumulation of secreted proteins. Our findings serve as a reference to study GARP and EARP complexes and provide a comprehensive overview of the lysosomal proteome. We demonstrate that a systematic phenotypic analysis of trafficking mutants is necessary to understand the pathological consequences of their dysfunction