Quantitative proteomic analysis of temporal lysosomal proteome and the impact of the KFERQ-like motif and LAMP2A in lysosomal targeting.

ABSTRACT

Autophagic pathways are regulated mechanisms that play important roles in lysosome-mediated cellular degradation. Yet, the contribution of different autophagic pathways in lysosomal targeting, and characterization of the extent and specificity in their degradome remains largely uncharacterized. By undertaking a multiplex quantitative mass spectrometry approach, we have previously analyzed the lysosomal proteome during chaperone-mediated autophagy (CMA)-stimulated conditions in cancer cells. Here, we have extended our multiplex quantitative mass spectrometry and bioinformatics analysis on the proteome from isolated lysosomes to gain a comprehensive view of the temporal enriched lysosomal content upon non-macroautophagy-activated conditions. In parallel, we describe the functional dependency of LAMP2A on, and to what degree the presence of KFERQ-like motifs in proteins influences, their lysosomal targeting. These findings establish a framework for a better understanding of the degradome mediated by autophagic pathways beyond macroautophagy, and present characterization of the impact of LAMP2A in lysosomal targeting in cancer cells.Abbreviations CMA chaperone-mediated autophagy; ER endoplasmic reticulum; EIF4A1 eukaryotic translation initiation factor 4A1; eMI endosomal microautophagy; FC fold change; GO gene ontology; ISR integrated stress response; LAMP2A lysosomal associated membrane protein 2A; MA macroautophagy; MI microautophagy; MS mass spectrometry; PCA principal component analysis; TAX1BP1 Tax1 binding protein 1.