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

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

Autophagy (2021-01-16)
Merve Kacal, Boxi Zhang, Yuqing Hao, Erik Norberg, Helin Vakifahmetoglu-Norberg
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Spautin-1, ≥98% (HPLC)
Sigma-Aldrich
Anti-TAX1BP1 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Chloroquine diphosphate salt, powder or crystals, 98.5-101.0% (EP)
Sigma-Aldrich
Anti-VPS4 antibody produced in rabbit, ~1.0 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Lysosome Isolation Kit, sufficient for 25 g (tissue), sufficient for 20 mL (packed cells), enrichment of lysosomes from tissues and packed cells