Maintaining homeostasis of metabolites such as amino
acids is critical
for cell survival. Dysfunction of nutrient balance can result in human
diseases such as diabetes. Much remains to be discovered about how
cells transport, store, and utilize amino acids due to limited research
tools. Here we developed a novel, pan-amino acid fluorescent turn-on
sensor, NS560. It detects 18 of the 20 proteogenic amino acids and
can be visualized in mammalian cells. Using NS560, we identified amino
acids pools in lysosomes, late endosomes, and surrounding the rough
endoplasmic reticulum. Interestingly, we observed amino acid accumulation
in large cellular foci after treatment with chloroquine, but not with
other autophagy inhibitors. Using a biotinylated photo-cross-linking
chloroquine analog and chemical proteomics, we identified Cathepsin
L (CTSL) as the chloroquine target leading to the amino acid accumulation
phenotype. This study establishes NS560 as a useful tool to study
amino acid regulation, identifies new mechanisms of action of chloroquine,
and demonstrates the importance of CTSL regulation of lysosomes.