Isotope Tracing of Amino Acids into Cellular Protein Reveals Unexpectedly Efficient Transfer from Lysosomes to Ribosomes

Abstract

KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) tumors are nutrient poor and rely on macropinocytosis of extracellular protein to provide building blocks for future biosynthesis. Despite the importance of protein scavenging as an alternative nutrient acquisition mechanism, little is known about the mechanistic details underlying the pathway. In particular, the roles of lysosomal protein degradation, mTOR signaling, and amino acid sensing are poorly understood. Here, we show that the previous integrated experimental-computational method of amino acid metabolism was biased and poorly estimated the rate of amino acid incorporation into newly synthesized protein. Using a refined protein purification protocol, we were able to more accurately determine the labeled fraction within newly synthesized protein. With the added protocol, the model demonstrated a preferential incorporation of lysosome-derived amino acids into new protein in nutrient deprived conditions. We hypothesize that the transfer of amino acids from the lysosome to the ribosome results from localization of amino acyl tRNA synthetases to the lysosome. Additionally, through this localization, amino acyl tRNA synthetases sense amino acids and enable cells to maintain protein translation to decrease cellular stress within these nutrient-starved conditions. This quantitative investigation sheds light on the possibility of amino acyl tRNA synthetases functioning as amino acid sensors and suggests a complex signaling pathway that recruits the synthetases to the lysosome in pancreatic cancer.