ABSTRACT
The delivery of newly synthesized soluble lysosomal hydrolases to the endosomal system is essential for lysosome function and cell homeostasis. This process relies on the proper trafficking of the mannose 6-phosphate receptors (MPRs) between the trans-Golgi network (TGN), endosomes and the plasma membrane. Many transmembrane proteins regulating diverse biological processes ranging from virus production to the development of multicellular organisms also use these pathways. To explore how cell signaling modulates MPR trafficking, we used high-throughput RNA interference (RNAi) to target the human kinome and phosphatome. Using high-content image analysis, we identified 127 kinases and phosphatases belonging to different signaling networks that regulate MPR trafficking and/or the dynamic states of the subcellular compartments encountered by the MPRs. Our analysis maps the MPR trafficking pathways based on enzymes regulating phosphatidylinositol phosphate metabolism. Furthermore, it reveals how cell signaling controls the biogenesis of post-Golgi tubular carriers destined to enter the endosomal system through a SRC-dependent pathway regulating ARF1 and RAC1 signaling and myosin II activity.
Subject(s)
Cell Membrane/enzymology , Endosomes/enzymology , High-Throughput Nucleotide Sequencing/methods , RNA Interference , Receptor, IGF Type 2/metabolism , trans-Golgi Network/enzymology , ADP-Ribosylation Factor 1/genetics , ADP-Ribosylation Factor 1/metabolism , Cluster Analysis , Gene Expression Regulation, Enzymologic , Gene Regulatory Networks , HeLa Cells , Humans , Phosphatidylinositol Phosphates/metabolism , Protein Interaction Maps , Protein Transport/genetics , Receptor, IGF Type 2/genetics , Signal Transduction , Transfection , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
We introduce a universal metabolic labeling strategy using elemental heavy 36Sulfur (36S) called 36Sulfur stable isotope labeling of amino acids for quantification (SULAQ). In the proof of principle experiment, Pseudomonas putida KT2440 was grown in defined minimal medium with sodium benzoate or sodium succinate as the sole carbon and 32S- or 36S-sodium sulfate as the sole sulfur sources. Quantification using mass spectrometry resulted in 562 proteins with 1991 unique peptides. SULAQ technology can be a valuable alternative strategy for the quantitative comparisons in MS-based proteomics approaches characterizing bacterial and other biological samples in different growth conditions.
