Si trova su / Altri legami
© 2021 American Chemical Society.Precise spatiotemporal regulation of protein complex assembly is essential for cells to achieve a meaningful rely of information flow via intracellular signaling networks in response to extracellular cues, whose disruption would lead to disease. Although various attempts have been made for spatial and/or temporal analysis of protein complexes, it is still a challenge to track cell–wide dynamics of a particular protein complex under physiological conditions. Here we describe a workflow that combines endogenous expression of tagged proteins, organelle marker distribution–directed subcellular fractionation, scaffold protein–mediated receptor complex purification, and targeted proteomics for spatiotemporal quantification of protein complexes in whole cell scale. We applied our method to investigate the assembly kinetics of EGF–dependent ErbB receptor complexes. After fractionation using the density gradient centrifugation and organelle assignment based on organelle markers, endogenous ErbB complex in different subcellular fractionation was efficiently enriched. By using targeted mass spectrometry, ErbB complex components that expressed medium to low level was precisely quantified with in–depth coverage, simultaneously in time and subcellular spaces. Our results revealed a sophisticated scheme of complex behaviors characterized by multiple subcomplexes with distinct molecular composition formed across subcellular fractions enriched with cytosol, plasma membrane, endosome, or mitochondria, implying organelle–specific ErbB functions. Remarkably, our results demonstrated for the first time that activated ErbB receptors might increase their signaling range through promoting a cytosolic, receptor–free subcomplex, consisting of Shc1, Grb2, Arhgef5, Garem1, and Lrrk1. These findings emphasize the potential of our strategy as a powerful tool to study spatiotemporal dynamics of protein complexes.
