Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling
82 Pages Posted: 12 May 2020 Sneak Peek Status: Under ReviewMore...
All proteins interact with other cellular components to fulfill their function. While tremendous progress has been made in the identification of protein complexes, their assembly and dynamics remain difficult to characterize. Here we present a high-throughput strategy to analyze the native assembly kinetics of protein complexes. We apply our approach to characterize the co-assembly for 320 pairs of nucleoporins (NUPs) constituting the ~ 50 MDa nuclear pore complex (NPC) in yeast. Some NUPs co-assemble fast via rapid exchange whereas others require lengthy maturation steps. This reveals a hierarchical principle of NPC biogenesis where individual subcomplexes form on a minute-time-scale and then co-assemble from center to periphery in a ~1-hour-long maturation process. Intriguingly, the NUP Mlp1 stands out as joining very late and associating preferentially with aged NPCs. Our approach is readily adaptable beyond the NPC, making it possible to analyze intracellular dynamics of a variety of multiprotein assemblies.
Keywords: maturation kinetics, macromolecular complex biogenesis, assembly order, nuclear pore complex, data independent acquisition, DIA mass spectrometry, INST-MFA, RITE, pulse-SILAC AP-MS, metabolic labeling
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