Discovery of AAA+ Protease Substrates through Trapping Approaches.

ABSTRACT

Proteases play essential roles in cellular proteostasis. Mechanisms through which proteases recognize their substrates are often hard to predict and therefore require experimentation. In vivo trapping allows systematic identification of potential substrates of proteases, their adaptors, and chaperones. This combines in vivo genetic modifications of proteolytic systems, stabilized protease-substrate interactions, affinity enrichments of trapped substrates, and mass spectrometry (MS)-based identification. In vitro approaches, in which immobilized protease components are incubated with isolated cellular proteome, complement this in vivo approach. Both approaches can provide information about substrate recognition signals, degrons, and conditional effects. This review summarizes published trapping studies and their biological outcomes, and provides recommendations for substrate trapping of the processive AAA+ Clp, Lon, and FtsH chaperone proteolytic systems.