Protein Synthesis and Degradation
A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of proinflammatory interleukin 12 family cytokines
Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and β subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate β subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone–client complexes for mass spectrometry.Single molecule microscopy reveals diverse actions of substrate sequences that impair ClpX AAA+ ATPase function
AAA+ (ATPases Associated with diverse cellular Activities) proteases unfold substrate proteins by pulling the substrate polypeptide through a narrow pore. To overcome the barrier to unfolding, substrates may require extended association with the ATPase. Failed unfolding attempts can lead to a slip of grip, which may result in substrate dissociation, but how substrate sequence affects slippage is unresolved. Here, we measured single molecule dwell time using total internal reflection fluorescence microscopy, scoring time-dependent dissociation of engaged substrates from bacterial AAA+ ATPase unfoldase/translocase ClpX.
