Proteome-Wide Analyis of Chaperonin-Dependent Protein Folding in Escherichia coli

In Escherichia coli, the cylindrical chaperonin GroEL and its
cofactor GroES promote the folding of a fraction of newly
synthesized polypeptide chains by acting as an Anfinsen cage. GroEL
recognizes substrate proteins with its apical domains of the
tetradecameric structure. Exposed hydrophobic side chains in
non-native proteins interact with GroEL and bound substrates are
subsequently encapsulated under the GroES lid, where they can fold
in a protected environment. Despite the detailed knowledge about
structural and mechanistic features of GroEL and GroES, little is
known about its genuine in vivo substrate proteins. Here, the
nearly complete set of GroEL interacting proteins in vivo was
identified and quantified by an approach using affinity
chromatography for the isolation of GroEL/GroES/substrate complexes
and subsequent analysis by mass spectrometric methods. GroEL
substrate proteins were analyzed with respect to their fold types
and functional classes, revealing a preference for proteins which
fold into the versatile TIM barrel fold to interact with GroEL.
Further in vivo and in vitro experiments with individual proteins
identified as GroEL substrates verified the data obtained by the
proteomic approach and allowed conclusions on the usage of the
other main chaperone system in E. coli: DnaK/DnaJ/GrpE. Taken
together, the results culminated in the classification of GroEL
interacting proteins according to their dependence on chaperones
for folding. Class I proteins are largely independent of chaperones
but their folding yield can be increased by chaperone interaction.
Class II proteins do not refold efficiently in the absence of
chaperones in vitro, but can utilize either the DnaK or the
GroEL/GroES systems for folding. Class III substrates are fully
dependent on GroEL. DnaK can bind class III proteins and thus
prevent their aggregation, but folding is achieved only upon
transfer to GroEL.