Protein Folding in Archaea

Chaperonins are a specific class of barrel-shaped chaperones,
present in almost all organisms. Newly synthesized proteins
encapsulated by the chaperonin can attain their native structure
unimpaired by aggregation during repeated cycles of ATP-dependent
binding and release. Chaperonins are generally divided into two
groups. Group I chaperonins, such as the barrel-shaped GroEL
oligomer, are found predominantly in bacteria and cooperate with
cofactors of the Hsp10 familly (i.e. GroES). The Group II
chaperonins, on the other hand, do not require a Hsp10- cofactor
and are found in the eukaryotic cytosol and in archaea. The
function of GroEL is understood in great detail and the substrate
interaction proteome has been recently identified. In contrast, our
knowledge about the natural substrates of Group II chaperonins is
deficient and as a consequence, mechanistical studies on Group II
chaperonins have been limited to using the eukaryotic model
substrates actin and tubulin as well as heterologous model
substrates. In the present study, the complete substrate spectrum
of a Group II chaperonin, the thermosome (Ths) of the mesophilic
archaeon Methanosarcina mazei (M. mazei), was analysed for the
first time. In addition, the unique coexistence of both the goup I
and the group II chaperonins in M. mazei, which was confirmed in
the initial part of the study, provided the opportunity to obtain
new insights into how the substrate selection differs between the
two chaperonin groups. For these purposes, the chaperonin
substrates were isolated by immunoprecipitation of the
chaperonin-substrate complexes and identified by liquid
chromatography coupled mass spectrometry (LC-MS) using three
different approaches: LC-MS after separation of the proteins (i) by
classical 2D-PAGE, (ii) by difference gel electrophoresis (Ettan
DIGE) and (iii) by 1D-PAGE. Analysis of substrates of both the
thermosome (MmThs) and GroEL/GroES (MmGroEL, MmGroES) of M. mazei
revealed that each chaperonin handles a defined set of substrates,
and both chaperonins contribute to the folding of ~17% of the
proteins in the archaeal cytosol. Bioinformatic analysis revealed
that the chaperonin specificity is governed by a combination of a
various physical properties (hydrophobicity, net charge and size),
structural features (i.e. the domain fold), and less concrete
characteristics like the evolutionary status and, in this context,
the phylogenetic origin of the substrate.