Chaperones, Molecular
497
correctly. Coimmunoprecipitation experi-
ments with antibodies against GroEL have
also shown that a large number of differ-
ent proteins (about 10–15% of all proteins
in
E. coli
) bind to GroEL shortly after their
synthesis is complete. It is thus thought
that GroEL, together with GroES, acts to
promote the correct folding of a subset of
the proteins present in the bacterial cytosol
that would otherwise not fold correctly. Af-
ter heat shock, an increased association
of proteins with GroEL is seen, and this
implies that GroEL also acts to help refold
proteins, which become wholly or partly
denatured by an increase in temperature.
The Hsp60 proteins present in mitochon-
dria and chloroplasts are also involved in
promoting the folding of some of the pro-
teins that have been newly imported into
the organelle after synthesis in the cytosol
as well as some of those that are syn-
thesized within the organelle. It was the
recognition (in the late 1980s) of the unex-
pectedly high sequence homology between
the chloroplast Hsp60 protein (the major
function of which is the folding of the large
subunit of rubisco, encoded by the chloro-
plast genome) and the
E. coli
GroEL protein
(identiFed originally as a protein required
for the folding of a component of bacterio-
phage lambda), which helped to galvanize
research into the role and mechanism of
action of these proteins.
The role of the Group II proteins is
less clear. In archaea, the proteins are
heat shock inducible, but not in eukary-
otes. Archaea encode one, two, or three
proteins in the Group II family, and these
assemble into large complexes of two rings
with eight- or ninefold symmetry. Eukary-
otes encode eight proteins in the Group
II family, which assemble into a double-
ring complex with eight-fold symmetry.
All eight genes are essential in yeast.
The protein complex has been referred
to variously as CCT, TRiC, and TCP-1.
The principal role of this complex in eu-
karyotes appears to be the folding of the
cytoskeletal proteins actin and tubulin, and
genetic evidence shows that when the func-
tion of the CCT complex is compromised
with conditional mutants, cells show dis-
organized actin, defects in morphogenesis
and cell division, and acute sensitivity to
microtubule-depolymerizing compounds.
Although many other proteins are also
substrates for the complex, the complete
range of cellular substrates has yet to
be deFned. Moreover, actin and tubulin
are not found in archaea, and the sub-
strates for the archaeal Hsp60 proteins
are completely unknown. The Group II
Hsp60 proteins do not require a cofactor
homologous to the Hsp10 protein used
by the Group I proteins, but substrates
are delivered to them by a totally unre-
lated chaperone called GimC or prefoldin,
which again is found in both archaea and
eukaryotes but not in bacteria.
4.1.3
Mechanisms of Action of the Hsp60
Family
The mechanism of action of the Group
I proteins, as typiFed by the
E.
coli
GroEL protein, has been very intensively
studied, and yet there remain considerable
uncertainties about exactly how the protein
works.
In vitro
experiments show that
if certain proteins are denatured (by
chemical or heat treatment) and are then
allowed to refold, the yield of refolded
protein is very low, but can be increased to
nearly 100% by incubation with GroEL,
GroES, and ATP (±ig. 2). This process
requires a complex reaction cycle, which is
discussed more fully in the next section.
During the reaction cycle, the unfolded
protein is transiently bound in the cavity
formed on one ring by the capping of
the ring by the GroES protein. There are
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