Biology Reference
In-Depth Information
translation in a eukaryotic cell extract (such as that derived from rabbit reticulocyte
lysate) of the same sequences that yield insoluble material in
E. coli
results in the
generation of soluble tubulin that is functional in terms of its ability to polymerize into
microtubules (
Cleveland, Kirschner, & Cowan, 1978
). This posed the following
paradox: tubulin translated in a prokaryotic cell context does not fold and leads to
the production of inclusion bodies, while translation of the identical sequences in
eukaryotic cells leads to the generation of functional tubulin heterodimers.
The deposition of insoluble
-tubulin in
E. coli
cells has been successfully
exploited in order to develop an
in vitro
folding assay for these proteins (
Cowan,
1998
). The method depends on the ability to label the recombinant protein in the pro-
karyotic hostwithout labeling any host cell proteins. This is done using a vector inwhich
the expression of recombinant sequences is driven by a T7 promotor: in the presence of
35
S-methionine and rifampicin (a drug that inhibits
E. coli
RNA polymerase, but not T7
polymerase), only the recombinant protein is labeled (
Studier, Rosenberg, Dunn, &
Dubendorff, 1990
). The labeled inclusion bodies can be relatively easily purified be-
cause of their extreme insolubility, and the recombinant proteins unfolded in 8 M urea.
This procedure yields probes of sufficiently high purity and specific activity (i.e.,
>
-and
a
b
10
6
cpm/
g) that they can be used in
in vitro
folding assays to identify factors that
are required for productive folding. The products of such
in vitro
folding reactions
can be readily identified by their characteristic mobility on native polyacrylamide gels.
Our development and use of this assay led to the discovery and purification of
the cytosolic chaperonin (
Gao, Thomas, Chow, Lee, & Cowan, 1992
) (termed
CCT, for
C
ytosolic
C
haperonin containing
T
-complex polypeptide 1; also termed
TriC, for
T
-
ri
ng
C
omplex). This is a large, ribosome-sized multimolecular complex
assembled from eight different (though related) polypeptides into a structure that is
readily visible in the electron microscope as a double toroid. CCT polypeptides are
distantly related to those of GroEL, the chaperonin that is present in
E. coli
and that
functions in the facilitated folding of a significant proportion (estimated to be at least
5%) of newly synthesized proteins (
Hartl & Hayer-Hartl, 2002; Lorimer, 1996;
Young, Agashe, Siegers, & Hartl, 2004
). All chaperonins, including CCT, function
by providing a sequestered environment within the toroidal cavity where folding can
take place in the absence of nonproductive interactions with other proteins. Cycles of
ATP hydrolysis and ADP/ATP exchange result in allosteric changes in the chaper-
onin that govern the binding and release of the target protein (
Spiess, Meyer,
Reissmann, & Frydman, 2004; Valpuesta, Martin-Benito, Gomez-Puertas,
Carrascosa, & Willison, 2002
). In the case of
m
-tubulin, interaction with
CCT is an obligatory part of the folding reaction (
Cowan & Lewis, 2001
). Moreover,
while GroEL participates in the facilitated folding of a wide range of proteins in
E. coli
cells, it cannot facilitate the productive folding of actins or tubulins, the two
principle targets of CCT (
Tian, Vainberg, Tap, Lewis, &Cowan, 1995b
). This explains
why it is not possible to produce soluble tubulins via their expression in
E. coli
.
Unlike actin and other obligate targets of CCT, neither
a
- nor
b
-tubulin can par-
tition to the native state as a result ofATP-dependent interactionwithCCT alone (
Gao,
Vainberg, Chow, & Cowan, 1993
). Rather, tubulin subunits released from CCT are
assembled into
a
- and
b
/
heterodimers by interaction with several proteins known as
a
b
