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other, forming a multimolecular complex; TBCC enters this complex, forming a
supercomplex that releases native tubulin heterodimers upon E-site GTP hydrolysis.
The hydrolysis of GTP by
-tubulin in the supercomplexmay be thought of as a switch
for the release of heterodimers because cofactors have amuch lower affinity for GDP-
tubulin than for GTP-tubulin (
Tian et al., 1999
). Importantly, in addition to participat-
ing in the generation of
de novo
tubulin heterodimers, TBCC, TBCD, and TBCE can
act together as a GTPase activator (GAP) for native tubulin. The biological signifi-
cance of this activity has not been clearly established
in vivo
, but it may serve as a
quality-control mechanism that continually checks for the ability of native heterodi-
mers to hydrolyze GTP. It may also contribute toward modulating microtubule dy-
namics by influencing the size of the pool of GTP-bound tubulin (
Tian et al., 1999
).
Not surprisingly, the tubulin-specific chaperone-dependent reaction that
assembles the heterodimer can be driven in reverse: incubation
in vitro
of native het-
erodimers with a molar excess of TBCD or TBCE results in heterodimer disruption
and (at least in the case of TBCD) the formation of the TBCD/
b
b
complex (the TBCE/
a
complex does not appear to exist as a stable entity). These reactions (which we
refer to as the back reaction, shown as purple arrows in
Fig. 11.1
) are also apparent
in vivo
: when cells are transfected with plasmids engineered for the overexpression
of TBCD or TBCE, their microtubule network is destroyed (
Fig. 11.2
). A noteworthy
feature of tubulin destruction by TBCD is that it is modulated by interaction of
TBCD with the small Ras family member GTPase Arl2 (
Bhamidipati et al., 2000
).
This chapter describes methods for the preparation and purification of the
tubulin-specific chaperones. All five of these proteins were originally isolated from
a tissue source (bovine testis) via multiple chromatographic dimensions in which the
protein of interest was assayed via CCT-driven
in vitro
tubulin folding reactions.
These laborious and time-consuming protocols have now been superseded by the
production of the corresponding biochemically active recombinant proteins in a va-
riety of host/vector systems, and it is these methods that are described below. The
sequences of TBCA-E are well conserved among mammals, so the protocols we pro-
vide should be applicable for their purification via expression using cloned cDNAs
from any mammalian species. The methods we describe are for the proteins in unmo-
dified form. This is because the effect on biological activity (if any) of the addition of
a tag to facilitate affinity purification is uncertain. The yield of recombinant protein
in the host systems we have developed is variable (depending on the chaperone in
question), but the chromatographic methods we describe are for the most part
straightforward. The
in vitro
CCT-dependent folding assay used to demonstrate
TBC activity has been described in detail elsewhere (
Cowan, 1998
).
11.1
METHODS
11.1.1
cDNA clones and vectors
Full-length cDNA clones encoding TBCA, TBCB, TBCC, TBCD, and TBCE are
available a number of commercial sources (e.g., Origene Inc., Cambridge Biosci-
ences Inc., Invitrogen Inc., GeneCopoeia Inc.). For expression, the vector of choice
