Biology Reference
In-Depth Information
c.
Analyze a small proportion of the fractions contained within this conductivity
range by 8% SDS-PAGE to determine the purity and yield of the product.
11.1.7.5
Size exclusion chromatography
A final purification step is done as described in
Section 1.5.4
, except that the optimal
gel filtration column for TBCD is Superose 6 rather than Superdex 200. TBCD
emerges from this column as a symmetrical peak with an apparent molecular mass
of about 120 kDa. This material can be concentrated using a Millipore Centricon 30
concentration device, flash frozen in small aliquots and stored at
70
C.
11.2
DISCUSSION
The ultimate test of the quality of the tubulin-specific chaperones prepared by the
methods described here is their performance in
in vitro
folding reactions. In these
reactions, highly labeled unfolded
-tubulins made by expression in
E. coli
are presented by sudden dilution from denaturant into an aqueous reaction containing
CCT, ATP, GTP, and one or more of the tubulin-specific chaperones prepared as
described earlier. Detailed protocols describing the preparation of labeled tubulin
probes, the purification of CCT, and the assembly of the folding reaction are de-
scribed in
Cowan (1998)
. Alternatively, in the case of TBCD and TBCE, the activity
of the purified proteins may be tested via their ability to disrupt the native tubulin
heterodimer in the back-reaction. In either case, the reaction products are analyzed
by electrophoresis on nondenaturing (ND) polyacrylamide gels, a procedure de-
scribed in detail by
Fanarraga, Carranza, et al. (2010)
. An example of the complexes
formed by TBCA, TBCC, TBCD, and TBCE either alone or in combination and their
migration properties is shown in
Fig. 11.3
. Such analyses (among others) were es-
sential for the elucidation of the overall tubulin heterodimer assembly pathway
shown in
Fig. 11.1
, but they have also proved useful in the analysis of spontaneously
occurring human mutations in
TUBA1A
and
TUBB2B
. These mutations result in dev-
astating defects in the neuronal migration events that accompany normal brain de-
velopment during late embryogenesis. For example, some of the disease-causing
mutations result in defective interactions of CCT-generated folding intermediates
with either TBCB (in the case of
TUBA1A
)(
Tian et al., 2008
) or TBCA (in the case
of
TUBB2B
)(
Jaglin et al., 2009
). Mutations in the human gene encoding TBCE are
also known to cause the rare inherited disease HRD (hypoparathyroidism, mental
retardation, and facial dysmorphism) (
Parvari et al., 2002; Tian et al., 2006
).
It is likely that the tubulin-specific chaperone-dependent reactions that partici-
pate in the
de novo
assembly of the
a
-or
b
-tubulin heterodimer occur
in vivo
in free
solution in a manner that does not depend on microtubules or microtubule dynamics.
This is because the heterodimer is assembled upon transcription/translation of
a
/
b
-or
b
-tubulin in rabbit reticulocyte lysate, a cell-free cocktail in which the endogenous het-
erodimer concentration (about 0.1 mg/ml) is well below the critical concentration (
C
C
)
required for polymerization into microtubules. However,
a
there is accumulating
