M. Caplow et J. Shanks, EVIDENCE THAT A SINGLE MONOLAYER TUBULIN-GTP CAP IS BOTH NECESSARY AND SUFFICIENT TO STABILIZE MICROTUBULES, Molecular biology of the cell, 7(4), 1996, pp. 663-675
Evidence that 13 or 14 contiguous tubulin-Gm subunits are sufficient t
o cap and stabilize a microtubule end and that loss of only one of the
se subunits results in the transition to rapid disassembly (catastroph
e) was obtained using the slowly hydrolyzable GTP analogue guanylyl-(a
,b)-methylene-diphosphonate (GMPCPP). The minus end of microtubules as
sembled with GTP was transiently stabilized against dilution-induced d
isassembly by reaction with tubulin-GMPCPP subunits for a time suffici
ent to cap the end with an average of 40 subunits. The minimum size of
a tubulin-GMPCPP cap sufficient to prevent disassembly was estimated
from an observed 25- to 2000-s lifetime of the GMPCPP-stabilized micro
tubules following dilution with buffer and from the time required for
loss of a single tubulin-GMPCPP subunit from the microtubule end (foun
d to be 15 s). Rather than assuming that the 25- to 2000-s dispersion
in cap lifetime results from an unlikely 80-fold range in the number o
f tubulin-GMPCPP subunits added in the 25-s incubation, it is proposed
that this results because the minimum stable cap contains 13 or 14 tu
bulin-GMPCPP subunits. As a consequence, a microtubule capped with 13-
14 tubulin-GMPCPP subunits switches to disassembly after only one diss
ociation event (in about 15 s), whereas the time required for catastro
phe of a microtubule with only six times as many subunits (84 subunits
) corresponds to 71 dissociation events (84-13). The minimum size of a
tubulin-GMPCPP cap sufficient to prevent disassembly was also estimat
ed with microtubules in which a GMPCPP-cap was formed by allowing chan
ce to result in the accumulation of multiple contiguous tubulin-GMPCPP
subunits at the end, during the disassembly of microtubules containin
g both GDP and GMPCPP. Our observation that the disassembly rate was i
nhibited in proportion to the 13-14th power of the fraction of subunit
s containing GMPCPP again suggests that a minimum cap contains 13-14 t
ubulin-GMPCPP subunits. A remeasurement of the rate constant for disso
ciation of a tubulin-GMPCPP subunit from the plus-end of GMPCPP microt
ubules, now found to be 0.118 s(-1), has allowed a better estimate of
the standard free energy for hydrolysis of GMPCPP in a microtubule and
release of Pi: this is +0.7 kcal/mol, rather than -0.9 kcal/mol, as p
reviously reported.