From the Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom
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ABSTRACT |
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The molecular chaperone activities of the only
known chaperonin in the eukaryotic cytosol (cytosolic chaperonin
containing T-complex polypeptide 1 (CCT)) appear to be relatively
specialized; the main folding substrates in vivo and
in vitro are identified as tubulins and actins. CCT is
unique among chaperonins in the complexity of its hetero-oligomeric
structure, containing eight different, although related, gene products.
In addition to their known ability to bind to and promote correct
folding of newly synthesized and denatured tubulins, we show here that
CCT subunits Molecular chaperones are a diverse group of proteins that assist
the correct folding and intracellular targeting of newly synthesized
polypeptides (1) and can modulate the oligomerization and
polymerization of folded native proteins (e.g. Ref. 2). The
chaperonins are a family of molecular chaperones that are characterized
by their oligomeric structure, namely a double torus of ~60 kDa
subunits (3) enclosing a central cavity within which the folding
substrate may be sequestered (4-6). Chaperonin-assisted protein
folding proceeds by ATP-driven, alternating cycles of substrate binding
and release, ultimately resulting in a native, or near-native, protein
that is no longer recognized by the chaperonin (7, 8). The cytosolic
chaperonin containing T-complex polypeptide 1 (CCT)1 is the only known
chaperonin in the cytosol of eukaryotes (9, 10). The eight-membered
rings of the CCT double torus consist usually of eight distinct but
related (~30% identity) gene products, CCT In view of the close functional relationship between CCT subunits and
the synthesis and assembly of tubulins, it seemed appropriate to
determine whether CCT subunits could be detected as
microtubule-associated proteins (MAPs). MAPs are defined operationally
as proteins that copurify with tubulins to a constant stoichiometry
during microtubule assembly (18, 19). Several MAPs appear to fulfill
structural roles in microtubule function, e.g. by modulating
the stability of microtubules and by forming side-arm structures
important for maintaining the integrity of the cytoskeleton
(e.g. Refs. 20-22). Such structural MAPs commonly promote
the in vitro assembly of tubulin into microtubules. Other
MAPs are more transiently associated with the microtubule, such as the
ATP-driven motor molecules kinesin and cytoplasmic dynein, which are
required for the microtubule-based motility so critical for the
functioning and proliferation of eukaryotic cells (23, 24). The
association of these latter MAPs with microtubules is often detected
only in vitro when microtubules are polymerized in the
presence of a non-hydrolyzable ATP analog such as AMPPNP (25). Indeed,
a number of stringent experimental criteria have been developed over
the years by which a protein may be defined as a MAP. Two of these
criteria, copolymerization through temperature-dependent
cycles of microtubule assembly and disassembly (18, 19) and
copurification with Taxol-assembled microtubule protein prepared and
extracted under carefully defined centrifugation conditions (26), have
been used in this investigation of the association of CCT subunits with microtubules.
Initially, we detected CCT subunits in standard mammalian brain MAP
preparations and have since examined in more detail the CCT content of
MAPs in the P19 cell line. This pluripotent mouse embryonal carcinoma
cell line has the ability to differentiate into a variety of phenotypes
(27). Of particular interest to us, in view of the presence of CCT
subunits in standard brain MAP preparations, is the neuronal
differentiation pathway that is induced by aggregation in the presence
of submicromolar levels of retinoic acid. The use of this cell line, in
conjunction with the plant alkaloid Taxol (28) to induce microtubule
assembly, permitted a much greater flexibility in experimental
conditions, including microtubule polymerization in the absence of
added nucleotide. We report here that CCT subunits do indeed copurify
with microtubules. The subunit proportions of the
microtubule-associated CCT subunits differ from those in the parental
tissue/cell lysate and from those in the bulk 20 S chaperonin particles
purified therefrom. These microtubule-associated CCT subunits are not
assembled in a chaperonin-sized particle. We discuss the possible role
free CCT subunits may play in the molecular chaperone activities of the
eukaryotic cytosolic chaperonin and identify a conserved sequence in
CCT subunits that may contribute to their binding to microtubules.
Cells--
The P19 mouse embryonal carcinoma cell line was
obtained from Prof. Peter Andrews (University of Sheffield, Sheffield,
United Kingdom) and maintained at 37 °C in 5% CO2 in
Dulbecco's modified Eagle's medium (Life Technologies, Inc., Paisley,
UK) supplemented with 2 mM glutamine, 100 IU/ml penicillin,
0.1 mg/ml streptomycin, and 10% (v/v) fetal bovine serum
(heat-inactivated; Sigma, Poole, UK). Cells were radiolabeled overnight
(17 h) by replacing normal maintenance medium with 5 ml of medium
containing only a 5% normal methionine and cysteine content and
supplemented with 100 µCi of [35S]methionine/cysteine
(Tran35S-label, ICN). Retinoic acid-induced differentiation
to the neuronal phenotype (P19N) was as reported elsewhere (29), and
neurons were harvested 6 days post-plating following aggregation in
retinoic acid. P19EC and P19N cells were washed with phosphate-buffered saline and scraped into and washed in ice-cold extraction buffer (0.09 M PIPES-NaOH, pH 6.9, containing 2 mM EGTA and
1 mM MgCl2 (90% PEM buffer)) containing the
protease inhibitors leupeptin (5 µg/ml), pepstatin (1 µg/ml), and
phenylmethylsulfonyl fluoride (0.2 mM). The washed cell
pellets were resuspended in 2-3 volumes of extraction buffer and
homogenized by 20 passes in a glass-glass homogenizer. Homogenates were
then centrifuged at 100,000 × g for 90 min at 4 °
in a Beckman TL100 benchtop ultracentrifuge. The resulting supernatants
are referred to as the cell extracts.
20 S CCT Particle Purification--
Chaperonin particles were
purified as described (17) by fractionation of a sucrose gradient
(10-40%) resolution of P19 cell and rat testis extracts. Sucrose
gradient fractions containing 20 S particles were then concentrated and
separated from proteasomes by anion-exchange chromatography over
Resource Q (Amersham Pharmacia Biotech), eluting bound CCT with 300 mM NaCl in column buffer (17).
MAP Purification from Taxol-induced Microtubules Assembled from
Cell Extracts--
1-ml aliquots of cell extract (containing 5-6
mg/ml protein) were made 20 µM Taxol (paclitaxel,
Sigma).Where indicated, nucleotides to a final concentration of 1 mM or phosphocellulose-purified rat brain tubulin (see
below) to a final concentration of 0.7 mg/ml was also added. Each
polymerization mixture was underlaid with a 500-µl cushion of 10%
(w/v) sucrose in extraction buffer containing protease inhibitors, 20 µM Taxol, and, where appropriate, 1 mM
nucleotides. After incubation at 35 °C for 15 min, microtubules (verified by electron microscopy of negatively stained samples) were
pelleted by centrifugation at 30,000 × g for 30 min at
25 °C in a Beckman TL100 ultracentrifuge. Supernatants were
carefully removed, and pellets were washed with warm assembly buffer
(extraction buffer containing 20 µM Taxol) before
resuspension in 200 µl of warm assembly buffer. MAPs were dissociated
from these Taxol-stabilized microtubules either by addition of 5 mM MgATP and incubation at 35 °C for 10 min or by
addition of 20 µl of 4 M NaCl in assembly buffer.
Microtubules were sedimented at 30,000 × g for 30 min at 25 °C, and the resulting supernatants were retained as MAPs.
Tubulin and MAP Purification from Thrice-cycled Rat Brain
Microtubules--
Rat brains were homogenized in an equal volume of
ice-cold 0.1 M PIPES-NaOH, pH 6.9, containing 2 mM EGTA, 1 mM MgCl2, and 1 mM GTP and centrifuged at 130,000 × g for
1 h at 4 °C. The resulting extract was mixed with an equal
volume of the same buffer containing 8 M glycerol and
warmed at 35 °C for 30 min. Microtubules were harvested at
130,000 × g for 1 h at 25 °C. Microtubules
were then taken through two more cycles of this glycerol-aided,
temperature-dependent assembly/disassembly (30) before
being resolved into tubulin and MAPs by chromatography over
phosphocellulose (31). Limited proteolysis of phosphocellulose-purified
tubulin with subtilisin (protease type XXIV, Sigma ) was as described
by Mejillano and Himes (32). Copolymerization of purified CCT with
phosphocellulose-purified tubulin or with subtilisin-digested tubulin,
in the presence of 20 µM Taxol and 0.1 mM
GTP, was at 35 °C in 90% PEM buffer for 15 min. Polymerization
mixtures were then underlaid with 500 µl of 10% sucrose in the
appropriate buffer and centrifuged at 30,000 × g for
30 min at 25 °C. After removal of the supernatant and careful
washing, the pellet was resuspended for further analysis. CCT binding
to dimeric tubulin was assessed by gel filtration on a Superose 6 sizing column (Amersham Pharmacia Biotech) equilibrated with 90% PEM
buffer containing 0.1 mM GTP.
Antibodies--
Rabbit polyclonal antibodies were raised against
keyhole limpet hemocyanin conjugates (33) of peptide sequences taken
from COOH-terminal sequences of murine CCT subunits (11) and from a
sequence near the COOH terminus of the constitutive form of hsp70
(hsc70) (34). Sera were affinity-purified over the appropriate immobilized peptide (17).
Other Procedures--
Specimens for electron microscopy were
applied to Formvar- and carbon-coated grids and were negatively stained
with 4% (w/v) aqueous uranyl acetate prior to examination in a Philips
Model 410 electron microscope operating at 80 kV. SDS-PAGE was carried out according to Laemmli (35), and immunoblot analysis was as described
(17). Protein concentrations were measured by the method of Bradford
(36) with bovine serum albumin as the standard.
CCT Subunits Are Present in Rat Brain MAP
Preparations--
Microtubules purified by three cycles of
temperature-dependent assembly/disassembly were largely
composed of MAPs from P19 Cells Contain CCT Subunits--
Brain tissue is
peculiarly amenable to purification of microtubule proteins by
self-assembly because of its high concentrations of tubulin and
microtubule-stabilizing MAPs such as MAP2 and tau. The conditions
required for purification of microtubule proteins by self-assembly are,
however, very restrictive and can seldom be applied without
modifications to other tissues or to cultured cells. Because we wished
to determine the association of CCT subunits with microtubules under a
wider variety of conditions, we examined MAPs purified with the aid of
Taxol from the P19 mouse embryonal carcinoma cell line. Taxol is a
plant alkaloid that stabilizes microtubules by binding to
Although P19N cell extracts were able to support self-assembly of
microtubules in the presence of GTP, presumably due to the induction in
these cells of microtubule assembly-promoting MAPs such as MAP2 and
tau, Taxol was required for microtubule formation in embryonal
carcinoma cell extracts (Fig. 2,
a and b, lanes 2 and
7). Since it is generally held that a single round of
Taxol-induced polymerization using defined centrifugation conditions
yields microtubules at least as pure as those obtained after two or
three cycles of the temperature-dependent
assembly/disassembly procedure (26), we examined microtubules isolated
from P19 cells by a single Taxol-induced assembly. This premise was
confirmed by comparing MAPs prepared by the Taxol procedure from rat
brain extract (Fig. 2a, Br lane) with MAPs
isolated from thrice-cycled brain microtubules (Fig. 1a,
lane 4), which contained a similar array of
polypeptides. MAPs were readily liberated from these Taxol-stabilized
microtubules by exposure to mild (0.36 M) salt treatment.
Proteins displaced from Taxol-purified P19EC and P19N microtubules are
shown in Fig. 2a (lanes 3 and
8, respectively). There were marked differences in the MAP
profiles from P19EC and P19N cells, and the compositions of both these
MAP preparations were very different from the parental cell lysate
profiles (Fig. 2a, lanes 1 and
6, respectively). Both these observations point to the
selective nature of this MAP isolation procedure. Immunoblot analyses
of these MAP preparations demonstrated the presence of CCT subunits
(Fig. 2, c-i, lanes 3 and
8), although, as with brain MAP preparations, the relative
contents of the CTT
The above P19 cell MAP preparations were from microtubules prepared in
the absence of added nucleotides. Since all CCT subunits are
ATP-binding proteins, it was considered of interest to determine whether nucleotides modulated CCT subunit-microtubule associations. Polymerization in the presence of the non-hydrolyzable ATP analog AMPPNP increased the amounts of CCT subunits associated with P19EC and
P19N microtubules (Fig. 2, c-i, lanes
4 and 9), even though the amounts of tubulin
polymerized were somewhat lower under these conditions (Fig.
2b). It should also be noted that polymerization in the
presence of AMPPNP led to marked changes in the MAP profiles (Fig.
2a, lanes 4 and 9), with
the prominent appearance of kinesin heavy chain (asterisks,
identified only on the basis of size and properties) and possibly
cytoplasmic dynein (arrows). Additionally along with
kinesin, CCT subunits were displaced from the Taxol-stabilized P19
microtubules by exposure to MgATP alone (Fig. 2, c-i,
lanes 4 and 9); subsequent exposure to
mild salt MAP-eluting treatment displaced additional MAP polypeptides
(Fig. 2a, lanes 5 and 10), al though CCT subunits were not among these (c-i,
lanes 5 and 10). The amounts of CCT
subunits associating with microtubules could also be increased by
introducing exogenous phosphocellulose-purified brain tubulin into the
P19 extract polymerization mixtures (data not shown). Hsc70 was also
detected in P19 cell MAP preparations (Fig. 2j). However,
hcs70 association with tubulin was not increased by the presence of
AMPPNP. Furthermore, hcs70 was not so readily displaced from
microtubules by ATP as were CCT subunits (Fig. 2, c-j,
compare lanes 4 and 5 and
lanes 9 and 10). This latter observation is similar to the partial release of hsp70 from flagellar axonemes by ATP reported by Bloch and Johnson (40).
CCT Subunits in MAP Preparations Are Not Assembled in
Chaperonin-sized Oligomers--
The levels of CCT subunits associated
with polymerizing microtubules were non-stoichiometric compared with
those in either cell extracts or purified P19 20 S CCT chaperonin
particles (see below). This raised the question of whether the CCT
subunits in MAP preparations were in the form of a chaperonin particle
of unusual subunit composition or free subunits or smaller oligomers. Electron microscopic examination of negatively stained P19EC cell MAP
preparations (Fig. 3b) failed
to identify any characteristic ring structures so easily discerned in
partially purified (~20 S sucrose gradient fractions) P19EC
chaperonin particles (Fig. 3a), although many unidentified
protein oligomers/aggregates were present in the MAP preparation.
Electron microscopy of phosphocellulose-purified rat brain MAPs (Fig.
3c) revealed a similar mixture of unidentified protein
aggregates, but again, no chaperonin-sized rings. Chaperonin particles
were rarely detected (Fig. 3d, arrows) in
micrographs of cell extracts containing Taxol-induced microtubules
(i.e. in microtubule polymerization mixtures), perhaps not
surprisingly in view of the low abundance of CCT (15), but also showing
that 20 S CCT particles were not particularly concentrated in the
vicinity of microtubules under these circumstances. To confirm that
chaperonin particles would be detectable in the presence of
microtubules if present in abundance, sucrose gradient-purified P19EC
chaperonin was added to salt-extracted, Taxol-purified P19EC
microtubules (i.e. tubulin-only microtubules) (Fig.
3e). Chaperonin rings were readily observed in such a
mixture (Fig. 3f).
The absence of normal CCT chaperonin particles in MAP preparations was
confirmed by sucrose gradient analysis. The 20 S chaperonin particle
normally fractionates at 20-22% sucrose on our 10-40% (w/v) sucrose
gradient resolutions (17, 41). Exposure of P19EC cell extract to the
salt concentrations used to displace CCTs from Taxol-stabilized
microtubules (0.36 M) did not cause any change in this
fractionation position (Fig.
4a, arrow).
Immunoblot analyses of equivalent sucrose gradient fractionation of MAP
preparations clearly showed the great majority of CCT subunits in MAPs
migrating at the top of the gradient (Fig. 4, b-d), as did
the majority of MAP polypeptides (Fig. 4e). Small amounts of
the CCT Subunits of Purified CCT Cosediment with Microtubules Polymerized
from Pure Tubulin--
The presence of CCT subunits in MAP
preparations could be due to direct interaction with tubulin or, more
indirectly, to interaction with other MAPs, including folding cofactors
(43, 44). To distinguish between these two possibilities, sedimentation
analysis of polymerization mixtures, using rat brain
phosphocellulose-purified tubulin with purified P19EC and rat testis
CCTs, was carried out. The components of the polymerized material from
such incubations are shown in Fig.
5a. Under the centrifugation
conditions employed, P19 20 S CCT particles did not sediment (Fig.
5a, lane 2). The presence of CCT in
the polymerization mixture appeared to make little or no difference to
the yield of pelleted microtubules (Fig. 5a, compare
lanes 3 and 4). Silver staining (Fig.
5a) and immunoblot analysis (Fig. 5, b-d)
revealed selected CCT subunits cosedimenting with the microtubules
formed. However, CCT
The selective association of CCT subunits with tubulin is dependent on
polymerization. In gel filtration analysis of CCT/dimeric tubulin
mixtures with tubulin levels below the critical concentration for
microtubule assembly, only CCT
In addition to some CCT subunits, several other polypeptides present in
small amounts in purified P19EC CCT chaperonin were concentrated into
the assembled microtubule pellets (Fig. 5a, lane
4, arrows). The identities of these polypeptides
at 183, 167, 154, 102, 92, and 40 kDa and the doublet centered at 72 kDa are not known. Neither band in the 72-kDa doublet was recognized by
our rabbit anti-hsc70 polyclonal antibody. A similar range of
polypeptides, also present in small amounts in the CCT preparation but
strikingly concentrated into assembled microtubules, was detected in
CCT purified from rat testis (data not shown).
The binding of MAPs such as MAP2, tau, and cytoplasmic dynein to
tubulin involves the highly acidic COOH termini of tubulins. Removal of
this domain by limited subtilisin digestion abolishes binding of MAP2
and cytoplasmic dynein without diminishing the ability of the residual
tubulin to polymerize (e.g. Ref. 32). When
subtilisin-digested tubulin was polymerized in the presence of CCT, the
subunits associated with the microtubules formed (Fig. 6). However, the selectivity in the
associating CCT subunits, which we had found with whole tubulin, was no
longer operative. The CCT In pilot studies to confirm and extend the findings of Brown
et al. (16), we observed, by immunofluorescence microscopy, both CCT The presence of partially denatured tubulin molecules in standard
microtubule preparations might be expected to attract the attentions of
molecular chaperones, particularly the CCT chaperonin, and this could
explain their behavior as MAPs. However, the tubulin/actin folding
activity of CCT is understood to require the 20 S chaperonin particle
containing all eight CCT subunits (6, 12, 15, 42). The data in this
present report suggest that removal of the tubulin COOH termini
(residues 439-451 and 434-445 for The dissociation constants for the binding of selected CCT subunits to
polymerized tubulin are <0.1 µM (e.g. Fig.
5f). Since the concentration of CCT in the cytosol is
between 1 and 2 µM (9, 50, 51), there should be
significant CCT subunit binding to microtubules in the cell, even if
only 5% of CCT is in the form of free subunits (42); we have more
recent evidence, however, that in cells, the proportion of CCT in the
form of free subunits is likely to be substantially
>5%.2 The low stoichiometry
of CCT subunit binding to microtubules is not without precedent among
MAPs (for example, the STOP protein (52)). Restriction of binding to a
specific subset of tubulin molecules, e.g. GTP-tubulin or
some post-translationally modified tubulin, could explain the low
stoichiometry observed. At present, we can only speculate on the
functions of these free CCT subunits. The observation that most are
associated only with polymerizing tubulin may indicate a role for these
subunits in facilitating tubulin polymerization in the intracellular
environment, a role already suggested by the requirement for CCT If some CCT subunits can associate with microtubules in a way other
than via the proposed subunit apical domains that recognize unfolded
protein substrates including tubulins (50), what region of the subunits
might bind to microtubules? Using synthetic oligonucleotides devised
for the purpose of probing Tetrahymena genomic libraries for
MAP-encoding sequences, Soares et al. (53) identified the gene encoding the Tetrahymena ortholog of CCT,
,
, and
also associated with in
vitro assembled microtubules, i.e. behaved as
microtubule-associated proteins. This nucleotide-dependent association between microtubules and CCT polypeptides
(Kd ~ 0.1 µM CCT subunit) did not
appear to involve whole oligomeric chaperonin particles, but rather
free CCT subunits. Removal of the tubulin COOH termini by subtilisin
digestion caused all eight CCT subunits to associate with the
microtubule polymer, thus highlighting the non-chaperonin nature of the
selective CCT subunit association with normal microtubules.
INTRODUCTION
Top
Abstract
Introduction
References
, -
, -
, -
,
-
, -
, -
, and -
(11). In yeast, these eight subunits are
encoded by essential genes, and mutations in individual subunits lead
to defects in the functioning of the cytoskeleton, most commonly
manifested as arrest in mitosis (reviewed in Ref. 12). There is both
in vivo (10) and in vitro (13-15) evidence that
major substrates of the cytosolic chaperonin are tubulins and actins.
In addition to assisting folding of newly synthesized tubulins and
actins, the CCT
subunit appears to be a component of the centrosome
and essential for nucleated microtubule assembly from this organelle
(16). That this process can take place in a permeabilized cell system,
in the absence of protein synthesis, suggests that CCT
at least may
also be involved in facilitating the polymerization of fully folded
tubulins; which, if any, other CCT subunits are required remains to be
determined. Indeed, whether CCT subunits always and only exist in cells
as components of a single type of 20 S oligomeric particle or whether
free subunits and assemblies of variable subunit composition have some
functional roles is not yet resolved. We have presented data that
support the latter possibility (17).
MATERIALS AND METHODS
RESULTS
- and
-tubulins and the high molecular mass MAPs
characteristic of brain microtubules (Fig.
1a, lane
2). The MAPs persisting through three microtubule assembly/disassembly cycles were further purified away from tubulins (Fig. 1a, lane 3) by phosphocellulose
chromatography. This MAP preparation (Fig. 1a,
lane 4) was predominantly the high molecular mass
MAP groups MAP1 and MAP2, but also contained small amounts of many
other proteins. The Coomassie Blue-stained SDS-polyacrylamide gel
resolution of these minor components was very different from the
profile of polypeptides present in the original brain extract (Fig.
1a, compare lanes 1 and 4).
Thus, their presence in this highly purified MAP preparation signified
their selective purification along with tubulins during microtubule
assembly/disassembly cycles. Several subunits of the cytoplasmic
chaperonin CCT were detectable by immunoblotting in this MAP
preparation (Fig. 1, b-f, lanes 3). However, when compared
with the CCT subunit profiles of the original brain extract (Fig. 1,
b-f, lanes 1), the CCT
(Fig. 1c) and CCT
(Fig. 1e) subunits were depleted,
whereas CCT
(Fig. 1f, lower band) appeared
relatively enriched in the MAP preparations. This indicated the
selective partitioning of particular CCT subunits as MAPS. No CCT
subunits were detected in phosphocellulose-purified tubulin (Fig. 1,
b-f, lanes 2). The 70-kDa heat shock
cognate protein (hsc70) has long been known as a ubiquitous
cytoskeleton-associated protein also termed
-internexin (37),
and the starting point for its purification from brain is the isolation
of MAPs (38). We confirmed the presence of this polypeptide in our
brain MAP preparation (Fig. 1f, lanes
1 and 3, upper bands). A comparison of
the relative abundance of hsc70 in MAPs and brain extract with that of
CCT subunits (Fig. 1, b-f, compare lanes
1 and 3) indicated that CCT
, -
, -
, and
-
were MAPs, at least to the same extent as hsc70.
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Fig. 1.
CCT subunits are present in MAPs prepared
from thrice-cycled rat brain microtubule protein. Shown are
SDS-PAGE resolutions on 9% acrylamide gels detected by the following.
a, Coomassie Blue staining of 15 µg of rat brain extract
protein (lane 1), 5 µg of thrice-cycled microtubule
protein (lane 2), 5 µg of phosphocellulose-purified
tubulin (lane 3), and 5 µg of MAPs (lane 4).
b-f, immunoblot detection of CCT (b), CCT
(c), CCT
(upper band) and CCT
(d), CCT
(e), and hcs70 (upper
band) and CCT
(f) in 20 µg of rat brain extract
protein (lanes 1), 5 µg of phosphocellulose-purified
tubulin (lanes 2), and 5 µg of MAPs (lanes 3).
Molecular mass markers are 205, 116, 94, 68, 45, and 29 kDa.
-tubulin
in such a way as to span adjacent protofilaments in the tubule wall
(39) and has been widely used to promote microtubule assembly in
non-neuronal tissue and cell extracts. The two major advantages of the
Taxol procedure are that it can be performed with small amounts of
tissue or cells and under a wide variety of buffer conditions,
including the absence of nucleotides. In this study, we examined MAPs
purified both from the rapidly proliferating, undifferentiated P19EC
cells and from post-mitotic neuronal cultures (P19N) that were in fact
>90% neurons with the residue <10% fibroblast-like cells. At the
stage of differentiation examined, these neurons expressed many
neuron-specific proteins, including the characteristically neuronal
MAPs, MAP2, and tau, none of which were detectable in the
undifferentiated embryonal carcinoma cells (data not shown).
and CTT
subunits were reduced compared
with the corresponding cell lysates (Fig. 2, c-i,
compare lanes 3 with lanes
1 and lanes 8 with lanes 6). Once
again, the levels of most CCT subunits in P19 cell MAPs were similar,
relative to cell lysate content, to those of hsc70 (Fig.
2j). As a percentage of their total cell extract content
(P19EC cells), the amounts of CCT subunits copurifying with MAPs varied
from 0.4% for CCT
to 3% for CCT
, with other CCT subunits at
~2%. In the case of P19 neurons, these percentages were
approximately doubled because the cell lysate content of most CCT
subunits in P19 neurons was less than half that in embryonal carcinoma
cells.
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Fig. 2.
P19 cell MAP preparations contain CCT
subunits. Shown are SDS-PAGE resolutions on 9% acrylamide gels of
MAP preparations (a and c-j) and corresponding
tubulin pellets (b) from P19EC cells (lanes
1-5), from P19 neurons (lanes 6-10),
and from rat brain (Br lanes). Lanes 1, 10 µg
of embryonal carcinoma cell extract proteins; lanes 2,
embryonal carcinoma cell MAPs isolated without Taxol; lanes
3, Taxol-polymerized embryonal carcinoma cell MAPs eluted with
0.36 M NaCl; lanes 4, Taxol-polymerized
embryonal carcinoma cell MAPs isolated in the presence of AMPPNP and
then eluted with 5 mM ATP; lanes 5,
Taxol-polymerized embryonal carcinoma cell MAPs isolated in the
presence of AMPPNP and eluted with 0.36 M NaCl subsequent
to ATP in lanes 4; lanes 6, 20 µg of P19N
extract proteins; lanes 7-10, MAPs prepared from P19N cells
under conditions corresponding to lanes 2-5. Gel loadings
in a and c-j (lanes 2-5
and 7-10) represent 10% of the total MAPs, and those in
b represent 1.7% total tubulin, purified under the
specified conditions from 1 ml of cell extract. c-j are
immunoblots corresponding to lanes 1-10 in
a probed for CCT (c), CCT
(d),
CCT
(upper band) and CCT
(e), CCT
(f), CCT
(g), CCT
(h), CCT
(i), and hcs70 (j). Molecular mass markers are
205, 116, 94, 66, and 45 kDa.
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Fig. 3.
Chaperonin rings cannot be detected in MAP
preparations by electron microscopy. Shown are electron
micrographs of a negatively stained 20 S fraction from a sucrose
gradient fractionation of P19EC cell extract (a), MAPs
salt-eluted from P19EC microtubules purified by the Taxol procedure
(b), phosphocellulose-purified rat brain MAPs
(c), Taxol-induced microtubule polymerization in a P19EC
cell extract (arrows indicate chaperonin particles)
(d), salt-extracted microtubules purified from P19EC cells
by the Taxol procedure (e), and P19EC 20 S chaperonin mixed
with salt-extracted P19EC microtubules (f). a-c
were dialyzed into 90% PEM buffer prior to grid preparation.
Bar = 50 nm.
subunit and, on prolonged exposures for ECL detection, very
small amounts of the CCT
and CCT
subunits could be detected in
the 20 S particle position of the gradient. We have noticed a tendency
for free CCT subunits to reassemble into 20 S particles (data not
shown) over a time span similar to the centrifugation time involved in the sucrose gradient resolution, and so these small amounts of ~20 S
particles may have re-formed from free CCT subunits in MAPs during the
course of the experiment. However, it would appear that the bulk, and
possibly all, of the CCT subunits isolated as MAPs were as free
subunits, or "microcomplexes" (42), rather than as chaperonin
particles.
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Fig. 4.
Sucrose gradient resolution of CCT subunits
in MAP preparations. 200-µl samples were fractionated on 12-ml
continuous 10-40% (w/v) sucrose gradients (17) into 1-ml fractions.
Gradient loads were 1.23 mg of P19EC cell extract exposed to 0.36 M NaCl for 30 min at 25 °C and then dialyzed into
sucrose gradient buffer (a) and 120 µg of MAPs salt-eluted
from P19EC microtubules purified by the Taxol procedure and then
dialyzed as described for a (b-e). Proteins in
15-µl (a) and 32-µl (b-e) aliquots of
sucrose gradient fractions were resolved by SDS-PAGE on 9% acrylamide
gels and detected by Coomassie Blue staining (a); by
immunoblotting with anti-CCT antibody (b), anti-CCT
(upper band) and anti-CCT
antibodies (c), and
anti-CCT
antibody (d); and by silver staining
(e). The 20 S position is indicated by the arrow.
Molecular mass markers are 205, 116, 94, 68, 45, and 29 kDa.
did not cosediment with tubulin under these
conditions (Fig. 5c). The cosedimentation of certain CCT
subunits with microtubules was therefore due to their binding to
tubulin rather than to other MAPs. The binding of purified CCT to
polymerized phosphocellulose-purified tubulin was quantified both by
measuring binding of radiolabeled CCT to microtubules and by
quantitative immunoblot detection of particular subunits bound to
microtubules. The resulting Scatchard plots yielded a dissociation
constant (Kd) of 0.145 µM for
radiolabeled CCT (Fig. 5e), presumably an averaged
Kd for the various subunits, and
Kd values for CCT
and CCT
of 0.072 and 0.065 µM, respectively, from immunoblot analysis (e.g. Fig. 5f). The stoichiometries of binding
determined from the Scatchard plots were in the range 1:30-40 CCT
monomer/tubulin dimer.
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Fig. 5.
Selected CCT subunits cosediment with
polymerized pure tubulin. a-d, shown are SDS-PAGE
resolutions on 9% acrylamide gels of sucrose gradient-purified P19EC
CCT (lanes 1) and of material sedimented at 30,000 × g for 30 min at 25 °C following incubation of 200 µg/ml
P19EC CCT alone (lanes 2), 920 µg/ml
phosphocellulose-purified tubulin alone (lanes 3), and 200 µg/ml P19EC CCT plus 920 µg/ml tubulin at 35 °C for 15 min with
20 µM Taxol (lanes 4). Gel loadings were 0.1 µg of CCT (lanes 1) and 1% of the total pelleted material
(lanes 2-4). Proteins were detected by silver staining
(a) and by immunoblotting and probing for CCT
(b), CCT
(upper band) and CCT
(c), and CCT
(d). Molecular mass markers are
205, 116, 94, 68, 45, and 29 kDa. e and f,
Scatchard analyses of CCT-microtubule binding. e,
radiolabeled P19 CCT (11,300 dpm/µg of protein), purified by sucrose
gradient and anion-exchange resolutions, at 8-45 µg/ml was incubated
with 240 µg/ml tubulin and 20 µM Taxol at 35 °C for
15 min. Radioactivity in the washed and resuspended microtubule pellets
(125 µg/ml protein) was determined. f, purified P19 CCT at
13.5-67.5 µg/ml was incubated with 125 µg/ml tubulin and 20 µM Taxol for 15 min at 35 °C. CCT
content in the
washed and resuspended microtubule pellets (24 µg/ml protein) was
measured by immunoblot analysis together with a standard range of CCT
concentrations.
could be detected in the tubulin dimer-containing fractions (data not shown).
and CCT
subunits, which did not
associate with polymerizing whole tubulin, were associated with
subtilisin-digested tubulin polymers to the same extent as the other
CCT subunits. It may be the case that removal of the tubulin
COOH-terminal domains generates a tubulin or microtubule species
recognizable as a folding substrate by the oligomeric assembly of CCT
subunits, i.e. by the CCT chaperonin particle, required for
the correct folding of newly synthesized tubulins. We further note
that, in contrast to the lack of effect of CCT on the yield of whole
tubulin polymer, subtilisin-digested tubulin polymerization was
significantly enhanced by the presence of CCT (Fig. 6a).
This and the loss of selectivity in microtubule-associated CCT subunits
after subtilisin digestion of tubulin highlight the
non-chaperonin-like nature of the selective association of
CCT subunits with polymerizing whole tubulin and also argue against the
observed association merely arising from CCT whole complex binding to
denatured tubulin in the preparation.
View larger version (27K):
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Fig. 6.
All CCT subunits cosediment with polymerized
subtilisin-digested tubulin. Rat testis CCT purified by sucrose
gradient fractionation and anion-exchange chromatography at 150 µg/ml
was incubated at 35 °C for 15 min with 20 µM Taxol
alone (lanes 2), plus phosphocellulose-purified
rat brain tubulin at 600 µg/ml (lanes 3 and
4), and plus subtilisin-digested tubulin at 520 µg/ml
(lanes 5 and 6). Material sedimented
by subsequent centrifugation at 30,000 × g for 30 min
at 25 °C was resolved by SDS-PAGE on 9% acrylamide gels with gel
loadings of 0.1 µg of CCT (lanes 1) and 1.6% of the total
pelleted material (lanes 2-6). Proteins were detected by
Coomassie Blue staining (a); by silver staining (b); and by
immunoblotting and probing for CCT (a), CCT
(d), CCT
(e), and CCT
(f).
DISCUSSION
and CCT
as components enriched at the centrosome (data not shown) and, by immunoblotting, CCT components in brain MAP preparations. This report addresses the basic question of whether or
not CCT subunits are MAPs. Some CCT subunits certainly do fulfill the
generally accepted biochemical criteria for defining proteins as MAPs.
The association of CCT subunits with microtubules is, in several ways,
similar to that between microtubules and the hsc/hsp70 family of
molecular chaperones (37, 38, 45). Neither molecular chaperone (CCT nor
hcs70) is quantitatively removed from cell extracts by assembling
microtubules (Ref. 38 and this report). Immunofluorescence detection of
both chaperones produces diffuse staining in the cytoplasm (9, 16, 17,
40) rather than a microtubular fibrous staining exhibited by antibodies
to some of the structural MAPs (e.g. MAP4 in Ref. 46) or the
punctate, vesicular cytoplasmic staining reported for the motor protein kinesin (e.g. Ref. 47). Neither CCT subunits nor hcs70
stimulates tubulin assembly in vitro (Ref. 38 and this
report); and finally, both chaperones are dissociated from microtubules
by ATP (Refs. 38 and 40 and this report), although we found CCT
subunits to be more readily displaced than hsc70. We therefore conclude that certain CCT subunits behave as MAPs in a similar way to members of
the hsc/hsp70 family that are already classified as MAPs (37, 38,
45).
- and
-tubulins,
respectively) (48) by subtilisin generates a form of tubulin that is
indeed recognized as a folding substrate for the CCT chaperonin
containing all eight subunits. Dobrynski et al. (49) have
similarly reported that in vitro translation of
-tubulin
mRNA lacking 27 residues from the COOH terminus causes arrest of
the resulting polypeptide on the CCT complex. Thus, the presence of
selected CCT subunits in MAP preparations possibly points
to functions of some CCT subunits in association with microtubules other than, or in addition to, those undertaken by these subunits when
they are incorporated into the core 20 S chaperonin particle. Indeed,
we have shown here that the CCT subunits associated with microtubules
appear to be free subunits or microassemblies rather than 20 S
oligomeric CCT, although from their fractionation position in sucrose
gradients, it seems more likely that these microtubule-associated CCT
subunits are actually free subunits rather than the microassemblies described by Liou and Willison (42).
in
centrosome-nucleated microtubule growth (16). Alternatively, the
association of CCT subunits with microtubules may relate to
microtubule-based transport or motility, a function already suggested
for microtubule-associated hsc70 (38).
. They
defined a motif (-(P/A)GGG-) common to the microtubule-binding repeats
of tau and MAP2 and to some CCT subunits. Table
I is an alignment of murine CCT subunit
sequences (11) compared with this conserved motif in MAPs, and it
should be noted that, in the sequences for CCT
and CCT
, the P/A
preceding the triple G is replaced by tyrosine. This nonconservative
substitution could explain the weaker association of these two subunits
with microtubules when compared with the other subunits. Since the
analysis by Kim et al. (50) and our own modeling
studies3 locate this (P/A)GGG
motif near the ATP-binding site hinge region, conformation changes in
the CCT subunits consequent to ATP hydrolysis could well affect
accessibility of this motif for binding to microtubules. However, the
synthetic peptide VPGGGA did not compete with CCT subunits for binding
to microtubules (data not shown), and so if this motif is involved in
CCT subunit association with microtubules, other as yet unidentified
regions of the CCT subunits must also be required for binding to occur.
Alternatively, as we noted with polymerization of pure tubulin in the
presence of purified CCT, certain polypeptides in these CCT
preparations other than CCT subunits were quite strikingly concentrated
into assembling microtubules (Fig. 5). It is quite plausible that some
of these proteins, which may be naturally associated with the CCT
chaperonin, are the actual mediators of interactions between CCT
subunits and microtubules.
MAP sequence motif implicated in microtubule binding is present in
some CCT subunits
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ACKNOWLEDGEMENTS |
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We thank Julie Grantham and Anthony Baines (University of Kent, Canterbury) for stimulating discussions and Jo Roobol for fine secretarial assistance.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-122-776-4000 (ext. 3212); Fax: 44-122-776-3912; E-mail: A.Roobol{at}ukc.ac.uk.
The abbreviations used are: CCT, cytosolic chaperonin containing T-complex polypeptide 1; MAP, microtubule-associated protein; AMPPNP, adenyl-5'-yl imidodiphosphate; P19N, P19 neuron; P19EC, P19 embryonal carcinoma; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); PAGE, polyacrylamide gel electrophoresis.
2 A. Roobol and M. J. Carden, manuscript in preparation.
3 H. C. Whitaker, M. Lobell, and M. J. Carden, unpublished data.
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REFERENCES |
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