Heterologous expression and folding analysis of a b-tubulin isotype
from the Antarctic ciliate
Euplotes focardii
Sandra Pucciarelli
1,2
, Cristina Miceli
1
and Ronald Melki
2
1
Dipartimento di Biologia Molecolare, Cellulare e Animale, Universita
`
di Camerino, Camerino, Italy;
2
Laboratoire d’Enzymologie
et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France
Mammalian tubulins and actins attain their native con-
formation following interactions with CCT (the cytosolic
chaperonin containing t-complex polypeptide 1). To study
the b-tubulin folding in lower eukaryotes, an isotype of
b-tubulin (b-T1) from the Antarctic ciliate Euplotes
focardii, was expressed in Escherichia coli. Folding analysis
was performed by incubation of the
35
S-labeled, denatured
b-T1 in the presence, or absence, of purified rabbit CCT
and cofactor A, a polypeptide that stabilizes folded
monomeric b-tubulin. We show for the first time in
protozoa that b-tubulin folding is assisted by CCT
and requires cofactor A. In addition, we observed that
E. focardii b-T1 competes with human b5 tubulin isotype

lished results].
The efficient biogenesis of tubulins, as well as of actins,
depends on the eukaryotic chaperonin referred to as CCT
(cytosolic chaperonin containing TCP-1), TCP-1 complex,
TRiC or Ct-cpn60 [7,8]. Similar to its prokaryotic counter-
part GroEL, CCT has a double ring structure. However
CCT has an eightfold symmetry and is composed of seven
to nine distinct polypeptides (designated as a, b, c, etc.) [8]
whereas GroEL has a sevenfold symmetry and is made of a
single polypeptide chain. In addition, and in contrast to
GroEL, CCT binds and folds a small range of misfolded
polypeptides, most of which are components of the
eukaryotic cytoskeleton. Misfolded actin and tubulin
appear to bind to the apical domain of the CCT ring
[9,10] in a nearly native conformation [10,11]. Substrate
binding to CCT is accompanied by a change in the
conformation of CCT [9,10,12]. Nucleotide exchange and
hydrolysis also induce a change in the conformation of
CCT, which increases the affinity for misfolded target
proteins (as reviewed in Melki [13]). Once released from
CCT, a-andb-tubulin chains interact with additional
cofactors (five were discovered in mammals, denoted A–E)
to constitute the native tubulin heterodimer, i.e. the
functional component of microtubules [14–16]. Cofactor
A (Cof A) has been shown to stabilize the b-tubulin
polypeptide chain [15]. Homologues of the mammalian
postchaperonin tubulin folding cofactors have been identi-
fied in two species of yeast, and their function is correlated
with the biogenesis of a-andb-tubulin and the constitution
of functional microtubules [17,18].

three distinct isotypes of b-tubulin have been identified,
named b-T1, b-T3 and b-T4 [15,24,25]. b-T1 appears to be
the most conserved as it shows the highest degree of amino
acid identity (96%) with the Euplotes b-tubulin consensus
sequence, identified by the alignment of tubulin sequences
of non cold-adapted congeneric species available in the
GenBank database. By contrast, b-T3 and b-T4 appeared
quite divergent, as the percentage of identity of both
isotypes compared to the Euplotes consensus sequence is
86% (S. Pucciarelli and C. Miceli, unpublished results.).
Thesynthesisofthreedifferentb-tubulin isotypes may be
an adaptive strategy of E. focardii to generate an hetero-
geneous pool of molecules, each one with peculiar
properties, to allow microtubule polymerization at the
stringent temperature conditions of the Antarctic environ-
ment [25,26, S. Pucciarelli and C. Miceli, unpublished
results]. Whether tubulin folding assisted by CCT plays a
role in microtubular cold-adaptation has not been inves-
tigated so far.
Here we present the first in vitro characterization of the
folding mechanism of E. focardii b-tubulin. In this study we
use the most conserved isotype, b-T1, with the long-term
objective to compare the folding of all the E. focardii
b-tubulin isotypes with those of non cold-adapted organ-
isms. We show for the first time that b-tubulin from lower
eukaryotes needs the assistance of CCT complex to attain its
native tridimensional structure, in a manner similar to
mammalian b-tubulin. Moreover, Cof A is required to
stabilize folded b-T1. These results support the hypothesis
that the CCT-mediated folding evolved early in the

EGTA, 5 m
M
EDTA, 2 m
M
phenyl-
methanesulfonyl fluoride, 2 m
M
o-phenanthroline,
10 mgÆmL
)1
pepstatin A, 5 mgÆmL
)1
leupeptin), and soni-
cated twice for 5 s each (14/30 amplitude microns, Soniprep
150). Each suspension was centrifuged at 14 100 g and the
supernatant used as cytoplasmic extract.
SDS/PAGE was performed according to the method of
Laemmli [26]. After electrophoresis, the gels (10% acryl-
amide) were blotted as described previously [25]. Immuno-
blotting was performed using polyclonal antibodies directed
against the a subunit of hamster CCT (anti-TCP-1a [12]),
peroxidase–conjugated secondary antibodies (Bio-Rad) and
then detected by enhanced chemiluminescence (ECL,
Amersham).
Subcloning of
E. focardii
b-T1 into the expression
vector pET 11a
The complete b-T1 gene sequence is available on Gene-
Bank

agarose were pooled and concentrated by ultrafiltration
(Centricon 30, Amicon Inc., Beverly, MA, USA). Approxi-
mately 250 lL of the concentrated material was applied to a
Superose 6 column (HR 10/30, Pharmacia) equilibrated in
80 m
M
Mes, pH 6.8, 1 m
M
EGTA, 1 m
M
MgCl
2
and 1 m
M
dithiothreitol. Mt-cpn60 and Cof A purifications were
performed as described previously [15,28].
In vitro
folding assays and competition experiments
In vitro folding assays and analysis of the reaction products
on nondenaturing PAGE were performed in folding buffer
(80 m
M
Mes, 1 m
M
EGTA, 1 m
M
MgCl
2
,1m
M

CCT is in the cytoplasm of
E. focardii
The presence of the CCT among the cellular components of
E. focardii was shown by cellular fractionation and subse-
quent immunoblotting analysis using anti-(TCP-1a) poly-
clonal Igs [12]. Two bands with apparent molecular weight
close to that of the rabbit CCT a-subunit were recognized
by the antibodies in the cytosolic fraction of E. focardii
(Fig. 1). One of the polypeptides presumably corresponds
to the a-subunit of E. focardii CCT because it has a
molecular weight identical to that of the a-subunit of rabbit
CCT; the second polypeptide may correspond to another
CCT subunit of E. focardii that contains epitope(s) similar
to that/those of the a-subunit. These results indicate that a
chaperonin containing two vertebrate TCP-1a related
polypeptide chains is present in the cytosol of E. focardii.
Properties of recombinant
E. focardii
b-T1
A number of tubulin polypeptide chains from various
exotic origins [29,30] have been successfully expressed in
E. coli as soluble proteins. In contrast, recombinant tubulin
polypeptide chains from various vertebrates [31–33] form
inclusion bodies within E. coli presumably because they are
misfolded.
To determine whether E. focardii b-T1 is soluble when
expressed in E. coli
35
S-labeled b-T1 was synthesized as
reported by Gao et al. [31] using the construct described in

,1m
M
GTP, 1 m
M
ATP). The
reaction products were analyzed on nondenaturing PAGE
as described [31], immediately after dilution or after 1 h of
incubation at 30 °C. b-T1 behaviour was similar to that of
b5 tubulin [33]. At early times, the bulk of the radioactivity
migrates as a broad band with a slower mobility than folded
b5 tubulin run under identical conditions. After 1 h,
virtually all of the input radioactivity was found at the
origin, i.e. at the top of the gel (data not shown). We
conclude that b-T1 aggregates rapidly in solution under the
conditions used in our folding assays, as does b5 tubulin
[33]. We further conclude that b-T1 folding properties differ
significantly from that of Giardia duodenalis and Reticu-
lomyxa filosa tubulins [29,30] and are instead similar to that
of higher vertebrate b-tubulin.
Folding of
E. focardii
b-T1 requires CCT and Cof A
Up to now, the folding pathway of protozoan tubulins has
never been established. In Tetrahymena, coexpression of
genes encoding CCT subunits and tubulin during cilia
recovery has been demonstrated [34,35]. However, no direct
evidence for the participation of the CCT complex and
Cof A with b-tubulin folding process has been documented.
As a first analysis of the protozoan tubulin folding,
labeled, urea-denatured E. focardii b-T1 was diluted in

massie stained. Molecular size markers are indicated on the right.
Ó FEBS 2002 Folding of b-tubulin from Euplotes by CCT (Eur. J. Biochem. 269) 6273
a-tubulin/b-T1 tubulin heterodimer or to a slower mobility
of the b-T1/Cof A complex, labeled denatured b-T1 was
folded in vitro by purified rabbit CCT in the presence or in
the absence of Cof A, and the reaction products were
analyzed on nondenaturing PAGE. Similar reactions where
human b5 tubulin was substituted to b-T1 were run in
parallel, as a control. The results are presented in Fig. 2B. In
the absence of Cof A, b-T1 binds to CCT (upper arrow).
No folded products are generated as described [14]. In the
folding reaction containing both CCT and Cof A, an
additional band is generated (middle arrow). A similar band
(lower arrow) corresponding to b5/Cof A complex [15] is
generated in the equivalent b5 tubulin control folding
reaction. Therefore, the middle band probably corresponds
to folded b-T1 in complex with Cof A. The slightly lower
mobility of the b-T1/Cof A complex as compared to that of
the b5/Cof A complex is probably due to the difference in
the isoelectric point of b-T1 and human b5, calculated from
their respective primary sequences (4.55 and 3.91, respect-
ively, determined using the
WINPEP
software [39]). Finally, in
a manner similar to that observed for b5 tubulin, no folded
b-T1 tubulin is generated in the folding reaction containing
Cof A but lacking CCT. We conclude from these data that
CCT is required for the folding of b-T1. We further
conclude that folded b-T1isstabilizedbyCofA,asisb5
tubulin.

following its interaction with cpn60, in a manner similar to
mammalian actin and tubulins [40].
b-T1 and human b5 tubulin compete for
the same site on CCT
The results obtained by the folding assay described above
clearly show that b-T1 and CCT form a complex. To
determine whether b-T1 binds to CCT in a manner similar
to b5 tubulin despite the difference in the primary structure,
we performed a competition experiment using rabbit CCT,
labeled b-T1 and unlabeled b5, as described in Melki et al.
[32]. In a control reaction, a competition experiment was
performed using labeled and unlabeled b5. The reaction
products were analyzed on nondenaturing PAGE and
quantified using a phosphorimager. As shown in Fig. 3, the
radioactive signal in the CCT/tubulin complex (arrow)
decreases with increasing amount of unlabeled b-tubulin.
This result suggests that the binding of b5 to CCT particles
hinders that of b-T1 and vice versa (not shown). A
quantitative analysis of the relative yields of the CCT/
b-tubulin complex formed revealed that CCT has a similar
affinity for b-T1 and b5 tubulin, although slightly lower for
b-T1 (compare the intensities at given unlabeled b5 : labeled
b-T1 and unlabeled b5 : labeled b5 ratios). The slight
difference in affinity is more apparent when the amount of
bound labeled tubulin is plotted as a function of the
unlabeled : labeled b-tubulin ratio, assuming that the higher
value obtained is 100% binary complex. The comparison of
the slopes of unlabeled b5 : labeled b-T1 and unlabeled
b5 : labeled b5 competition experiments (Fig. 3b) reveals
that b-T1 binds to CCT with a twofold lower affinity than

of b-tubulin, involved in its binding to CCT [22]. Polypep-
tide P259–T372 is among these regions. It is considered the
most important as it binds to CCT with the strongest
affinity [22]. Based on the tubulin 3D model [45,46], this
polypeptide includes strand S7, that has been demonstrated
to be involved in the stabilization of the native tubulin
monomer by establishing an intramolecular interaction with
the N-terminus, and helix H10, that is implicated in a/b
interdimeric longitudinal contacts (underlined by L, in
Fig. 4) [45]. It also includes amino acid residues involved in
lateral contacts between tubulin heterodimers in microtu-
bule walls (underlined by M, in Fig. 4): the helix H9, the
H10–S9 loop, and the S7–H9 loop, defined as the ÔM loopÕ
[45,46]. Comparison of the primary structure of E. focardii
b-T1 polypeptide P259–T372 with the same region of the
Euplotes consensus sequence, obtained from the alignment
of the known b-tubulin sequences for Euplotes species
(accession numbers P20365 and Q08115, and S. Pucciarelli
and C. Miceli, unpublished results) and three vertebrate
tubulins (including human b5) (Fig. 4), revealed 18
amino acid positions where substitutions occur. The P268I
substitution (shown in bold italics) is unique to E. focardii
and can be correlated with microtubule cold-stability. It
affects a residue in a proline-rich motif. Site-directed
mutagenesis in this tubulin motif results in a weaker affinity
of the mutant tubulin for CCT [43]. Additional substitutions
in b-T1 that are specific to protozoa are indicated by
asterisks in Fig. 4. The substitution A352S is located before
the VCDIP motif (boxed in Fig. 4) that is involved in the
interaction between b-tubulin and CCT [22]. This and other

ulin isotypes were identified in E. focardii. They are denoted
as b-T3 and b-T4. Comparison of the primary structure of
b-tubulin isotypes from E. focardii to those of non-Antarc-
tic organisms, revealed numerous amino acid substitutions
that probably accumulated in order to allow microtubules
polymerization and stability at the low temperature of the
Antarctic sea water ([6], S. Pucciarelli and C. Miceli,
unpublished results). An analysis of the putative role of
these amino acid substitutions in cold adaptation by
mapping them on the 3D structure of tubulin (carried out
in collaboration with E. Nogales, University of California,
Berkeley, USA) will be presented elsewhere. The substitu-
tions located in b-T1, b-T3 and b-T4 regions implicated in
the binding to CCT [22,42,44] (Fig. 4) may have coevolved
with CCT surface areas, allowing the interaction of
Antarctic b-tubulin isotypes with CCT in the adverse
energetic conditions of the Antarctic habitat. The role of
these amino acid substitutions in the folding process of
E. focardii b-tubulin isotypes should therefore be investi-
gated further.
ACKNOWLEDGEMENTS
This work was supported by the Italian ÔProgramma Nazionale di
Ricerca in AntartideÕ,bytheÔCentre National de la Recherche
ScientifiqueÕ and by the ÔAssociation pour la Recherche sur le CancerÕ.
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Ó FEBS 2002 Folding of b-tubulin from Euplotes by CCT (Eur. J. Biochem. 269) 6277