Jpn. J. Protozool. Vol. 37, No. 1. (2004)

11

Original
Identification of an XMAP215-related 60 kDa polypeptide as a major
microtubule-associated protein in Tetrahymena pyriformis
Takanori MATUI1,*, Kiyotaka TOKURAKU2, Yuka SHIMAZAKI1 and Susumu
KOTANI1
1

Department of Biochemical Engineering and Science, Faculty of Computer Science and Systems
Engineering, Kyushu Institute of Technology, Fukuoka 820-8502, Japan, 2Department of Chemical
Science and Engineering, Miyakonojo National College of Technology, Miyazaki 885-8567, JapanSUMMARY

INTRODUCTION

We have investigated the microtubuleassociated proteins (MAPs) of Tetrahymena pyriformis. Three polypeptides, with apparent molecular masses of 90 kDa, 60 kDa, and 37 kDa, cosedimented with Taxol-stabilized mammalian
microtubules. Partial amino acid sequencing of
the 60kDa MAP revealed that it was a newly identified Tetrahymena protein. The sequence washomologous to Xenopus XMAP215, suggestingthat the 60kDa MAP was a member of the
XMAP215 family. The purified 60kDa MAP promoted the polymerization of mammalian tubulin in
vitro. The 37kDa MAP was identical to Tetrahymena glyceraldehyde-3-phosphate dehydrogenase.
The 90 kDa polypeptide was not characterized further, because of its low abundance.

The ciliated protozoan Tetrahymena has
elaborate microtubular structures. During amitosis, microtubules assemble inside the macronucleus, and between the macronucleus and the cortex (Fujiu and Numata, 2000), while various intracytoplasmic microtubular structures are observed
during conjugation. Microtubules also associatewith the contractile vacuole pore and the oral apparatus (for review, see Frankel, 2000). Tetrahymena is one of the best experimental systems forstudying the functions of cellular microtubules.
Microtubule dynamics are regulated by various microtubule-associated proteins (MAPs), andthis is probably also true for Tetrahymena. In
many eukaryotic cells, XMAP215, originally identified in amphibian cells, and its related proteinsplay a key role in regulating microtubule dynamics(for review, see Ohkura et al., 2001). In Tetrahymena, microtubule-dependent motor moleculesand molecular chaperones reportedly bind to cortical microtubules (Frankel, 2000), and one of themwas shown to mediate the oral apparatus formation(Frankel et al., 2001), possibly by interacting with*Corresponding author
Tel: + 81 948 29 7840
Fax: + 81 948 29 7801
E-mail: uu0012tm@bse.kyutech.ac.jp
Received: 29 Oct 2003; Accepted: 5 Dec 2003.

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Tetrahymena microtubule-associated proteins

cortical microtubular structures. Yet, most of thefunctions of Tetrahymena cellular microtubules
have remained unclear, in part because the molecules that modulate microtubule dynamics havenot been characterized.
We aimed to clarify the cellular microtubule
functions by investigating how MAPs influence
microtubule dynamics in Tetrahymena. For thispurpose, Tetrahymena MAPs were biochemicallycharacterized in this study. We successfully isolated a novel Tetrahymena MAP with potent
microtubule assembly activity. The relationshipbetween this newly identified MAP and
XMAP215 is discussed.
MATERIALS AND METHODS
Materials
Taxol was generously supplied by Dr. N.
Lomax, of the Natural Products Branch, National
Cancer Institute, Bethesda, MD. Anti-MAP1, antiMAP2, anti-MAP4, and anti-tau antibodies wereprepared against mammalian MAP1, MAP2,
MAP4, and tau as described previously (Kotani etal., 1986). Anti-(AP sequence) antibody was prepared against a bacterially expressed MAP4 fragment as described (Tokuraku et al., 2003). Anti(chick α-tubulin) monoclonal antibody (clone no.
DM1A) was purchased from Sigma-Aldrich Japan
K.K. Anti-(Tetrahymena GAPDH) antiserum was
generously supplied by Dr. A. Soukri (the University of Hassan II, Morocco). All other materials
were of reagent grade.
Protein preparations
Twice-cycled microtubule protein and tubulin were purified from bovine or porcine brain as
described previously (Kotani et al., 1986). The
Tetrahymena pyriformis strain W was cultured according to Watanabe et al. (Watanabe et al., 1994).
To prepare the cell extract, the cells were harvested, washed twice with reassembly buffer (RB)(100 mM 2-(N-morpholino)ethanesulfonic acid
(MES) (pH 6.8), 0.1 mM EGTA, and 0.5 mM
MgCl2), suspended in RB supplemented with 0.5
mM GTP and protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 mM 1chloro-3-tosylamido-7-amino-L-2-heptanone, 0.1
mg/ml leupeptin, and 0.02 mg/ml pepstatin A),
homogenized, and centrifuged at 100,000 × g for
60 min. For the isolation of MAPs, Taxolstabilized mammalian microtubules were added tothe cell extract, and the mixture was centrifuged at
100,000 x g for 60 min at 2°C through a cushion of
10% sucrose in RB. The subsequent proceduresfor the Taxol-dependent preparation of MAPs, andthe heat-treatment of the MAP fraction, were as
described (Kotani et al., 1986).
The 60kDa MAP was purified as follows.
The Tetrahymena extract was heat-treated, applied
to a DEAE-cellulose column equilibrated with abuffer (20 mM MES (pH6.8), 0.1 mM EGTA, and
0.5 mM MgCl2) containing 0.5 mM PMSF, andeluted with a linear gradient of NaCl (from 0 to 0.5
M). The 60kDa MAP-rich fractions were collected, and subjected to the Taxol-dependent MAP
preparation procedure (Kotani et al., 1986). The
residual tubulin in the MAP fraction was removed
by heat-treatment.
The 37kDa MAP (Tetrahymena glyceraldehyde-3-phosphate dehydrogenase (GAPDH))
was prepared from the Tetrahymena extract by themethod of Hafid et al. (Hafid et al., 1998), exceptthat we used a single phosphocellulose column
chromatography step, instead of the series of Blue
Sepharose CL-6B and Polybuffer Exchanger PBE94 column chromatographies.
Assay and other procedures
Microtubule assembly was monitored byeither the change in absorbance at 350 nm or theamount of polymerized tubulin, as described(Tokuraku et al., 2003). Samples for electron microscopy were negatively stained and observed asJpn. J. Protozool. Vol. 37, No. 1. (2004)

13

Fig.1. Isolation and characterization of Tetrahymena
MAPs. A: Taxol-dependent MAPs preparation.
Tetrahymena soluble extract (lane 1) was mixed withtaxol-stabilized mammalian microtubules and centrifuged, and the microtubule pellet (lane 2) was obtained. The pellet was washed with RB to remove
the non-specifically bound proteins, and was separated into the supernatant (lane 3) and the pellet (lane4). The second microtubule pellet (lane 4) waswashed with RB containing 0.35 M NaCl to release
the MAPs, and was separated into the MAPcontaining supernatant (crude MAPs) (lane 5), andthe remaining microtubule pellet (lane 6). The
Taxol-stabilized microtubules were mixed again with the dialyzed crude MAPs fraction (lane 5), collected by centrifugation, and washed with the high salt buffer to release the final MAP fraction (lane 7).
Samples were electrophoresed and stained with Coomassie Brilliant Blue (CBB). The molecular masses(kDa) are indicated on the left side of the figure. Arrows indicate the three MAPs that appeared reproducibly in the final MAP fraction. The arrowhead indicates degraded tubulin (see text). B: Heat stabilityof Tetrahymena MAPs. The crude MAPs fraction (prepared similarly to lane 5 of A) (lane 1) was heattreated, and the heat-stable fraction was electrophoresed (lane 2). The arrow indicates the 60kDa MAP.
C: Immunoreactivity of the 37kDa MAP with anti-(Tetrahymena GAPDH) antiserum. The purified
37kDa MAP was electrophoresed, transferred onto a polyvinylidene fluoride membrane, and stained with
CBB (lane 1), or immunodetected with anti-GAPDH antiserum (lane 2).

described (Tokuraku et al., 2003). Preparation of
the rabbit anti-(60kDa MAP) antiserum and immunoblotting were according to Katsuki et al.
(2000). The protein concentration was determinedby the method of Lowry et al. (1951), using bovine
serum albumin as the standard. Polyacrylamide
gel electrophoresis in the presence of sodium dodecyl sulfate was according to Laemmli (1970).
In-gel digestion of proteins was done by themethod of Cleveland et al. (1977). The amino acidsequences were determined by automated Edman
degradation with a PROCISETM protein sequencer(Applied Biosystems, USA).

RESULTS
Isolation of Tetrahymena MAPs
Although the concentration of the intrinsic
Tetrahymena tubulin was too low for the Taxol-

dependent MAPs preparation procedure (data notshown), we successfully identified the proteins that
coprecipitated with Taxol-stabilized mammalian
microtubules (Fig. 1A, lane 2). Since the crude
MAPs fraction (Fig. 1A, lane 5) still contained
many protein bands, some of which were not reproducible, the fraction was subjected to an additional coprecipitation-desorption cycle. The resultant MAP fraction (Fig. 1A, lane 7) reproducibly
contained three major polypeptides, with apparent
molecular masses of 90 kDa, 60 kDa and 37 kDa(Fig. 1A, arrows). The prominent 50 kDa bandand the 45 kDa band (arrowhead) were tubulin andits degradation product, since they reacted with theanti-(chick α-tubulin) monoclonal antibody (datanot shown). None of the 90 kDa, 60 kDa, and 37
kDa polypeptides were reactive to anti-MAP1,
anti-MAP2, anti-MAP4, anti-tau, or anti-(AP sequence) antibodies (data not shown). Among thedetectable bands in the crude MAP fraction (Fig.

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Tetrahymena microtubule-associated proteins

1A, lane 5), only the 60kDa MAP was heat-stable(Fig. 1B).
Amino acid sequence analysis

Fig. 2. Partial amino acid sequences of the 60kDa
and 37kDa MAPs. The two proteins were proteolyzed, and the N-terminal portions of the two
60kDa MAP-derived peptides and the single
37kDa MAP-derived peptide were sequenced.
Homologous regions of XMAP215 (EMBL accession number AJ251130) and Tetrahymena
GAPDH (Zhao et al., 1997) are aligned. Residue
numbers are indicated in parentheses. Asterisks
indicate identical residues.

Fig.3. Fractionations of the 60kDa MAP. A: Subcellular localization of the 60kDa MAP. Tetrahymena cell homogenate (lane 1) was centrifuged,
and separated into soluble (lane 2) and insoluble(lane 3) fractions. Samples were electrophoresedand either stained with CBB (upper panel) or detected with anti-(60kDa MAP) antiserum (lower
panel). B: Electrophoretic patterns of fractions
obtained during the purification of the 60kDa
MAP. Lane 1, Tetrahymena crude extract; lane 2,
fraction after DEAE-cellulose chromatography;
lane 3, the purified 60kDa MAP. The 60kDa
MAP is indicated by an arrow.

The N-terminal amino acid sequences of theintact 60kDa and 37kDa MAPs could not be analyzed, so their N-termini may have chemical modifications. Consequently, the two MAPs were subjected to in-gel digestions, and the internal sequences were determined (Fig. 2). As shown in
Fig. 2, two sequences of the 60kDa MAP werehighly homologous to Xenopus XMAP215 (EMBL
accession number AJ251130), while the sequenceof the 37kDa MAP was identical to Tetrahymena
GAPDH (Hafid et al., 1998), one of the glycolytic
enzymes. Tetrahymena GAPDH shows an apparent molecular mass of 36 kDa (Hafid et al., 1998),
and the anti-(Tetrahymena GAPDH) antiserum(Hafid et al., 1998) reacted with the 37kDa MAP(Fig. 1C). In addition, mammalian GAPDH reportedly binds to microtubules (for review, see Sirover,1999). Consequently, the 37kDa MAP was considered to be identical to Tetrahymena GAPDH. Tetrahymena proteins homologous to the 60kDa MAP
have not been reported so far. The amino acid sequence of the 90kDa MAP was not determined,
because the content of this protein was too low fora sequence analysis.
Characterizations of the 60kDa MAP and the
37kDa MAP (GAPDH)The 60kDa MAP existed abundantly in thesoluble fraction, rather than the membranous one(Fig. 3A). From the soluble fraction, it was purified to homogeneity (Fig. 3B).
The 60kDa MAP enhanced both the initialrate and the plateau level of bovine tubulin polymerization, in a concentration-dependent manner(Fig. 4A). Electron microscopic observations
showed that the assembled structures were normal
microtubules (Fig. 4B). Sedimentation assays(Tokuraku et al., 2003) also revealed that theJpn. J. Protozool. Vol. 37, No. 1. (2004)

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Fig.4. Effects of the 60kDa, and the 37kDa
MAPs on microtubules. A: Microtubule assembly induced by the 60kDa MAP. Bovine
brain tubulin (1.50 mg/ml) was mixed with
0.00 mg/ml (curve 1), 0.05 mg/ml (curve 2),0.10 mg/ml (curve 3), and 0.20 mg/ml (curve4) of the 60kDa MAP. B: Electron micrographs of microtubules reconstituted from1.50 mg/ml of tubulin and 0.20 mg/ml of the
60kDa MAP. Bars indicate 200 nm. C:
Microtubule bundling induced by the 37kDa
MAP. The sample represented in curve 1
contained the 37kDa MAP alone (0.80 mg/
ml). Bovine brain tubulin (1.50 mg/ml), in
the absence (curves 2 and 3) or presence(curves 4-9) of 30 µM Taxol, was mixed with
0.00 mg/ml (curves 2 and 4), 0.10 mg/ml(curve 5), 0.20 mg/ml (curve 6), 0.40 mg/ml(curve 7), and 0.80 mg/ml (curves 3, 8, and
9) of the 37kDa MAP. In curve 9, 1 mM
ATP was also present. D: Electron micrographs of microtubules and the 37kDa MAPinduced bundles. The conditions for the left,
center, and right panels were the same as
those of curve 4, curve 8, and curve 9 of C,
respectively. Bar indicates 200 nm.

amount of polymerized tubulin and the microtubule-bound 60kDa MAP increased with higher
concentrations of the 60kDa MAP (data notshown).
Mammalian GAPDH bundles microtubules,
rather than assembling them (Sirover, 1999).
Since the homology between mammalian and Tetrahymena GAPDH is low (61%) (Zhao et al.,1997), and the effects of Tetrahymena GAPDH on
microtubules were not tested, we purified the
37kDa MAP and analyzed its activity on microtubule assembly (Fig. 4C, D). The 37kDa MAP
did not promote bovine microtubule assembly inthe absence of Taxol (Fig. 4C, curves 1-3). Whenthe assembly was initiated by Taxol, the turbidityof the microtubules steadily increased, depending

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Tetrahymena microtubule-associated proteinsFig.5. Sequence comparison of the 60kDa MAP and
XMAP215 family members. A: Partial amino acidsequence alignments of the 60kDa MAP, Xenopus
laevis XMAP215 (EMBL accession number
AJ251130), Homo sapiens ch-TOG (EMBL accession number X92474), Drosophila melanogaster
Msps (EMBL accession number AJ249115), Caenorhabditis elegans Zyg-9 (EMBL accession number
AF035197), Arabidopsis thaliana Mor1 (EMBL accession number AF367246), and Dictyostelium discoideum DdCP224 (EMBL accession number
AJ012088). Residues identical to XMAP215 areshadowed. Residue numbers are indicated in parentheses. B: Schematic diagrams. In each of the molecules, the black box and the hatched line box designate the regions homologous to QRIERLEE andAPTKEVEEK of the 60kDa MAP, respectively,
while the horizontal-striped box indicates the microtubule-target motif (Popov et al., 2001), and the graybox shows a region highly conserved among family
members (Popov et al., 2001). The region of
XMAP215 that putatively corresponds to the 60kDa
MAP is underlined .

on the 37kDa MAP concentration (Fig. 4C, curves
4-8). The microtubule-bound 37kDa MAP also
increased in a concentration-dependent manner(data not shown). Many microtubule bundles wereobserved in the presence of the 37kDa MAP (Fig.
4D). The turbidity increase was inhibited by adding ATP (Fig. 4C, curve 9), which was a characteristic similar to the mammalian GAPDH-induced
microtubule bundling (Sirover, 1999).

DISCUSSION
In this study, we identified three MAPs (90kDa, 60 kDa and 37 kDa) in a Tetrahymena extract. Two of them, the 60kDa and 37kDa MAPs,
were further characterized. The 60kDa MAP wasa newly identified Tetrahymena MAP that facilitated tubulin polymerization. The 37kDa MAP
was Tetrahymena GAPDH. Its activities on

microtubules were similar to those of mammalian
GAPDH (Sirover, 1999).
The molecular mass and the heat-stability of
the 60kDa MAP were similar to those of the major
heat-stable mammalian MAP, tau, but no immunocrossreactivity between them was detected (datanot shown). In addition, the partial amino acidsequences of the 60kDa MAP were not homologous to those of tau; instead, they were homologous to those of XMAP215. XMAP215 belongs toa well-known, evolutionarily conserved MAP family, whose members are regulators of microtubule
dynamics (Ohkura et al., 2001). As shown in Fig.
5A, the sequence homologous to QRIERLEE isconserved among the family members, while
APTKEVEEK is conserved only in XMAP215 andch-TOG; the two proteins are homologous along
the entire length (Popov et al., 2001). Judging
from the molecular masses of the sequenced peptides, the 60kDa MAP approximately corresponds

Jpn. J. Protozool. Vol. 37, No. 1. (2004)

to the C-terminal third of XMAP215 (Fig. 5B, underline). The marked difference in the molecularsize is a unique feature of the 60kDa MAP, as amember of the XMAP215 family (Fig. 5B). Since
the N-terminus of the 60kDa MAP is chemicallymodified (see Results), it is not a degradation
product of a larger molecule. Popov et al. (2001)
reported that XMAP215 contains a microtubuletarget motif, on its C-terminal region. The putative 60kDa MAP homologous region on
XMAP215 includes this motif (Fig. 5B, horizontal-striped box).
XMAP215 family members are usually associated with the elongation of centrosome-nucleatedmicrotubules and the formation of the mitotic apparatus (Ohkura et al., 2001). Although Tetrahymena does not contain typical mitotic apparatus
observed in vertebrate cells, functionally similar
structures were reported, such as the perimicronucleus microtubules of Tetrahymena thermophila(Gaertig and Fleury, 1992), and intramacronucleus
microtubules which appear during cell division(Fujiu and Numata, 2000). It is probable that the
60kDa MAP regulates the formation of these microtubular structures during nuclear division. In
addition, Tetrahymena contains elaborate cytoplasmic microtubule networks (see introduction).
Since the 60kDa MAP was mostly recovered fromthe cytoplasmic protein fraction (Fig. 3A), and it
promoted microtubule assembly under centrosome-free conditions (Fig. 4A), the 60kDa MAP
may also regulate the dynamics of cytoplasmic
microtubules.
Full length sequencing of the 60kDa MAP is
now in progress, in order to reveal further similarities and differences between the 60kDa MAP and
XMAP215.
ACKNOWLEDGEMENTS
We wish to thank Dr. O. Numata (University ofTsukuba, Japan) for providing the Tetrahymena,

17

and Dr. A. Soukri (University of Hassan II, Morocco) for the anti-(Tetrahymena GAPDH) antiserum. Thanks are also due to Dr. T. Takeda(Columbia University, USA) for valuable advice,and to Dr. B. Guthrie (SKYBAY Scientific Editing) for editing the manuscript. This work was
supported in part by the Sasakawa Scientific Research Grant from The Japan Science Society.

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