Jpn. J. Protozool. Vol. 36, No. 2. (2003)

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Original
Localization of the 65-kDa Phosphoprotein in the Ciliary Axoneme ofParamecium caudatum

Tokushige OGAWA and Munenori NOGUCHI*
Department of Environmental Biology and Chemistry, Faculty of Science, Toyama University,
Toyama 930-8555, Japan.

SUMMARY
The cAMP-dependent phosphorylation of the29-kDa and 65-kDa axonemal proteins is closely
correlated with the control mechanism of ciliary
beat direction. Localization of the 65-kDa protein
is still unknown whereas the 29-kDa protein has
been identified as a light chain of the outer-arm 22Sdynein. The localization of the 65-kDa phosphoprotein was determined by two-dimensional electrophoresis using a native gel in the first run. In thenative gel, the 65-kDa phosphoprotein co-migrated
with dynein heavy chains. This suggests that the65-kDa phosphoprotein plays an important role incAMP-dependent ciliary control as an intermediatechain of a dynein complex.
INTRODUCTION
The locomotor behavior of Paramecium de-*Corresponding author
Tel: +81-076-445-6675
Fax: +81-76-445-6549
E-mail: noguchi@sci.toyama-u.ac.jp
Received: 2 June 2003; Accepted: 4 Aug 2003.

pends on the ciliary beat direction and beat frequency. The beat direction is controlled by intracellular Ca2+ concentration (Naitoh and Kaneko,1972, 1973; Naitoh and Sugino, 1984) and presumably by cyclic nucleotide concentration(Nakaoka and Ooi, 1985; Majima et al., 1986;
Bonini and Nelson, 1988). Hamasaki et al. (1991)
reported that cAMP-stimulated phosphorylation of
the 29-kDa axonemal protein regulated the velocity of microtubule translocation and the swimming
speed in Paramecium. Barkalow et al. (1994)
reported that the 29-kDa protein functioned as a22S dynein regulatory light chain. They showed a22S dynein that was reconstituted with the phosphorylated 29-kDa protein translocated microtubules significantly faster than controls. We havereported that the cAMP-dependent phosphorylation of the 29-kDa and 65-kDa axonemal proteins
was closely correlated with the control mechanismof ciliary beat direction (Noguchi et al., 2000).
Cilia on the cortical sheets from Tritonglycerol-extracted Paramecium lose their ability tochange their orientation in response to cAMP andto removing Ca2+ by tryptic digestion within 1 min(Noguchi et al., 2000). The brief digestion removes axonemal cAMP-dependent protein kinaseactivity within 10 sec. Following the digestion,

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Axonemal 65-kDa Phosphoprotein

not only the phosphorylated 29-kDa but also the65-kDa axonemal proteins disappeared in 1 min(Noguchi et al., 2000). The disappearance of thekinase activity and phosphorylated proteins accompanied the disability of ciliary responses. The29-kDa phosphoprotein has been thought to be
involved in the ciliary response through controlling the sliding velocity of outer doublet microtubules. The 29-kDa phosphoprotein has beenshown to co-purified with 22S dynein in a sucrose
density gradient centrifugation and to be a lightchain of the outer-arm 22S dynein (Bonini andNelson, 1990; Hamasaki et al., 1991). On theother hand, the 65-kDa phosphoprotein is a strong
candidate that sets the direction of ciliary beatantagonizing the effect of Ca2+ (Noguchi et al.,
2000; 2003). However, the 65-kDa phosphoprotein has not been shown to co-purified with dynein
heavy chains in a sucrose density gradient centrifugation and the localization of the 65-kDa phosphoprotein is still unknown (Bonini and Nelson, 1990;
Hamasaki et al., 1991).
In this study, we tried to clarify the localization of the 65-kDa phosphoprotein in the ciliary
axonemes from Paramecium caudatum. Twodimensional electrophoresis using a native gelelectrophoresis as a first run revealed that the 65kDa phosphoprotein was presumably associatedwith a dynein complex.
MATERIALS AND METHODS
Isolation of axonemes
Paramecium caudatum (stock G3) was cultured
in a hay infusion. Cells were grown to the latelogarithmic phase at 25ºC. Collected cells werewashed three times with a washing solution (2 mM
KCl, 2 mM CaCl2, 0.3 mM phenylmethylsulfonyl
fluoride (PMSF), 10 mM Tris-maleate, pH 7.0).
Cells were deciliated by the dibucaine treatment
following the method of Mogami and Takahashi(1983) with slight modifications. Cilia were iso-lated from cell bodies by centrifugation repeated
twice at 600 g for 5 min. The pellets were discarded.
The supernatant was centrifuged at 7,700 g for 10
min to pellet the cilia. The pellet was resuspended
in TMKE solution (10 mM Tris-maleate, pH 7.0, 5mM MgCl2, 20 mM potassium acetate, 1 mMEGTA) containing 0.3 mM PMSF and centrifuged.
The pellet was rewashed with the TMKE solution.
The isolation of cilia was monitored by dark-fieldmicroscopy. These cilia were then treated with ademembranation solution containing 0.1% Triton
X-100 in TMKE solution for 10 min at 0ºC. The
Triton was removed by washing twice withHKMED solution (30 mM HEPES, pH 7.3, 20 mM
potassium acetate, 5 mM MgSO4, 1 mM EGTA, 1mM dithiothreitol (DTT), 0.3 mM PMSF, 10 µg/ml
leupeptin).
Phosphorylation of the axonemes
In vitro phosphorylation of the axonemes wasperformed following the procedure described in
our previous paper (Noguchi et al., 2000). The
reaction mixture contained 0.15 mg axonemes in
80 µl of TMKE solution as well as 10 µM cAMP.
Phosphorylation by endogenous protein kinases
was started by addition of 20 µl of γ-[32P]ATP toachieve a final concentration of 1 µM ATP. The
ATP concentration of the γ-[32P]ATP was 5 µM,
and radioactivity was adjusted to 10 or 20 µCi withadenosine 5'-γ-[32P]triphosphate (specific activity
6000 Ci/mmol from Du Pont-New England Nuclear). Immediately after 10 min incubation at0ºC, the reaction mixture was centrifuged at
10,000g for 10 min. The pellet of the axonemes
was used for the extraction of crude dynein.
Extraction of crude dynein
Extraction of dynein was carried out by themethod of Larsen et al. (1991) with slight modifications. The pellet of axonemes was resuspendedin HKMED solution containing 0.6 M KCl. Thesuspension was kept on ice for 30 min to extractJpn. J. Protozool. Vol. 36, No. 2.
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the dyneins. Axonemes were separated fromextracted dynein by centrifugation. The high-salt
extract (crude dynein) was divided into two parts.
One part was immediately diluted with a buffer
solution (HEPES, pH 7.3) to achieve a KCl concentration of 20 mM and concentrated using Centricon-10 (low-salt sample). Another part wasconcentrated without diluting the KCl concentration using Centricon-10 (high-salt sample).
Two-dimensional electrophoresis using nativegel and SDS-PAGE
Two-dimensional electrophoresis was performed using agarose polyacrylamide slab gelelectrophoresis (AG-PAGE) as a first run (Takayaet al., 1995). The concentrated crude dynein wasmixed with 1/2 volume of 20% glycerol containing
1% bromophenol blue. The sample was thenapplied on the gel. The first run of a native gelelectrophoresis was performed using the AGPAGE (gel size, 16 × 16 × 0.1 cm). Electrophoresis was run at 5 mA and terminated when bromophenol blue ran to the end of the gel. A gel stripe
was cut out of the gel, incubated in an SDS sample
buffer (2% SDS, 5% 2-mercaptoethanol, 12%glycerol, 62.5 mM Tris-HCl, pH 6.8) for 7 min x2, and then placed on a stacking gel of SDSPAGE. The second run using SDS-PAGE wascarried out in 3-15% linear gradient acrylamide gel
containing a 0-19% glycerol gradient following amodification of the procedure of Laemmli (1970).
Molecular-weight standards were obtained from
Boehringer-Mannheim and Bio-Rad. The gelswere stained with silver (Blum et al., 1987). To
produce the autoradiograms, Fuji medical X-rayfilm RX (Fuji, Kanagawa) with intensifying screen(Kasei Optonix, Tokyo) was placed over the driedgels for 1 to a few days and developed.
RESULTS AND DISCUSSION
The localization of the 65-kDa phosphoprotein

Fig. 1. Two-dimensional electrophoresis of phosphorylated crude dynein (low-salt sample). Extracted
crude dynein from phosphorylated axonemes wasdiluted with a buffer solution (HEPES, pH 7.3) toachieve a KCl concentration of 20 mM and concentrated using Centricon-10 (low-salt sample). Stain.:
Silver stained gel. Autorad.: Corresponding autoradiogram. HC and T indicate the position of heavy
chains and tubulin in the second run. Markers at left
indicate (from top) 200, 116, 97, 66, 45, 31, and 21.5
kDa. Both 29- and 65-kDa protein were electrophoresed in conjunction with heavy chains in the first run.

has not been clarified. The 65-kDa protein isextracted together with the 29-kDa protein(Hamasaki et al., 1989), dyneins, and A-kinases.
However, the 65-kDa protein is not co-purified
with dynein heavy chains (HCs) in sucrose density
gradient centrifugation.
We tried to clarify the localization of 65-kDa

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P.Axonemal
tetraurelia65-kDa
Autogamy-related
Phosphoprotein
GenesFig. 2. Two-dimensional electrophoresis of phosphorylated crude dynein (high-salt sample). In thiscase, the high-salt extract was concentrated without
diluting the KCl concentration. Stain.: Silver stainedgel. Autorad.: Corresponding autoradiogram. HCand T indicate the position of heavy chains and tubulin
in the second run. Markers at left indicate (from top)
200, 116, 97, 66, 45, 31, and 21.5 kDa. Both 29- and65-kDa protein were not electrophoresed in conjunction with heavy chains in the first run in the high-saltcondition.

phosphoprotein by another method. We analyzed
using two-dimensional electrophoresis. In thisstudy, the first run of the two-dimensional electrophoresis was performed with native AG-PAGE gelelectrophoresis (Takaya et al.,1995). Using anative gel in the first run, the conformation ofdynein complexes were expected to keep theirconformation intact. The native gel electrophoreses were performed using both high-salt and low-salt samples. The high-salt extract from axonemesthat had previously been phosphorylated with γ[32P]ATP in the presence of cAMP was immediately diluted to achieve a KCl concentration of 20
mM and concentrated (low-salt sample). In another case, the high-salt extract was concentrated
without diluting the KCl concentration (high-saltsample).
Dynein HCs migrated as relatively compact
bands when the low-salt sample was electrophoresed in a native gel (Fig. 1, STAIN). On the other
hand, the dynein HCs of the high-salt sample
migrated as wider bands (Fig. 2, STAIN). In theautoradiograms from each two-dimensional electrophoresis, the mobility of the labeled phosphoproteins in the first run was different.
Identified phosphorylated bands corresponded
to 29-kDa, 65-kDa, and about 42-kDa proteins in
the SDS-PAGE (indicated by arrows in Fig. 1 and2, AUTORAD). These phosphorylated bands
migrated with dynein HCs in the native gel of thelow-salt sample (Fig. 1). This indicates that 65kDa phosphoprotein co-migrates with dynein
heavy chains as 29-kDa phosphoprotein in thelow-salt condition (Fig. 1). On the other hand, thephosphorylated bands migrated far from dynein
HCs in the case of the high-salt sample (Fig. 2).
These results suggest that the 65-kDa phosphoprotein was released from the dynein complex with along-time exposure to 0.6 M KCl (at least 2 hduring the concentrating procedure). The 65-kDa
phosphoprotein did not co-sedimented with dynein
HCs in sucrose density gradient centrifugation(Bonini and Nelson, 1990; Hamasaki et al., 1991).
This also may be due to a long time exposure to0.6 M KCl before loaded on a sucrose density
gradient and following overnight centrifugation.
The 65-kDa phosphoprotein presumably attach to some dynein complex in vivo and may playa significant role in cAMP-dependent regulationcooperating with 29-kDa light chain. In addition,
42-kDa phosphoprotein comigrated with dynein

Jpn. J. Protozool. Vol. 36, No. 2.
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heavy chains. The 42-kDa protein might be involved in the regulation of ciliary movement.
ACKNOWLEDGMENTS
We thank Professor K. Nakamura of Hiroshima Women's University for providing a protocol of the agarose polyacrylamide gel electrophoresis. We also thank Professor M. Takahashiof the University of Tsukuba for providing P.
caudatum (G3).
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