Jpn. J. Protozool. Vol. 37, No. 2. (2004)

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Original
ND7 gene involved in the membrane fusion in regulated exocytosis isconserved in three species of Paramecium
Terue HARUMOTO1,*, Makiko MIZOGUCHI2 and Mayumi SUGIURA1
1

Graduate School of Human Culture, Nara Women’s University, Kitauoya-nishi-machi,Nara, 630-8506, Japan, 2School of Medicine, Nagasaki University, Nagasaki, 852-8501, JapanSUMMARY

INTRODUCTION

A Paramecium tetraurelia ND7 gene homolog
was cloned and sequenced in P. caudatum. The
predicted protein possessed well-conserved regions, a transmembrane helix and two coiled-coildomains possibly involved in protein-protein interaction, as previously suggested for P. tetraurelia.
Southern hybridization revealed that the ND7 gene
homolog was also present in P. multimicronucleatum, suggesting that the ND7p is well-conserved
and plays an important role in regulated exocytosis
in Paramecium species. We treated cells with lysozyme to discharge most of the trichocysts andincubated cells to regenerate them. The ND7mRNA was present only in a trace amount in cellswith abundant trichocysts, while, it drasticallyincreased in trichocyst-regenerating cells. This
work also provides a useful system to isolate genes/
proteins which are involved in trichocyst discharge
in Paramecium.

Regulated exocytosis is a particular phenomenon in which secretory products are released in
response to specific extracellular stimuli, through acomplex mechanism involving a large number ofproteins. This process is restricted to specialized
cell types, such as neuronal and endocrine cells inmetazoa, and the mechanism has not been wellelucidated. The ciliate Paramecium, a unicellularorganism, undergoes the well-known regulated
exocytosis. The secretory granules called trichocysts are synthesized in the cytoplasm, transported
to the cell surface, and embedded at the specificdocking site in the plasma membrane. The specificstimuli, such as attack of the predators (Harumoto& Miyake, 1992), chemicals e.g. saturated picricacid (Adoutte, 1988, for review), aminoethyl dextran (Plattner et al. 1985) and lysozyme (Harumoto& Miyake, 1992), and certain physical or electricalstimuli, trigger the fusion of the plasma membrane
and the trichocyst membrane, and finally the contents are exocytosed to outside of the cell. The
mechanism of this regulated exocytosis in Paramecium has been well investigated morphologicallyand genetically. The specialized docking sites arecharacterized by intramembranous particles or‘rossette’, in the plasma membrane just above the*Corresponding author
Tel: + 81 742 20 3421
Fax: + 81 742 20 3421
E-mail: harumoto@cc.nara-wu.ac.jp
Received: 4 Dec 2004; Accepted: 18 Dec 2004.

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ND7 gene for regulated exocytosis in Paramecium

trichocyst tip, and the fibrous structure, ‘connecting
material’ which links the plasma membrane and thetrichocyst membrane (Plattner et al., 1973; Beisson
et al., 1976). In P. tetraurelia, 23 recessive mutants(nd: non-discharge) have been isolated and themutations mapped 13 different loci. These mutants
affect the final step of exocytosis, including membrane fusion of the plasma membrane and thetrichocyst membrane. The genetic analysis revealed
that the rosette and the connecting materials wereinvolved in the membrane fusion. Microinjection
experiments also revealed the site of action of the
products of the ND genes; ND7 and ND9 geneproducts were localized in the trichocysts and thecytosol, respectively. The trichocysts of the nd7
mutant are normally docked under the plasma
membrane, but are not able to discharge. Imperfectarrangement of the rosette and the connecting materials were associated in this mutation.
Recently the ND7 gene in P. tetraurelia wasisolated by functional complementation, and sequenced (Skouri & Cohen, 1997). The ND7 geneproduct is 506 amino acids long with a predicted
molecular mass of 59.3 kDa consisting of 16-aminoacid sequences of a signal peptide in N-terminus,
followed by a short hydrophobic domain and remaining large portion of hydrophilic region, which
is characteristic to the type I integral membraneproteins. The nd7 mutation is the deletion of T1294
which creates a frameshift resulted in abnormal C
terminus after K431. No homologous gene has beenfound in the database, suggesting that ND7p is a
novel protein specific to the regulated exocytosis.
To elucidate the ND7p function, it would be interesting to see whether ND7 gene is conserved inother species of Paramecium.
In this study, we represent that two other species of Paramecium possess the ND7 gene homologs, and show the complete sequence of ND7
homolog in Paramecium caudatum, and then we
discuss the possible function of the ND7 geneproduct. This study also represents a useful method

to isolate genes/proteins involved in trichocyst
discharge.

MATERIALS AND METHODS
StocksStocks 51, nd7, nd9 of P. tetraurelia, stocks
NT1, 27aG3, KNZ-5 of P. caudatum and stock 49Bof P. multimicronucleatum were used. Stocks 51,
NT1, KNZ-5, 49B were wild-type, and nd7, nd9
and 27aG3 were trichocyst non-discharge mutants.
All these mutants possess trichocysts but are incapable of discharging them.
Culture conditions
Cells were grown at 25°C in wheat grass powder (Pines, Lawrence, KS) infusion, bacterized twodays before use with Enterobacter aerogenes(Takagi, et al. 1996).
Purification of genomic DNA
Cells in the early stationary phase were collected in the pear-shaped centrifuge tube and incubated with 2 volumes of low-EDTA lysing solution(10 mM Tris-HCl, 1% SDS, 50 mM EDTA, pH 9.5)
(Harumoto, 1986) at 65°C for 20 min. Dissolved
cells were treated with phenol, and purified DNA
was treated with RNase and Proteinase K.
Extraction of RNA and cDNA synthesis
Total RNA was obtained from 1×105 cells in
the early stationary phase. Cells were treated by
Trizol reagent (Gibco life technologies) and mixed
with chloroform, and RNA was extracted. PolyA+
RNA was isolated using oligotex-dT30 (super) kit(Roche Molecular Biochemicals), and cDNA wassynthesized using SMART RACE cDNA amplification kit (Clontech).
Plasmids
Plasmid pGemND7(+) was kindly provided by

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Table 1 Nucleotide sequences of primers used in this study—————–———————————————————————————
primernucleotide sequences————————–————————————————————————
ND7_043F
5’-TAGGATCCGCNCCNTAYGCNGCNATG-3’
ND7_093F
5’-TAGGATCCGAYGTNCAYGCNTTYATG-3’
ND7_144F
5’-TAGGATCCGARATHTTYGGNGTNTGG-3’
ND7_187F
5’-TAGGATCCMGNTTYYARMGNGTNATG-3’
ND7_266F
5’-TAGGATCCGARAAYTTYCCNCCICC-3’
ND7_448R
5’-TAGGATCCYTCYTRRTCYTCYTTRAA-3’
ND7_506R
5’-TAGGATCCYTTYTCYTCYTRYTTYTC-3’
ND7_F1
ND7_F2
ND7_R1
ND7_R25’-GCTGATGAAGATGCTGAGGGCAAATGGGGTG-3’5’-GAAGACCAAGATGAGAGAAAAGAAAAGGATGC-3’
5’-GGCTCCATCCCTCGCAGTTCCCTCC-3’
5’-CCTGTGTACACCAAACACATCCTTCTCTTG-3’

P.c_ACT153F 5’-AGCTTACGTTGGTGATGAGG-3’
P.c_ACT821R 5’-AGACCTGACACTTCAAGACC-3’———————————–—————————————————————
Primers ND7_043F, ND7_093F, ND7_144F, ND7_187F, ND7_266F, ND7_448R,
and ND7_506R are degenerate primers designed from ND7 gene of P. tetraurelia.
ND7_F1, ND7_F2, ND7_R1, ND7_R2 are identical primers designed from the sequenced
700-bp fragment (see Result section). P.c_ACT153F and P.c_ACT821R are identical primers
designed from actin gene of P. caudatum (AB070223).

Dr. Jean Cohen. The plasmid (5 kb) contains 1.5-kb
open reading frame of ND7 gene (Skouri & Cohen,1997). The ND7 gene with 5’- and 3’ flanking
region is integrated in pGemND7(+) as a 2-kb Sph I- Pst I fragment.
Southern hybridization
Genomic DNAs of P. tetraurelia, P. caudatum
and P. multimicronucleatum were digested by BclI,
electrophoresed, blotted and hybridized with DIGlabeled pGemND7(+) DNA. The probe was synthesized by DIG DNA labeling kit (Roche Molecular Biochemicals).
Dot hybridization
Five micrograms of total RNA was placed on
the nylon membrane, dried and treated by 50 mM
NaOH. The membrane was irradiated by UV for 10
min and hybridized with DIG-labeled 2 kb Sph I Pst I fragment of pGemND7(+).

PCR, cloning products, and sequencing
Table 1 shows the primers which we used inthis study. Degenerate primers were designed fromthe amino acid sequence of ND7 gene of P.
tetraurelia (Skauri & Cohen, 1997). The identical primers were designed from the sequenced 700bp fragment described in Result section. The PCR
amplification was carried out using Advantage 2
Polymerase Mix and PCR kit (Clontech) on a
Perkin-Elmer 9700 thermal cycler with a start at
95°C for 1 min followed by 35 cycles at 95°C (30sec), 49°C (30 sec) and 72°C (1 min). PCR products
were electrophoresed, purified from the gel andcloned in pCR2.1-TOPO vector (Invitrogen).
Sequence reaction was performed using ABI Prism
Big Dye terminator cycle sequencing ready reaction
kit.
Induction of trichocyst regeneration
Cells suspended in SMB- (Sugiura & Harumoto, 2001) were mixed with lysozyme (final conc.

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ND7 gene for regulated exocytosis in Paramecium

Fig. 1. Detection of ND7 homologs in P. caudatum and
P. multimicronucleatum. P.t: P. tetraurelia, stocks 51,
nd7 and nd9. P.c: P. caudatum, stocks NT1 and 27aG3.
P.m: P. multimicronucleatum, stock 49B. Asterisks
show the expected bands as ND7 gene homologs in P.
caudatum and P. multimicronucleatum. The filter was
hybridized at 50°C.

100 µg/ml) solution and incubated for 30 sec at theroom temperature. Lysozyme induces trichocyst
discharge without killing cells (Harumoto & Miyake, 1992). The cells were washed and the discharged trichocysts were removed by centrifugation with excess amount of SMB-. Cells were thenresuspended in fresh SMB- and incubated at 25°C
for trichocyst regeneration.

Fig. 2. Regeneration of trichocysts. Cells (P. caudatum,stock KNZ-5) treated with 100 µg/ml of lysozyme for
30 sec were incubated in SMB-. The regenerated
trichocysts were discharged by treating cells with saturated picric acid. The extent of trichocyst discharge wasclassified into four categories (Harumoto & Miyake,1992): – (no discharge), + (discharge of up to threetrichocysts), ++ (intermediate discharge), +++ (fulldischarge). In the full discharge (+++), a fixed cell isfully surrounded by discharged trichocysts. (++) is between + and +++. One hundred cells were counted ineach sample. Cells were examined at 0, 2, 4, 6, 8 hours,
and 1 day after the treatment with lysozyme. cont.: cells
without treatment.

sample.

RESULTS
Monitoring of exocytosis
The competence of exocytosis was evaluatedby the extent of discharging trichocysts. Cells weretreated with saturated picric acid, and the discharged trichocysts were observed under the stereomicroscope. Saturated picric acid is not only afixative but a strong secretagogue to induce most of
the trichocysts in the cell. The extent of trichocyst
discharge was classified into four categories(Harumoto & Miyake, 1992): – (no discharge), +(discharge of up to three trichocysts), ++(intermediate discharge), +++ (full discharge). In
the full discharge (+++), a fixed cell is fully surrounded by discharged trichocysts. (++) is between+ and +++. One hundred cells were counted in each

Identification of ND7 homolog in P. caudatum
and P. multimicronucleatum by Southern hybridization
We detected the expected band of ND7 gene
about the size of 5.5 kb (Skauri &Cohen, 1997) instocks 51, nd7 and nd9 in P. tetraurelia in thestringent condition (68°C). In P. caudatum and P.
multimicronucleatum, the corresponding band was
not detected when hybridized at 68°C and 55°C, but
it appeared at 50°C. The band was about the size of
6.5 kb in P. caudatum (Stocks NT1 and 27aG3) and4.5 kb in P. multimicronucleatum (stock 49B) (Fig.
1). The results suggested that P. caudatum and P.
multimicronucleatum possessed the ND7 geneJpn. J. Protozool. Vol. 37, No. 2. (2004)

155

Fig. 3. Dot hybridization of ND7 homolog mRNA. Total RNA (5µg/ml) extracted from cells pretreated with lysozyme and incubated in SMB- for 0, 2, 4, 6, 8 hours and 1 day, was dotted in a filter
and hybridized with the DIG-labeled ND7 gene prove. Graph represents the relative amount of ND7
homolog mRNA as the amount of mRNA in 0h is 1.

homologs.
Regeneration of trichocysts and expression of
ND7 gene homolog
Preliminary experiments using cDNA as templates, prepared from cells of P. caudatum in theearly stationary phase, with possible combinationsof degenerate primers failed to amplify any band by
PCR. The result might be due to insufficientamount of mRNA of the ND7 gene homolog in thecells. To obtain abundant mRNA of the ND7 gene
homolog, we attempted to increase the amount by
inducing trichocyst regeneration. We artificiallyremoved trichocysts from cells by treating themwith 100 µg/ml lysozyme for 30 sec. Fig. 2 shows
the time course of trichocyst regeneration in thetreated cells. Immediately after the treatment, mostof the cells lost the capacity to discharge trichocysts, and the capacity gradually recovered withtime. After 4, 6, 8 hours after the treatment, about

50%, 70% and 90% of cells possessed relatively
large amount of trichocysts (++ and +++), respectively. We extracted RNA from these cells andsubjected them for the dot hybridization (Fig. 3).
The dot was only faint in 0 and 2 hours, which
gradually became distinct in 4-8 hours, and faded
again in 1 day. It suggests that the mRNA of the
ND7 gene homolog is present only in a trace
amount in both untreated cells (cont.) and cells
incubated in SMB- for up to 2 hours after the treatment with lysozyme, and it gradually increases withthe time elapses and reaches the peak at about 6-8hours.
Cloning and sequencing of ND7 homolog
Total RNA was extracted from the lysozymetreated cells after 8-hours incubation in SMB-.
PCR reaction was performed using the synthesized
cDNA as a template with several possible combinations of degenerate primers (Table 1). PCR

156

ND7 gene for regulated exocytosis in Paramecium

Fig. 4. Amplification of ND7 gene homolog in P. caudatum. Arrow (lane 10) indicates the expected band about thesize of 700 bp. As a positive control (lane cont.), actin gene primers were used. The primers used in each lane are:(lane 1) ND7_043F, ND7_448R, (lane 2) ND7_043F, ND7_506R, (lane 3) ND7_093F, ND7_448R, (lane 4)
ND7_093F, ND7_506R, (lane 5) ND7_144F, ND7_448R, (lane 6) ND7_144F, ND7_506R, (lane 7) ND7_187F,
ND7_448R, (lane 8) ND7_187F, ND7_506R, (lane 9) ND7_266F, ND7_448R, (lane 10) ND7_266F, ND7_506R,(lane cont.) P.c_ACT153F and P.c_ACT821R, M: DNA size marker.

products were identified in several combinations ofprimers (Fig.4, lanes 1,2,3,4,5, 6,8,10). We
cloned and sequenced a single clearly-identified
band (lane 10) about the size of expected 700 bp.
The sequence of this band had homology to ND7gene of P. tetraurelia. The identical primers were
designed from the sequence of this 700 bpfragment, and finally 1521 bp of whole ND7 gene
homolog was sequenced.
Common properties of ND7 gene
The sequenced ND7 gene homolog of P. caudatum had 74.1% homology in nucleotides and75.9% homology in amino acids to ND7 gene of P.
tetraurelia (Fig.5). The predicted amino acid sequence of the ND7 gene homolog consists of 506
amino acids same as ND7 gene in P. tetraurelia
with a molecular mass of 59.1 kDa and an isoelectric point of 5.38. As shown in Fig.5, ND7p has a
16-amino acid sequence at N terminus which corresponds to a signal sequence, followed by a hydrophobic region predicted to be a single transmembrane helix. The result supports the previous
suggestion that the ND7p is a type I integral membrane protein, leaving the short N terminus on theluminal face and the large remaining C terminus on
the cytosolic face. The stretch of proline residues(PPPDPEPP) which is required for disruption of the
α-helical structure of the anchor in the type I integral membrane protein, was also found. The restof the polypeptide is highly charged (20.2% acidresidues and 19.1% basic residues) and two regionsof amphipathic α-helices and/or coiled-coil domains, possibly involved in protein-protein or protein-lipid interactions are well conserved.

DISCUSSION
This study revealed that the ND7 gene homolog is present in P. caudatum, and possibly in P.
multimicronucleatum. We cloned and sequenced
the ND7 gene homolog in P. caudatum and showedthat the amino acid sequence was well conserved in
Paramecium two species suggesting the importantrole of ND7p in trichocyst discharge.
Skouri and Cohen (1997) suggested the possible role of ND7p; 1) ND7p is a membrane protein,anchored by a single transmembrane helix, 2)
ND7p is possibly involved in assembly of connect-Jpn. J. Protozool. Vol. 37, No. 2. (2004)

P. c
P. t

1

61

121

181

241

301

361

421

481

157

MIQYILLVTLLVKVNG QADPAAQTASYAAKQQAA MASAMAQAAPYAAMGIPIT IKPQT PPMRKIIQLLLVVIKLVQ SQKEQANTAAYAAQQQGA MAAAIAQAAPYAAMGIPIT IKPQT PP** *** ** *** ** * ** * ************** ***** **PDPEPP SLGTEPEPSSYNDLFTYSGYFDPYQDDVHAFMINGKRRRRHRVNCGRDRKGRYC
PPPDPP SLGTEPEPSEYNDLFTYSGYFDPYNDDVHAFMINGKRRKRHRVNCGRDRKGRYC* * ** ********* ************** ************* ***************DFKRSHHQHGLYNRDCIHYGGSLEIYGLWQCGSRTQLSSGAIIRFLVELHELVLDYKQQKDFRRSHHQHGLYNRDCIHYAGSLEIFGVWQCGSRKQLSTGAVIRFLVELHELVLAYKQQK** **************** ***** * ****** *** ** ************ *****NDYALQRFQRIMDYNSQVYAELKRIFKKNMKKNIELVMDILEKAN QQVIENFQQILDMSNNDYAIQRFQRVMEYNRDLYAKLKRAFKANIKKNIEFVMEILNKTN EQVIQNFQQILDMSK**** ***** * **** *** ** * ***** ** ** * * *** *********
EILANIKKDRESELDELKSQLQELLESF PPPSSICQQLSECDLCLSREGCVWCTQEQNCQEILADIRKEREDELEELKSQLQELLENF PPPLSICQQLRECDSCLSREGCIWCTEEQTCQ**** * * ** ** *********** * *** ****** *** ******* *** ** **EGTARDGAFFNSCDIWVSGSECPDPSQLRVQKKVSVKGELDDYYNQQDLELDQKLYLASKEGNARDGAFFNSCDIWVSGSECPEESQYKLDKKVSYKDELEDYYNQPDLELDQKLYLASK** ******************** ****** * ** ***** *************QREMVQLKNQIENRRTIIDQMTQIERDLKRRSIQIIDEFTKNKEYDGEFLSQIADEDAEGQRDMIQLKNQIESRRVVIDQMISIERNLKQRSIQIIDEFAKNKEYDGEFLAKIVDEDAEG** * ******* ** **** *** ** ********* ********** * ******KWGEYKKETKEIYKEDQDERKEKDAEENEKKQIESEKQKEREEFEKKEYEKRNEATVINKQWSDYKKETKKLFKEDQEAEKLKDEEELEKRKQQSEKDKKREEFEKQELEKRNESTVINK* *********** ** ** ***** * ****** * ***** *****SKYRDRESVKNVLEKEEKKEEKKQGT
SKYKDRTSVEDVLDKEQGKLEKQEEK*** ** ** ** ** * **

60

120

180

240

300

360

420

480

506

Fig. 5. Deduced amino acid sequence of ND7 gene homolog of P. caudatum (P. c)aligned with P. tetraurelia (P. t). Characteristic regions were represented in theboxes. Open box: signal peptides, box with broken line: hydrophobic transmembrane
helix, box with dotted line: proline cluster, double-lined boxes: amphipathic helices
and/or coiled-coil domains.

ing materials and rosette particles, 3) ND7p is likely
to interact with other proteins. The results obtainedin this study also support the hypothesis, because
the sequence of ND7 homolog represented single
transmembrane helix, and regions of amphipathic α-helices and coiled-coil domains. It is interesting todelete or mutate well-conserved sequences to identify the region which is indispensable and necessary

for the protein function.
This work also represents a useful method toisolate genes involved in trichocyst discharge.
Trichocysts, once removed from the cell, regenerate
within several hours. We treated cells with lysozyme as a secretagogue to induce trichocyst discharge, incubated them for regeneration, and thenobtained sufficient amount of mRNA. The results

158

ND7 gene for regulated exocytosis in Paramecium

indicated that only a small amount of the ND7 gene
was transcribed in untreated cells, and the mRNA
became abundant during trichocyst regeneration at
4-8 hours after the treatment.
Trichocysts, the exocytotic organelles ofParamecium provide a useful system to isolate
genes/proteins involved in the exocytosis. The
additional information about ND7 gene and themethod represented in this study will be advantageous for further investigation.
ACKNOWLEDGMENTS
We appreciate Dr. Jean Cohen for providing
ND7 gene of P. tetraurelia. We thank Prof.
Yoshiomi Takagi of Nara Women’s University for
helpful suggestion, Dr. Takashi Miyata and Dr.
Naoyuki Iwabe of Kyoto University for helpful
support. This work was also supported by a grant
for research from Yamada Science Foundation andby a grant-in-aid for scientific research (No.
14580713) from the Japan Society for the Promotion of Science to T.H.

REFERENCES
Adoutte, A. (1988) Exocytosis: Biogenesis, transport and secretion of trichocysts. In: Paramecium. H.–D. Görtz, ed. Springer, Berlin,
pp.325-362.
Beisson, J., Lefort-Tran, M., Pouphile, M., andRossignol, M. and Satir, B. (1976). Genetic
analysis of membrane differentiation in Paramecium. Freeze-fracture study of the trichocyst
cycle in wild type and mutant strains. J. Cell

Biol. 69, 126-143.
Harumoto, T. (1986) Induced change in a nonMendelian determinant by transplantation ofmacronucleoplasm in Paramecium tetrareulia.
Mol. Cell. Biol., 3498-3501.
Harumoto, T. and Miyake, A. (1992) Defensivefunction of trichocysts in Paramecium. J. Exp.
Zool., 260, 84-92.
Plattner, H., Miller, F., and Bachman, L. (1973)
Membrane specializations in the form of regular membrane to membrane attachment sites in
Paramecium. A correlated freeze-etching andultrathin sectioning analysis. J. Cell Sci., 13,
687-719.
Plattner, H, Sturzl, R and Matt, H. (1985) Synchronous exocytosis in Paramecium cells. IV.
Polyamino compounds as potent trigger agentsfor repeatable trigger-redocking cycles. Eur. J.
Cell Biol. 36(1), 32-37.
Skouri, F. and Cohen, J. (1997) Genetic approach toregulated exocytosis using functional complementation in Paramecium: Identification of the
ND7 gene required for membrane fusion. Mol.
Biol. Cell, 8, 1063-1071.
Sugiura, M. and Harumoto, T. (2001) Identification, characterization, and complete amino acidsequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum. Proc. Natl. Acad. Sci., 98(25),14446-14451.
Takagi, Y., Nimura, K., Tokusumi, Y., Fujisawa, H.
and Kaji, K. (1996) Secretion of mitogenic
factor(s) from stocks of Paramecium
tetraurelia, P. caudatum and P. multimicronucleatum. Zool. Sci., 13, 89-96.