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Plant Physiol. (1999) 119: 873-884
Characterization of Chlamydomonas reinhardtii
Zygote-Specific cDNAs That Encode Novel Proteins Containing Ankyrin
Repeats and WW Domains1
Hideo Kuriyama*,
Hiroyoshi Takano,
Lena Suzuki,
Hidenobu Uchida,
Shigeyuki Kawano,
Haruko Kuroiwa, and
Tsuneyoshi Kuroiwa
Department of Biological Sciences, Graduate School of Science,
University of Tokyo, Hongo, Tokyo 113, Japan (H.K., H.T.,
S.K., T.K.); Department of Hygiene and Oncology, Graduate School of
Medicine, Tokyo Medical and Dental University, Yushima, Tokyo 113, Japan (L.S.); Institute of Biological Science, University of Tsukuba,
Tsukuba, Ibaraki 305, Japan (H.U.); and Department of Biology,
Kyoritsu Women's Junior College, Tokyo 102, Japan (H. Kuroiwa)
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ABSTRACT |
Genes that are expressed only in the
young zygote are considered to be of great importance in the
development of an isogamous green alga, Chlamydomonas
reinhardtii. Clones representing the Zys3 gene
were isolated from a cDNA library prepared using zygotes at 10 min
after fertilization. Sequencing of Zys3 cDNA clones resulted in the isolation of two related molecular species. One of them
encoded a protein that contained two kinds of protein-to-protein interaction motifs known as ankyrin repeats and WW domains. The other
clone lacked the ankyrin repeats but was otherwise identical. These
mRNA species began to accumulate simultaneously in cells beginning 10 min after fertilization, and reached maximum levels at about 4 h,
after which time levels decreased markedly. Genomic DNA gel-blot
analysis indicated that Zys3 was a single-copy gene. The
Zys3 proteins exhibited parallel expression to the Zys3
mRNAs at first, appearing 2 h after mating, and reached maximum
levels at more than 6 h, but persisted to at least 1 d.
Immunocytochemical analysis revealed their localization in the
endoplasmic reticulum, which suggests a role in the morphological
changes of the endoplasmic reticulum or in the synthesis and transport
of proteins to the Golgi apparatus or related vesicles.
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INTRODUCTION |
Chlamydomonas reinhardtii is a unicellular, isogamous
green alga, the sexual life cycle of which is controlled by genetically determined mating types consisting of two kinds of haploid cells that
are morphologically very similar, but contain a distinct locus on their
nuclear genome (Ferris and Goodenough, 1994 ). In sexual reproduction,
the gametes are induced independently from corresponding vegetative
cells in a nitrogen-starved environment. When they encounter cells of
the opposite mating type, they recognize their partner, begin to
agglutinate, and then fuse to become zygotes. After zygote formation, a
number of events ensue, including preferential digestion of
male-derived chloroplast nuclei (Kuroiwa et al., 1982), nuclear fusion
(Cavalier-Smith, 1970; Kuroiwa et al., 1982), flagellar degeneration,
and zygospore formation (Cavalier-Smith, 1976). All functional proteins
and their mRNAs directly involved in these phenomena are thought to be
synthesized only after cell fusion (Kuroiwa et al., 1983; Kuroiwa,
1991). Therefore, genes expressed specifically and relatively early in
zygotes should play important roles in the regulation of this complex
series of events.
Fertilization has been intensively studied in organisms such as the sea
urchin, the newt, and mammals. The gametes of these animals highly
differentiate to form sperm and eggs, and the egg already has the full
complement of mRNA necessary for the very early stage of embryonic
development, because the blocking of RNA synthesis has no effect on the
embryo until it reaches the blastula stage (Gilbert, 1988). In
contrast, a C. reinhardtii zygote undergoes a burst of gene
expression immediately after cell fusion. Zygote-specific genes of
C. reinhardtii have been isolated using differential
screening by several groups (Ferris and Goodenough, 1987; Wegener and
Beck, 1991; Uchida et al., 1993). Uchida et al. (1993) employed a cDNA
library prepared from mRNAs of zygotes 10 min after cell fusion, so
their clones may include fragments of essential genes that function
from a very early stage and regulate the developmental system of a
zygote. We report here molecular-biological and immunocytochemical
characterization of one of these genes previously denoted as
Zys3 (Uchida et al., 1993).
The deduced amino acid sequence of a full-length Zys3 cDNA
clone contained two ankyrin repeats and two WW domains, both of which
are known to be functional protein-to-protein interaction sites. The
ankyrin repeat was originally noted in the CDC10 gene of
Shizosaccharomyces pombe by Aves et al. (1985).
CDC10 and its homologs SWI6 and SWI4
in Saccharomyces cerevisiae function in cell proliferation
and mating-type switching as transcription complexes (Breeden and
Nasmyth, 1987; Andrews and Herskowitz, 1989). A number of related genes
have since been isolated, including Fem-1, a sex determinant
in the nematode (Spence et al., 1990); Lin-12,
Glp-1, and Notch, intrinsic membrane proteins
(Wharton et al., 1985; Yochem et al., 1988; Yochem and Greenwald,
1989); GABP , NF- B/p105, IB (MAD-3),
bcl-3, and Arabidopsis AKRP, transcription-factor subunits
or regulators of transcriptional systems (Bours et al., 1990, 1993;
Ghosh et al., 1990; Kieran et al., 1990; Ohno et
al., 1990; Haskill et al., 1991; Lamarco et al., 1991; Thompson et al.,
1991; Zhang et al., 1992); and ankyrin, a cytoskeletal protein found in
mammals, Drosophila, and nematodes (Lux et al., 1990;
Bennet, 1992).
The WW domain is found in a wide range of cytoskeletal, regulatory, and
signaling molecules, including dystrophin, a cytoskeletal protein (Ahn
and Kunkel, 1993) that stabilizes the membrane and generates
contractile force; the human Yap protein, a mediator of cell-growth
signals (Sudol et al., 1995); IQGAP1, a human GTPase-mediated, cytoskeleton-regulating protein (Hart et al., 1996); rat FE65, a
transcription-factor activator (Bork and Sudol, 1994); and tobacco DB10, a RNA helicase (Itadani et al., 1994). Although these genes may
have a diverse range of functions, they all probably work via
protein-to-protein interactions, and in the case of the WW domain, the
sequences of the specific ligands have also been determined (Einbond
and Sudol, 1996). To our knowledge, Zys3 is the first gene
that encodes sequences of both of these motifs. Another Zys3 mRNA that contains no complete ankyrin repeat was also present, but its
temporal expression pattern was almost the same as that described
above. These different mRNA species were suggested to be transcribed
from the same single-copy gene.
Immunoblotting with two polyclonal antibodies indicated that the Zys3
gene products were expressed within 2 h of fertilization and
persisted for at least 1 d. The Zys3 gene products began to accumulate at the extending ER of zygotes at 3 h after cell
fusion. This localization pattern suggested their role in the control of the ER systems in synthesis, sorting, or transport of proteins required for further zygote development.
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MATERIALS AND METHODS |
Strains and Culture Conditions
Chlamydomonas reinhardtii wild-type strain 137c,
mt+, and mt
were cultured as described by Uchida et al. (1993). Vegetative cells
were maintained on Snell's medium II (Snell, 1982) supplemented with
1.0% (w/v) agar. Cells were harvested and suspended in distilled water
at a concentration of 1.0 × 107 cells/mL.
One-hundred-milliliter volumes of cell suspensions were incubated in
Petri dishes under light for about 3 h, with occasional shaking to
induce differentiation into gametes. After confirmation of
gametogenesis, suspensions of mt+ and
mt gametes were mixed and sampled at the
appropriate times.
Construction and Screening of a cDNA Library
For preparation of a cDNA library, total RNA was extracted by
ultracentrifugation through a CsCl cushion (Sambrook et al., 1989).
Poly(A+) was obtained using latex beads with
immobilized oligo(dT)-cellulose (Oligotex, Nippon Roche, Kamakura,
Japan).
Double-stranded cDNA was synthesized from the
poly(A+) RNA of zygotes at 10 min after
conjugation using a cDNA-synthesis kit (TimeSaver, Pharmacia Biotech)
according to the manufacturer's instructions. An EcoRI
adaptor-linker containing cleavage sites for NotI was
ligated to the cDNA and then to  gt 10 DNA that had been digested
with EcoRI and dephosphorylated using a cDNA rapid-cloning module (no. RPN1257, Amersham). The DNA was packaged into phage particles using an in vitro-packaging module (no. RPN1258, Amersham), and propagated using Escherichia coli NM514 as a host
bacterium, which does not support growth of insert-free gt 10. Approximately 1.0 × 105 independent
recombinant phages were screened, and inserts in positive plaques were
subcloned according to the method of Sambrook et al. (1989).
Sequence Analysis
Unidirectional deletions in the cloned fragments were produced
using an exo/mung bean nuclease deletion kit (Stratagene). Single-stranded DNAs from selected deletion clones were purified from
PEG-precipitated helper phage R408. Nucleotide sequences were
determined with the dideoxyribonucleotide chain-termination method
(Sanger, 1981) using a DNA-sequencing system (373S, Applied Biosystems,
Foster City, CA) and a Taq terminator cycle-sequencing kit
(DyeDeoxy, Applied Biosystems) according to the manufacturer's instructions. Sequencing data were analyzed with DNASIS software (Hitachi Software Engineering, Yokohama, Japan) and the BLAST program
(Altshul et al., 1990).
Northern-Blot Hybridization and RNase Protection Assay
Total RNA was extracted according to the method of Kirk and Kirk
(1985). RNA (10 µg/lane) was glyoxylated and electrophoresed in 1.1%
(w/v) agarose gel (Agarose NA, Pharmacia Biotech) in 10 mM
sodium phosphate buffer, pH 7.0, at 3 V/cm for 4 h with rapid circulation. After electrophoresis, RNA was transferred to a nylon membrane (Biodyne B, Pall Corporation, Port Washington, NY) with a
vacuum-blotting apparatus (Vacugene, Pharmacia Biotech), and the
membrane was treated as described by Sambrook et al. (1989).
Fragments from positions 225 to 507 of clone T91 and 232 to 590 of
clone T106 were cut with SacII (Toyo Boseki, Osaka, Japan) and BamHI (Takara Biomedicals, Kyoto, Japan) and then
subcloned into the pBluescript SKII+ to generate riboprobes specific
for those clones. Extra parts of the multicloning site were deleted, and the resultant plasmid vectors were designated as pRPZ01 and pRPZ02,
respectively. In vitro transcription was performed on 2 µg of
template vector linearized with HincII (Takara Biomedicals) using T7-RNA polymerase (Stratagene) in a 20-µL reaction system consisting of T7 reaction buffer (Stratagene), RNase inhibitor (Toyo
Boseki), 1 mM ATP, CTP, and GTP (Boehringer
Manheim), 7.5 µM UTP (Boehringer Manheim), and
12.5 µM [-32P]UTP
(Amersham). Subsequent hybridization and digestion were performed
according to the manufacturer's instructions for the RPAII kit
(Ambion, Austin, TX) using 2 µg of C. reinhardtii total RNA. Samples were electrophoresed through a denaturing 5% (w/v) acrylamide gel using a Mini-PROTEAN II Cell (Bio-Rad) at 250 V for 30 min. The gels were sealed in a polypropylene bag and exposed to
radiographic film (X-Omat AR, Kodak) for 2 to 6 h at  80°C.
Genomic Southern-Blot Analysis
Total DNA extraction was performed by the modified method of Ohta
et al. (1992). The DNA (10 µg/lane) was digested with the restriction
enzymes AvaI, PstI, and PvuII. Each
restriction fragment was separated in a 1.1% (w/v) agarose gel,
transferred onto a nylon membrane, and incubated with a labeled probe
complementary to the full-length cDNA clone, as described previously
(Sambrook et al., 1989). The membrane was then washed twice in 300 mM NaCl, 30 mM trisodium
citrate, 0.1% (w/v) SDS at 65°C for 15 min, twice in 15 mM NaCl, 1.5 mM trisodium
citrate, 0.1% (w/v) SDS at 65°C for 15 min, and then
autoradiographed.
Preparation of Anti-Zys3 Protein Antibodies and
Western-Blot Analysis
Residues 262 to 275 of the deduced amino acid sequence of Zys3
proteins, MHPNRRWYNTATRE, were selected as an antigen for the preparation of rabbit anti-Zys3 protein antibody.
A fragment from 116 to 1115 of clone T106 was isolated with
Nae I (Takara Biomedicals), ligated in the SmaI
site of the pQE32 expression vector (Qiagen, Chatsworth, CA), and
introduced into E. coli XL-1-Blue competent cells according
to the method of Inoue et al. (1991). Subsequent extraction and
purification of the fusion protein were carried out as described in the
manufacturer's instructions. Two milligrams of purified fusion protein
was used for the immunization of rats.
The sera were used without further purification in the following
experiments and their specific antibodies were designated as
-Zyspept3 and -Zysfuse3.
Sampling of total proteins for western-blot analysis was performed
essentially according to the method of Arnburst et al. (1993). About
1.0 × 107 cells of each sample were
dissolved in 50 µL of 1× sample buffer (62.5 mM Tris-Cl,
pH 6.8, 10% [v/v] glycerol, 2% [w/v] SDS, 5% [v/v]
-mercaptethanol, and 0.01% [w/v] bromphenol blue) with boiling for 5 min, and 5 µL was used for western-blot analysis.
Samples were fractionated by SDS-PAGE through a 12% Laemmli gel
(Laemmli, 1970) at 200 V for 42 min using a Mini-PROTEAN II Cell
(Bio-Rad). We used a miniature transblot apparatus (Bio-Rad) for
electroblotting onto nitrocellulose membranes, in which proteins within
a gel could be transferred at 100 V for 1 h in the cooled transfer
buffer (20% [v/v] methanol, 25 mM Tris-HCl, and 192 mM Gly). Labeled proteins were detected with an assay kit
(Immune-Blot, Bio-Rad).
Immunofluorescence Microscopy and Electron Microscopy
About 1 × 107 C. reinhardtii cells were fixed according to the method of Armbrust
et al. (1993). After blocking in 5% (w/v) BSA, 0.05% (v/v) Tween 20 in 1× PBS for 1 h at 37°C, a 1:100 dilution of -Zysfuse3 was
added to specimens attached to coverslips, which were then incubated
for 12 h or more at room temperature. The sample was washed twice
in 1× PBS for 10 min, and then incubated again at 37°C for 1 h
with 5% (w/v) BSA, 0.05% (v/v) Tween 20 in PBS containing a 1:100
dilution of FITC-conjugated goat anti-rat IgG antibody. After
subsequent washing with 1× PBS twice for 10 min, the coverslip was
placed on a glass slide onto which DAPI and 50% (v/v) glycerol
containing n-propylgallate had been dropped. The signal was
observed by an epifluorescence microscope (model BHSRFC, Olympus,
Tokyo, Japan).
For electron microscopy, cells were fixed in 2% (v/v) glutaraldehyde
for 2 h at 4°C and with 2% (w/v) OsO4 for
2 h at room temperature, dehydrated through an ethanol series, and
substituted for propylene oxide. The samples were embedded in Spurr's
resin (Spurr, 1969), as described by Nozaki et al. (1994). After
polymerization, sections were cut with a diamond knife on an
ultramicrotome (model MT-6000 XL, RMC-Eiko, Kawasaki, Japan), and then
stained with uranyl acetate and lead citrate. Samples for
immunodetection were fixed only with 2% (v/v) glutaraldehyde and
embedded in London Resin White (London Resin Co., Hampshire, UK), as
described previously (Johnson and Rosenbaum, 1990).
Following incubation for 2 d at 50°C, samples were sliced and
collected on Formvar-coated nickel grids. Each mesh was inverted onto
drops of blocking solution (PBS, pH 7.4, and 1% [w/v] BSA) for 30 min, incubated on drops of anti-Zys3 protein antibody (diluted in
blocking solution) overnight at 4°C, and washed with PBS, pH 7.4, containing 0.01% (v/v) Tween 20 (Bio-Rad). They were then incubated on
drops of gold goat anti-rat IgG antibody (10-nm EM grade, Zymed, San
Francisco, CA) diluted to 1:100 in blocking solution, pH 8.2, for
2 h, washed with PBS, pH 8.2, plus 0.01% (v/v) Tween 20, and
finally with distilled water before being air dried. Sections were
contrasted with uranyl acetate and viewed on an electron microscope
(JEM-1200 EX, Jeol).
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RESULTS |
Nucleotide Sequences of Zys3 cDNAs
To obtain longer cDNA inserts with greater efficiency, a new cDNA
library was constructed using mRNA prepared from zygotes 10 min after
conjugation. This library was estimated to contain about 3.5 × 106 independent clones, and a total of 2 × 105 phages was screened. Thirty-nine positive
clones were isolated at the first screening, eight of which were
arbitrarily selected for a second round of screening. Based on insert
sizes, three inserts were expected to contain nearly full-length
Zys3 cDNA (data not shown) and were further processed for
sequence analysis. The restriction maps of two different clones are
shown in Figure 1A.
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| Figure 1.
A, Restriction maps of cDNA clones T106 and T91.
Solid lines and rectangles indicate the noncoding and coding regions of
these clones, respectively. The two ankyrin repeats in T106 are
indicated as shaded boxes and the WW domains as black boxes. The
corresponding region of T91 was partially deleted, and no ankyrin
repeat was present. B, Nucleotide (above) and deduced amino acid
(below) sequences of cDNA clones T106 and T91. Lowercase letters
indicate the sequences present only in clone T106. The amino acids
composing the N-terminal putative signal peptide, ankyrin repeats, and
WW domains are represented with broken lines, double underlines, and
wavy lines, respectively. Clone T91 lacks the amino acid sequences in
the open box, and thus contains no ankyrin repeat. A stop codon, TGA,
is represented with an asterisk. The putative polyadenylation signal of
C. reinhardtii (Youngblom et al., 1984) is underlined.
Accession numbers for clones T106 and T91 are AB004042 and AB004043,
respectively.
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Figure 1B shows the sequences of cDNA clones that contain the putative
full-length Zys3-coding region. In clone T106, 101 bp of the
5 untranslated region was followed by 1113 bp of an open reading
frame, 326 bp of a 3 noncoding sequence, and 20 bp of
poly(A+) tail. A consensus polyadenylation signal
of C. reinhardtii (Youngblom et al., 1984) could be found in
the 3 untranslated region at position 1529. The open reading frame
corresponding to a polypeptide of 371 amino acid residues with a
predicted molecular mass of 39.3 kD was recognized. A putative signal
peptide of 25 residues was found using the pSORT program
(http://psort.nibb.ac.jp/) at the N terminus of the proteins. A
repeated amino acid motif, ----D--G-TPLH-AA-------V--LL--GA-, which has
been described in a number of genes, was located from residues 56 to 89 and 90 to 120 of clone T106, although one residue of the second repeat
was deleted. This domain sequence is called an ankyrin repeat (Bennet, 1992) and is supposed to be involved in protein-to-protein
interactions. The alignment of ankyrin repeats in various genes is
shown in Figure 2A. In ankyrin repeat 1 of clone T106, 10 amino acids were well conserved relative to the
ankyrin consensus sequence and, in ankyrin repeat 2, 11 amino acids
were identical. A second DNA clone, T91, had extensive homology to T106
but lacked complete ankyrin repeats.
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| Figure 2.
A, Comparison of ankyrin repeats of a Zys3 protein
and other gene products. Consensus amino acids within repeats of a
particular gene product are shown on the right. Consensus amino acids
of all repeats are indicated in the bottom row. The sequence data
(except those of Zys3) are cited from Spence et al. (1990), Haskill et
al. (1991), and Zhang et al. (1992). B, Comparison of the WW domains of
Zys3 with other proteins described by Sudol et al. (1995) or Chan et
al. (1996).
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Two WW domains (Sudol et al., 1995) were apparent at residues 163 to
187 and 287 to 313 of clone T106 (Figs. 1 and 2B), matching the consensus sequences
W--------(-) (-)V(F,Y)F(Y,W)------(-)G(S,T,N,E)G(S,T,Q,C,R)F(Y,W)--P (BEAUTY search; Worley et al., 1995).
Characterization of the Zys3 Gene
Northern-blot hybridization carried out with a full-length
Zys3 cDNA probe showed that Zys3 was not
expressed in the vegetative cells and gametes of either mating type,
and that it began to accumulate at 10 min and reached a maximum level
at 4 h after zygote formation. The signal then decreased
dramatically after 6 h (Fig. 3A;
Uchida et al., 1993). No additional signals could be found at other
positions even with this full-length probe (Uchida et al., 1993). As
mentioned above, screening of the cDNA library led to the isolation of
two homologous mRNA species (Fig. 1A), both of which would be detected
by this probe. The RNase protection assay was used to distinguish
between the expression patterns of these two mRNAs. The T91-specific
probe (305 bases) from pRPZ01 and the T106-specific probe (383 bases) from pRPZ02, which protected 274- and 352-base fragments,
respectively (Fig. 3B), were applied to the total RNA samples from
zygotes at 4 h (Fig. 3C). In both lanes the fragments of the
expected sizes appeared with cleaved, smaller bands, indicating that
both types of transcripts are present in vivo. The T91-specific probe
was then used to examine the temporal expression of T91 (Fig. 3D). T91
transcripts were first detected 10 min after cell fusion, with the
highest level of expression at 4 h and a rapid decrease at 6 h (Fig. 3D). This pattern was similar to that obtained by northern
blotting, but in this case, more intense signals were visible at the
sites of fragments 178 and 96 bases in length. These smaller fragments
must represent the cleaved probe from pRPZ01, which was not protected
from RNase A because of its incomplete hybridization with C. reinhardtii Zys3 mRNA of the T106 type, and showed the same
temporal pattern of expression as T91. Another experiment with the
T106-specific probe resulted in confirmation of the same pattern.
Therefore, we conclude that both types of transcripts were synthesized
simultaneously, but that T106 mRNA was far more abundant than T91 mRNA
in the zygotes.
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| Figure 3.
A, Northern-blot hybridization using the
full-length T106 probe of 10 µg of total RNA of vegetative
mt+ (V+) and mt
(V) cells, mt+ (G+) and
mt (G) gametes, and zygotes at 0, 10, and 30 min and 1, 2, 4, and 6 h. B, Location of the ankyrin
repeats on clone T106 and of the deleted sequence on clone T91. The
region selected as specific probes for T91 or T106 and resultant
fragments degraded by RNase A are indicated as solid lines. C,
Difference in signal patterns in the RNase protection assay applied to
total RNA of the zygote at 4 h with either specific probe. D,
Survey of the temporal expression patterns of Zys3 mRNA
by the RNase protection assay using the partial sequence specific for
cDNA clone T91. HincII-digested  X174 DNA was used as
a molecular marker.
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Genomic DNA of C. reinhardtii was digested with
AvaI, PstI, or PvuII, and
electrophoresed. Figure 4 shows an
autoradiograph of the blotted membrane, which was incubated with the
probe complementary to the full-length cDNA clone T106 and washed in
high-stringency conditions. The probe selectively and strongly
hybridized to fragments of about 1.5 kb of AvaI-digested
total DNA, about 4.5 kb of PstI-digested total DNA, and
about 2.5 and 1.9 kb of PvuII-digested total DNA. As
indicated in the sequence data (Fig. 1B), there was one
PvuII site (5 CAG/CTG 3) on the Zys3 cDNA at
the middle position of 665 to 670 nucleotides. These results indicate
that Zys3 exists as a single-copy gene. Other related
sequences appeared in the digested C. reinhardtii nuclear
genome when Southern-blot analysis was performed with moderate washing
conditions (60°C; data not shown).
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| Figure 4.
Southern-blot hybridization analysis using the
probe complementary to the full-length cDNA clone T106. Separated
10-µg total DNA samples that had been digested with
AvaI, PstI, or PvuII were
blotted onto nylon membranes and hybridized with the labeled T106 cDNA
probe. StyI-digested DNA was used as a molecular
marker.
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Expression Pattern of Zys3 Proteins
The sequence from position 116 to 1115 of clone T106 was ligated
to pQE32, an expression vector that imposed six His residues on the N
terminus of the recombinant protein for affinity purification. By
inducing overexpression of the protein in bacteria, a distinct band
emerged at the apparent molecular mass position of 38 kD in their
lysate (Fig. 5). The amount of this
protein increased as the interval between induction and sampling of
E. coli was elongated (data not shown), and this protein
also appeared in the fraction of affinity-purified sample, indicating
that an expression system of Zys3 fusion protein was established.
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| Figure 5.
Specificity of -Zyspept3 against the
bacterially expressed fusion protein. Total proteins of noninduced
E. coli (IPTG ), E. coli induced with
IPTG (IPTG +), and affinity-purified fusion protein were
electrophoresed, blotted, and visualized by staining with Ponceau S
(left). The results of western blotting against -Zyspept3 using the
same membrane are shown on the right. -Zyspept3 specifically reacted
with the 38-kD fusion protein.
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We constructed two polyclonal antibodies against Zys3 proteins. The
first was designed against the partial polypeptide sequence of clone
T106 (see ``Materials and Methods'') of residues 262 to 275 and was
named -Zyspept3. The second was generated against the overexpressed
fusion protein and was named -Zysfuse3.
The -Zyspept3 antibody specifically reacted with the 38-kD fusion
protein in IPTG-induced E. coli cell lysate and the
affinity-purified sample (Fig. 5, lanes 3 and 4), but no such signal
could be seen in noninduced cells (lane 2).
Against the fractionated total protein of C. reinhardtii
vegetative cells, gametes, and zygotes sampled at the indicated times (Fig. 6), -Zyspept3 antibody was
allowed to react on the nitrocellulose membrane. This antibody
recognized proteins of C. reinhardtii that had migrated to
the apparent molecular mass site of 40 kD. Aside from the distinct main
signal, a minor signal was also detected as a slightly heavier
molecule. This may represent the Zys3 precursor still possessing the
signal peptide (Fig. 1B). The protein of the main signal began to
accumulate 2 h after zygote formation, reached a maximum level at
6 h, and persisted over 22 h.
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| Figure 6.
Temporal expression pattern of Zys3 proteins.
Total protein samples of C. reinhardtii vegetative
mt+ (V+) and mt
(V) cells, mt+ (G+) and
mt (G) gametes, and zygotes at 0, 10, and 30 min and 1, 2, 4, 6, and 22 h were fractionated, blotted,
and probed with -Zyspept3. Strong signals were detected in the
vicinity of 40 kD at 2, 4, 6, and 22 h. A slightly heavier minor
band that appeared at the same time may be the Zys3 precursor
containing the signal peptide.
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Subcellular Localization of Zys3 Gene Products
Figure 7 shows the results of
western analysis of C. reinhardtii total proteins and
-Zysfuse3 antibody and the localization of Zys3 products
in cells by epifluorescence microscopy. The specificity of
-Zysfuse3, an antibody raised against the fusion protein, was tested
in the same way (Fig. 5). This antibody also reacted with the
overexpressed 38-kD protein of E. coli (data not shown), as
well as with the endogenous 40-kD protein and its putative precursor of
C. reinhardtii zygotes at 6 h (Fig. 7A), but did not
react with total protein from C. reinhardtii zygotes at 0 min (Fig. 7A). The -Zysfuse3 was used to analyze the subcellular localization of Zys3 proteins to increase the sensitivity for signal
detection.
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| Figure 7.
A, Specificity of -Zysfuse3, an antibody raised
against affinity-purified, bacterially synthesized fusion protein of
Zys3 and a 6× His tag. The fractionated total protein of the C. reinhardtii zygote at 0 min and 6 h after mating by
SDS-PAGE was blotted onto the nitrocellulose membrane. The -Zysfuse3
antibody was shown by western-blot analysis to specifically react with
the 40-kD Zys3 proteins and the precursor in the zygote at 6 h. B,
Indirect fluorescence microscopy for analysis of localization of Zys3
products. -Zysfuse3 was used as the primary antibody in the
mt gamete (a and b), and in the zygotes at
1 h (c and d) and 6 h (e and f) after fertilization to detect
its selective reaction against Zys3 proteins. Preimmune serum was also
applied to zygotes at 6 h (g and h). Cells were observed under UV
light by staining with DAPI (a, c, e, and g) and under blue light to
detect the FITC signal (b, d, f, and h). Strong fluorescence of FITC
was observed only around the cell nucleus from zygotes 6 h after
fertilization. Bar = 5 µm. Red autofluorescence in b and d
resulted from remnant chlorophyll molecules of the specimens.
|
|
Specificity of -Zysfuse3 was enough for the immunocytochemical
analyses. As shown in Figure 7B, the -Zysfuse3 antibody bound strongly to a protein in the C. reinhardtii zygotes at
6 h after fertilization, but did not react with anything in
gametes or very young zygotes at 1 h. Preimmune serum of
-Zysfuse3 did not detect anything in zygotes at 6 h, confirming
the specificity of this antibody. Visualization of the cell and
chloroplast nuclei with DAPI (Fig. 7B, a, c, e, and g) indicated that
Zys3 gene products exist within the cytoplasm and encompass the fused
cell nucleus.
A general ultrastructural image of a C. reinhardtii zygote
is presented in Figure 8a. The nucleus is
surrounded by the rough ER, the Golgi apparatus, and a large
chloroplast. The vesicular derivatives from the Golgi apparatus can be
distinguished from the ER sac by the filamentous appearance of their
contents (Fig. 8a; Minami and Goodenough, 1978). Ribosomes also attach
to the outer surface of the nuclear envelope. Detection of Zys3
proteins was performed according to the method of Nozaki et al. (1994) using young (Figs. 8, b-d, and 9a) and premature zygotes (Fig. 9, b-d) that had been embedded in London
Resin White and sectioned. The signals were localized on the surface of
the ER membrane (Fig. 8b) or on the invaginated protrusions from the
cytosol (Fig. 8c) at the early stages of zygote development (3 and
6 h after mating; Fig. 8, b and c, respectively). They were then
localized specifically on the continuous, anastomosing ER membrane
system, which spread throughout the C. reinhardtii zygotes
6 h after fertilization (Fig. 8d).
View larger version (144K):
[in this window]
[in a new window]
| Figure 8.
Immunoelectron microscopic analyses with the
anti-Zys3 protein antibody -Zysfuse3. A general ultrastructural
image of a very young zygote of C. reinhardtii is
represented first as a control (a), and then at 3 h (b) and 6 h (c and d) after being reacted with -Zysfuse3. N, Nucleus; cp,
chloroplast; s, starch grain; G, Golgi apparatus; v, Golgi-related
vesicle. Arrowheads indicate undefined structures protruding from the
cytosolic side into the ER. Bars = 500 nm.
|
|
View larger version (142K):
[in this window]
[in a new window]
| Figure 9.
Localization patterns of Zys3 proteins were
investigated along the protein-secretion pathway of C. reinhardtii zygotes at 6 h (a) and 22 h (b-d). CW,
Cell wall. Other abbreviations are as in Figure 8. Bars = 500 nm.
|
|
As for the distribution of signals along the protein-secretion pathway,
the Zys3 proteins migrated to the vicinity of the cis side
of the Golgi stacks by 6 h (Fig. 9a). Only negligible gold
particles were detected in the Golgi sac and Golgi-derived vesicles at
this stage (Fig. 9a). Even at 22 h after fertilization, none of
particles moved to the Golgi apparatus, remaining instead on the
membrane of the ER (Fig. 9b). This organelle appeared to extend toward
the cell surface, but was distinct from the plasma membrane (Fig. 9c).
Signal was barely seen on the plasma membrane or in the cell wall even
at this later stage (Fig. 9d). In addition, there was no regularity in
the distribution of signal in the gametes and zygotes 1 h after
fertilization (data not shown). This localization pattern was supported
by previous results with epifluorescence microscopy, in which signals
were detected in the cytoplasm of premature zygotes, but no signal was
evident in gametes and very young zygotes (Fig. 7). The fluorescent
structure in Figure 7B, f, corresponded to that of the ER of a C. reinhardtii zygote.
| |
DISCUSSION |
C. reinhardtii is one of the simplest experimental
organisms for the study of sexual reproduction (Goodenough et al.,
1995). The mt+ and
mt cells are thought to differ only
within a narrow region of the nuclear genome (Ferris and Goodenough,
1994), and the experimental control of gametogenesis and fertilization
is far easier than in most other organisms. Gametes can be induced
separately by deprivation of a nitrogen source, and the efficiency of
mating can be elevated to more than 90% within 10 min after mixing
masses of homogeneous mating types. To understand the fundamental
features of fertilization and subsequent zygote development, the
central part of sexual reproduction, it is most effective to employ
this simple and efficient system.
This experimental system has enabled us to analyze many zygote-specific
genes. At least 16 such genes have been isolated so far from C. reinhardtii by differential screening (Ferris and Goodenough,
1987; Wegener and Beck, 1991; Uchida et al., 1993). The message of the
zymB and zymC genes does not significantly accumulate until zygote development has progressed 24 h after fertilization (Wegener and Beck, 1991), suggesting that these genes may
participate in sporulation or later events. The Class V gene was
expressed from 2.5 h after cell fusion, but was dependent on other
products for its transcriptional activation (Matters and Goodenough,
1992). The function of this protein is unknown, although it contains
sequences homologous to cell-surface receptors and protein kinases
(Matters and Goodenough, 1992). In contrast, mRNAs of Classes I to IV,
VI, and zys1-4 began to accumulate from a very early stage
and required no zygote-specific protein synthesis (Ferris and
Goodenough, 1987; Uchida et al., 1993). Woessner and Goodenough (1989)
suggested a structural role in the architecture of the zygote cell wall
for Class IV and VI genes. Armbrust et al. (1993) reported that
transcription of ezy-1 (Class III) was sensitive to UV light
and that its product had an almost identical electrophoretic mobility
to a protein previously detected by Nakamura et al. (1988). This
ezy-1 product is thought to bind directly to chloroplast
nuclei to preferentially digest those derived from the
mt gamete. Some of these zygote-specific
genes are likely to be involved in transcriptional regulation (Uchida
et al., 1993). C. reinhardtii begins to express the
Zys3 gene from the moment of cell fusion, suggesting its
involvement in zygote development from an early stage.
Sequence analysis indicated that the zygote-specific gene,
Zys3, encoding novel proteins with two kinds of repeat
sequences, ankyrin repeats and WW domains, was probably involved in
protein-to-protein interactions. Both yeast
CDC10/SWI4 and SWI6 genes and the
C. reinhardtii Zys3 gene contain the two ankyrin repeats,
but in the yeast genes they are located separately, in the middle of the sequence, and in C. reinhardtii they are located
tandemly, near the 5 end (Breeden and Nasmyth, 1987; Fig. 1B).
Transmembrane receptors have six ankyrin repeats on their internal side
(Wharton et al., 1985; Yochem et al., 1988; Yochem and Greenwald,
1989), and cytoplasmic IB and premature NF-B have five and six
repeats, respectively (Bours et al., 1990; Ghosh et al.,
1990; Kieran et al., 1990; Haskill et al., 1991). In
contrast, some ankyrins have more than 20 repeats, which is the largest
number of all known genes containing this motif (Lux et al., 1990;
Benett, 1992). This variety in the number of ankyrin repeats does not
reflect the number of binding sites but, rather, the proper
conformation for binding (Devarajan et al., 1996). Up to four WW
domains have been detected in a single gene (human Nedd4;
Sudol et al., 1995). Each WW domain has its specific ligand, XPPXY
(Einbond and Sudol, 1996), so the number of binding sites harbored in
the T106 and T91 transcripts can be estimated to be at least three and
two, respectively.
Ankyrin repeats and WW domains are present in a variety of
cytoskeletal, regulatory, and signaling protein molecules (Bennet, 1992; Bork and Sudol, 1994), but to date have never been reported together in a single gene. One WW-domain protein, dystrophin, also has
repeats that are characteristic of spectrin, a cytoskeletal protein
that ties together an actin filament and an ankyrin at the plasma
membrane of animal cells (Ahn and Kunkel, 1993). Recently, the WW
domain of this protein was proven to bind to a dystroglycan through its
Pro-rich motif and to help stabilize the plasma membrane. Some
mutations preclude this contact and thus cause muscular dystrophy (Einbond and Sudol, 1996). Organization of proteins by multiple functional protein-to-protein interaction domains helps to establish the complicated network of the membrane skeleton (Ahn and Kunkel, 1993).
Computer analyses suggested that Zys3 proteins have a signal peptide at
their N terminus that targets the outside of the cell and therefore
could be directed to the ER, the Golgi apparatus, and the Golgi-derived
vesicles. We speculate that Zys3 proteins play a role in the
organization of intracellular membrane conformation, in particular, in
the organization of membranous organelles working in the
protein-secretion system of the zygote through the protein-to-protein interaction.
Peters et al. (1995) studied the mouse ankyrin gene Ank3, a
major isoform of three ankyrins in kidney cells. Various mRNA species
are transcribed from a single gene, Ank3, some of which completely lack the ankyrin repeat. Transcripts were alternatively spliced depending on the tissue in which they were expressed. In
addition, proteins lacking the repeat domain were localized in the
cytoplasm, whereas those containing the repeats were attached to a
structure located directly below the plasma membrane, suggesting their
functional differentiation within a single cell. In Zys3 an
exact region existent only in one mRNA species did not cover the total
repeat domain (Fig. 1B); consequently, no complete ankyrin repeat is
present in the putative amino acid sequence of the other. The sequence
of the deleted region included the consensus characteristics of introns
that begin with "gt" and end with "ag" (Fig. 1B). These characteristics are also apparent in the intron of another C. reinhardtii gene (Matters and Goodenough, 1992). It is most likely that T106 and T91 mRNAs are transcribed from the same template via
alternative splicing (Figs. 1B and 4). The entire nucleotide sequence,
except for the ankyrin-repeat region and the 5 end of the 5
untranslated region of T106, was identical between the two species,
suggesting that differential posttranscriptional regulation of this
molecule occurs in C. reinhardtii. Since these species
accumulate within a single cell in the same pattern (Fig. 3D), they may
differ in their functions in vivo based on their ability to interact
with other proteins.
Genomic Southern-blot analysis (Fig. 4) indicated that Zys3
is a single-copy gene. The numbers and lengths of each band were identical between mt+ and
mt genomic DNA (data not shown), so we
can conclude that the Zys3 gene was not specific to either
mating type.
Western blotting with the -Zyspept3 antibody confirmed that these
proteins exhibited zygote-specific expression (Fig. 6), and that Zys3
proteins began to accumulate 2 h after fertilization and remained
in the cells for about 1 d. Nuclear fusion, flagellar degeneration, zygote wall formation, and chloroplast fusion are known
to occur 4 to 6 h after fertilization, when these proteins accumulate to maximum levels. The fact that the Zys3 products remained
for more than 22 h suggests a role in the regulation of the
progression of long-term zygote development. Since RNase protection
analysis revealed identical expression patterns of the two related mRNA
species, we expected multiple species of proteins of different
molecular masses. However, such signals could not be detected by
western-blot analysis, possibly due to the scantiness of the smaller
species as suggested by their mRNA levels (Fig. 3C).
Cytoplasmic localization of Zys3 products was demonstrated by indirect
immunofluorescence microscopy applied to whole-cell specimens. Figure
7B shows that the signal was detected exclusively in the zygote at
6 h after fertilization. Strong fluorescence was concentrated
around the nucleus. However, the structure of a C. reinhardtii cell is marked by its single, large chloroplast that
occupies nearly half the cell, and it was therefore not clear whether
Zys3 products were distributed uniformly throughout the cytoplasm or in
particular organelles. This problem was resolved by observation of
sectioned specimens via electron microscopy (below). DAPI fluorescence
(Fig. 7B, a, c, e, and g) confirmed previous reports (Kuroiwa et al.,
1982); 1 h after cell fusion the zygote contained cell and
chloroplast nuclei derived from both parents, but at 6 h it lacked
the male-derived chloroplast nuclei and had a single, fused cell
nucleus.
Electron microscopy revealed that most signals of gold particles were
first detected on the surface of the membrane of the ER sac, then near
the cis side of the Golgi apparatus (Figs. 8 and 9a). Later,
they continued to gather distinctly at the ER, but the form of this
organelle changed into the anastomosing type, which could not have been
recognized without this antibody (Figs. 8d and 9). As shown in Figure
8a, this form of ER was not discernible, even in the micrograph of
cells fixed with both OsO4 and glutaraldehyde soon after mating, and has never been reported at other haploid stages;
therefore, it must be formed in parallel with zygote development or
synthesis of this protein.
Many studies have shown that there are close relationships between ER
and actin filament organization (e.g. Quader et al., 1987; Staehelin,
1997; Zhou et al., 1997), strongly suggesting that some mechanism that
integrates the cytoskeleton with the membrane system exists on the ER,
as has been discovered at the plasma membrane of animal cells (Bennet,
1992; Zhou et al., 1997). Recently, one of the human ankyrin isoforms
was shown to colocalize with the ER and Golgi-matrix components
I * spectrin and -COP (Devarajan et al., 1996), suggesting its
functional relationship in the organization of intracellular membrane
systems. Although the total sizes of Zys3 proteins were far smaller
than those of mammalian, nematode, or Drosophila ankyrins,
the domain organization and localization pattern support the idea that
they function in the membrane skeleton for the regulation of ER
configuration (Figs. 2B, 8, and 9). Large, immunogenic, spectrin-like
molecules were also discovered in the vegetative C. reinhardtii cell (Lorenz et al., 1995), but it is not known
whether these proteins are also expressed in zygotes, or if there is
any relationship between them and other proteins such as the ankyrins.
The Zys3 proteins continued to associate with the membrane system for a
relatively long time. Figures 6 and 9 indicate that these proteins
could still be detected in the ER of zygotes 22 h after
fertilization. As pointed out by Minami and Goodenough (1978), young
C. reinhardtii zygotes have increasing secretory activity
through which glycoprotein fibers are transported through the Golgi
apparatus. Therefore, these Zys3 molecules may be synthesized for the
activation of ER functions in processing, sorting, and targeting of
rigid, zygote-specific cell wall materials and/or mucilaginous
extracellular matrices.
As noted by Cavalier-Smith (1976), a marked increase in the volume or
number of ER, Golgi, and related vesicles can be observed in the
zygote. Given that sexual reproduction was originally established against adverse environments (Goodenough et al., 1995), the formation of a rigid, desiccation-resistant cell wall may accompany the early
stage of zygospore formation. The development of a synthetic secretory
system represented by the ER and Golgi apparatus and related genes
underlies this wall construction. In this case a specific modification
of ER composition, and thus a modification of ER properties, was
controlled solely by the stimulus of fertilization (Arnburst et al.,
1993; Uchida et al., 1993) and was confined to the young zygote of
C. reinhardtii. A similar ER form can be seen in other
cells, such as those of the higher plants (e.g. Jones, 1980; Quader et
al., 1987), but their distribution and kinetics seem unlikely to be
directly related to sexual reproduction. For C. reinhardtii,
a drastic increase in secretory systems may be required only at the
time of zygote formation.
Although the origin and evolution of sexual reproduction remain
controversial (Graham, 1993), many scientists believe that the oogamous
reproductive system evolved from an isogamous system of ancestral
flagellates. C. reinhardtii should exhibit many fundamental aspects of fertilization and zygote development. Analyses of the regulatory mechanisms of the morphological change and activation of
the ER in phylogenically related organisms may help to clarify the evolution of this structure. Further studies, including a search
for Zys3 homologs in related species, promoter analysis, and
the determination of proteins that interact with Zys3, will reveal the
mechanisms of regulation of the intricate intracellular membrane system
during sexual reproduction of C. reinhardtii.
| |
FOOTNOTES |
1
This work was supported by a Grant-in-Aid for
Special Promoted Research (project no. 06101002 to T.K.) from the
Ministry of Education, Science and Culture of Japan.
*
Corresponding
author; e-mail kuriyam2{at}gol.com or kuriyama{at}biol.s.u-tokyo.ac.jp;
fax 81-3-3812-4929.
Received July 21, 1998;
accepted December 2, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DAPI, 4,6-diamidino-2-phenylindole.
FITC, fluorescein isothiocyanate.
IPTG, isopropyl-1-thio--D-galactopyranoside.
| |
ACKNOWLEDGMENTS |
We thank Dr. Soichi Nakamura of the University of
Ryukyus for his kind gift of C. reinhardtii strain 137c, and
Drs. Shigeyasu Tanaka and Takeshi Suzuki of Gunma University for their
kind advice on the design of the antigen region to which the peptide
antibody was raised. We are also very grateful to Drs. Sachihiro
Matsunaga and Chieko Saito of the University of Tokyo for their
technical instruction in many of these experiments.
| |
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Abstract
Introduction