1 Department of Biological Sciences, University of Maryland Baltimore County
(UMBC), Baltimore, MD 21250, USA
2 Laboratoire de Biologie Cellulaire 4, Université Paris-Sud, 91405
Orsay, France
3 Biologia MCA, Università di Camerino, 62032 Camerino, Italy
4 Kimball Research Institute, New York Blood Center, 310 E. 67th St, New York,
NY 10021, USA
* Author for correspondence (e-mail: kloetzel{at}umbc.edu)
Accepted 18 December 2002
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Summary |
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Key words: Signal sequence, Membrane skeleton, Alveolata, Protist, Epiplasm, Pellicle, Cortical cytoskeleton, Repetitive protein domains, Ciliate expression library
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Introduction |
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Ciliated protozoans, as large cells living in a wide range of potentially
disruptive environments, have evolved a variety of strategies for
strengthening and reinforcing their outermost surface. A monolayer of
flattened membranous sacs (termed `cortical alveoli') is characteristic of
ciliates, subtending the plasma membrane and effectively isolating the
cytoplasm from the environment by three membrane layers. Additionally, various
cytoskeletal elements are found in this outer cortical layer (often termed the
`pellicle'). Some are similar to cytoskeletal structures well represented in
other eukaryotes, such as microtubules, which are widely used supporting
elements in ciliate cortexes (Grim,
1982; Fleury and Laurent,
1995
; Adoutte and Fleury,
1996
). However, the other well-known eukaryotic supportive
elements, namely actinmyosin complexes and intermediate filaments, are
not commonly used to support the cortex. In their stead are often found layers
of microfilamentous material (Adoutte and
Fleury, 1996
). The protein composition of most such layers is
unknown; in a few, cytoskeletal proteins have been identified that are, to
date, well characterized only in protists (cf.
Bouck and Ngô, 1996
).
Examples of such novel cytoskeletal proteins are the tetrins, first
described in Tetrahymena (Honts
and Williams, 1990; Brimmer
and Weber, 2000
), and the epiplasmins of Paramecium
(Nahon et al., 1993
;
Coffe et al., 1996
) and other
protists (Huttenlauch et al.,
1998b
; Bouchard et al.,
2001
). Perhaps the most well characterized and widespread of
protist cytoskeletal proteins are the articulins. These proteins were first
described in the cortex of the euglenoid Euglena gracilis
(Marrs and Bouck, 1992
), where
they assemble into articulating strips below the plasma membrane.
Articulin-like proteins have also been identified beneath the plasma membrane
of the parasitic protists Plasmodium
(Stahl et al., 1987
;
Bowman et al., 1999
;
Tchavtchitch et al., 2001
) and
Toxoplasma gondii (Mann and
Beckers, 2001
) at certain life-cycle stages. Immunological
evidence indicates that similar proteins are found in dinoflagellates as well
(Bricheux et al., 1992
;
Huttenlauch et al., 1998b
).
Among ciliates, the clearest evidence for the presence of articulins is in
Pseudomicrothorax dubius. These cells possess a thick, continuous
filamentous layer termed the `epiplasm', situated in the cytoplasm immediately
below the cortical alveoli (Peck et al.,
1991
). The two major epiplasmic proteins in P. dubius
have been characterized (Huttenlauch et
al., 1995
; Huttenlauch et al.,
1998a
) and shown to have properties quite similar to the
articulins of Euglena. The hallmark of the articulins is a core of
numerous tandemly repeating 12-amino acid (a.a.) units, rich in valine and
proline (VP-rich).
The cortical cytoskeleton of euplotid ciliates is disposed in a different
fashion than in most other ciliates. In these cells, the surface is supported
by a monolayer of tightly abutted `alveolar plates' (APs;
Fig. 1), so called because the
individual polygonal scales of the assemblage occupy the spaces within the
membranous cortical alveoli (Ruffolo,
1976; Hausmann and Kaiser,
1979
; Geyer and Kloetzel,
1987
; Williams et al.,
1989
; Hausmann and
Hülsmann, 1996
). The major proteins making up these APs in
various species of Euplotes have been identified and partially
characterized (Williams et al.,
1989
; Williams,
1991
; Kloetzel,
1991
). Electrophoretic evidence suggests that at least three
subunit forms of these proteins exist in the plates. On the basis of peptide
mapping and genetic data, Kloetzel has proposed that each subunit is encoded
by a separate gene locus in Euplotes aediculatus
(Kloetzel, 1991
;
Kloetzel et al., 1992
), and
has termed the 125, 99 and 97/95 kDa electrophoretic variants the
-,
ß- and
-platein forms, respectively
(Kloetzel, 1993
). Confocal
immunofluorescence results reported in the present study show that these
platein forms, while co-localized within mature APs, display significant
differences in solubility.
|
In the work presented here, we have used anti-platein antibodies to screen
an expression library of Euplotes genes, and have isolated and cloned
a gene encoding one of the closely related ß- or -platein
subunits. Taking advantage of new peptide sequence information and a PCR-based
strategy, two additional platein genes have been cloned; these encode very
similar
-platein isoforms. The derived protein sequences of these three
genes indicate that the plateins display long tandem runs of VP-rich dodecamer
repeats, and clearly are members of the articulin class of cytoskeletal
proteins. However, distinct differences in amino acid composition and
arrangement indicate that the plateins make up a separate family within the
articulins. Moreover, all three plateins predict canonical
starttransfer sequences at their N-termini, which correlates well with
the final intra-alveolar location of the assembled skeletal plates. N-terminal
sequencing of a
-platein directly demonstrates that the predicted
signal peptide is removed from the mature protein. To our knowledge, the
plateins are the first cytoskeletal proteins from any eukaryotic cells
described to date that feature such N-terminal signal sequences.
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Materials and Methods |
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Preparation of cortical residues of Euplotes, as a means for
enrichment of plateins, basically followed the Triton-high salt protocol
(Williams et al., 1989) with
modifications reported elsewhere (Williams
and Honts, 1995
).
Antibody production and purification
The production of monoclonal antibodies (mAbs) against E.
aediculatus plateins has been described
(Kloetzel, 1991). The present
studies used mAb PL-5 (which recognizes all platein forms in this species) and
mAb PL-3 (recognizing only the ß and
isoforms of platein, but not
the
form).
To obtain an antibody specific for -platein, polyclonal antisera
were raised in rabbits against Euplotes cortical proteins separated
by SDS-PAGE and transferred onto nitrocellulose membranes, following methods
that have been described previously
(Kloetzel, 1991
).
Nitrocellulose strips containing the 125 kDa
-platein band were
excised, sonicated to a fine slurry in PBS, and used for immunizations. Whole
sera that reacted positively with the 125 kDa
-platein band in
immunoblots were affinity purified using
-platein bands blotted onto
PVDF membrane after electrophoretic separation, following described protocols
(Harlow and Lane, 1988
). The
final eluate (containing affinity-purified antibody that we designated AP-2)
yielded much lower backgrounds in immunoblotting and immunofluorescence
staining protocols than did whole serum.
Polyclonal antibodies (pAbs) against the two main electrophoretic plate
protein bands of Euplotes eurystomus (`anti-E' serum)
(Williams et al., 1989) were
kindly provided by Norman Williams.
Peptide sequencing
Euplotes cortical extracts were separated by SDS-PAGE, blotted
onto PVDF membranes, and stained with Coomassie Blue R-250. Strips of membrane
bearing individual platein bands were excised and incubated with
TPCKtrypsin for 24 hours at 37°C. The resultant peptides were
separated by HPLC and sequenced with an ABI 477A Protein Sequencer (for
details, see Matsudaira, 1993;
Fernandez et al., 1994
).
cDNA expression library construction
Messenger RNAs were isolated (Quick-Prep micro-mRNA purification kit;
Pharmacia) from a Euplotes culture allowed to grow slowly overnight
at 16°C in a dilute solution of dried milk (0.05% in mineral water) to
avoid interference from Tetrahymena food organisms. Double-strand
cDNAs were synthesized by random priming using a cDNA synthesis kit
(Amersham). The cDNA rapid adaptor ligation kit, cDNA cloning module
gt11 and
-DNA in vitro packaging module (Amersham) were used
to construct the library in the
gt11 vector. A total of
2.5x106 pfu recombinant phages were obtained.
Expression library screening and recombinant sequence analysis
The cDNA library was screened with an antibody raised against plateins
(E-band) from E. eurystomus, following standard procedures
(Sambrook et al., 1989).
0.8-1.0 x 105 plaques were screened; positive plaques were
excised and subjected to three further rounds of expression screening prior to
characterization of inserts. Amplification of positive
gt11 clones was
performed using 2.5 µl of phage suspension in 50 µl PCR reactions at pH
9.0. The amplification products were cloned into the SmaI site of the
vector pUC 18 for sequencing.
Some clones from the two gt11 libraries were sequenced by the
Genome Express company (Grenoble, France). Others were sequenced manually or
with the Vistra automatic sequencer (Amersham) using the DNA cycle sequencing
kit from Amersham. Sequences of
-platein genes were obtained by
automated DNA sequencing (ABI Prism Model 373A; PE Applied Biosystems) using
Big Dye methodology supplied by the manufacturer. The sequences obtained were
compared with the non-redundant sequence databases using the ExPASy interface
to the SIB BLAST network service (Altschul
et al., 1997
). The nucleotide sequences of the three platein genes
reported here have been submitted to GenBankTM, under accession numbers
AY124989 (
1), AY124990 (
2), and AY124991 (ß/
).
Southern and northern blot analyses
Blotting protocols with agarose-separated nucleic acids followed those
described (Sambrook et al.,
1989). ß/
-platein probes cut from recombinant plasmids
were labeled using the Megaprime DNA labeling kit (Amersham);
-platein
probes (from PCR reactions) were labeled using random hexanucleotide primers.
Hybridizations (using Hybond-N membranes; Amersham) were carried out overnight
at 65°C in 5x Denhardt's solution, plus 0.5% SDS, and 10 mM EDTA
(except Southern blots with ß/
-platein probes, which used 0.5 M
sodium phosphate pH 7.2, 7% SDS and 1 mM EDTA). 100 mg/ml denatured tRNA was
included in northern hybridizations.
Expression analysis
Total RNA was extracted from an actively growing vegetative culture of
Euplotes, since nucleic acids from the food organism
(Tetrahymena) do not cross-hybridize with platein probes.
PolyA+ RNA was prepared from other Euplotes cultures using
the QuickPrep mRNA Kit (Pharmacia). Reverse-transcript (RT-)PCR analysis was
performed in two separate reactions. cDNAs from total RNA reactions, using
M-MLV reverse transcriptase (Eurobio, France), were amplified with
ß/-platein-specific primers; amplification of polyA+
RNA reactions, using reverse transcriptase from Pharmacia Biotech, utilized
1- and
2-specific primers.
Indirect immunofluorescence
Immunolabeling was performed on permeabilized and fixed whole cells as
described previously (Fleury,
1991; Jeanmaire-Wolf et al.,
1993
). Ghosts were produced by treatment for 1 minute with 0.25-1%
Triton X-100 in PHEM buffer (Schliwa and
van Blerkom, 1981
), then fixed for 1 hour in 2% paraformaldehyde
(Sigma) in PHEM buffer at room temperature and washed three times in PBS, pH
7.4, containing 10 mM EGTA, 2 mM MgCl2, 3% BSA, 0.1% Tween 20 (this
buffer was used for all subsequent steps). The cells were incubated for 1 hour
in the presence of the different primary antibodies (working dilutions: 1/5
for PL-3 and PL-5 mAb supernatants; 1/100 for AP-2 affinity-purified pAb;
1/200 for Anti-E pAb). After two washes in the same buffer, the cells were
incubated for 1 hour with secondary antibody (Alexa Fluor 488-conjugated goat
anti-mouse or Alexa Fluor 568-conjugated goat anti-rabbit; Molecular Probes)
at 1/100 to 1/200 dilution. Following three washes, cells were mounted in
Citifluor medium (City University, London, UK) and observed with conventional
epifluorescence (Leitz) or with a Biorad MRC 1024 confocal microscope equipped
with a Nikon Diaphot 300 inverted microscope and a krypton/argon laser
(Service d'Imagerie Cellulaire, Orsay, France). Z-series acquisition was
obtained with a Nikon Plan Apo 60x oil immersion objective, using
522/DF35 and 598/40 filters for green and red light, respectively. Individual
focal plane projections were saved as separate files, then merged and
colorized using Adobe Photoshop (Adobe Systems, San Jose, CA).
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Results |
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Identification of a gene encoding ß/-platein in the
expression library
We took advantage of the availability of antibodies against plateins to
screen a E. aediculatus expression library. Five positives phages
were obtained in the first round of anti-platein screening with anti-E serum,
three of which (W1,
W2 and
W3) remained positive after
further rounds of immunoscreening. The nucleotide sequence of each cDNA insert
shows an open reading frame (ORF) that is in-frame with the
ß-galactosidase sequence; the three ORFs largely overlap, yielding an
assembled total sequence encoding 553 a.a. that is rich in valine and glutamic
acid and displays numerous internal repeats. Probing a Southern blot of total
cellular DNA with the insert of phage
W1 revealed a single band at
around 2 kb (data not shown), indicating the existence of a macronuclear gene
(`minichromosome') with a coding capacity of approximately 666 a.a. (74
kDa).
To obtain the entire sequence of this protein, we screened a macronuclear
genomic library of E. aediculatus with the W1 insert as
probe. One of the six positive clones found in the first round of screening
was further analyzed and corresponds to the complete macronuclear molecule.
The insert is 2069 bp long and has the typical features of E.
aediculatus `minichromosomes': a single ORF of 1935 nucleotides (n.t.),
with short adenine and thymine (AT)-rich 5' leader (46 n.t.) and
3' trailer (65 n.t.) sequences. Most of the 5' duplex
C4A4 telomeric repeats are missing. The deduced 644 a.a.
sequence included the previously determined partial sequence from the
expression library. The correspondence of the sequence obtained with several
ß- and
-peptides (cf. Fig.
3) indicates that this gene encodes either ß- or
-platein; the macronuclear gene sequence contains ß-fragments f50,
f52, f55 (with a single mismatch), f69 and f72 (with two mismatches: E instead
of SS) as well as
-fragments f52, f72 and parts of fragments f43 and
f71. Very interestingly, one
-peptide (f71) resembles the COOH-terminal
part of the
W2 clone (EPVWTQPVVVEPAWTNPA), whereas the
corresponding sequence of the genomic clone is EPVWTQPVVVEPAWTQPV.
This suggests that ß- and
-platein proteins differ near their
C-terminal extremities, and that the insert of phage
W2 derives
specifically from a
-platein gene. The genomic clone sequence
determined (GenBank AY124991) thus probably represents a ß-platein
gene.
|
Identification of two -platein genes using PCR
A different approach, independent of the expression library, was used to
identify the genes encoding -platein. We took advantage of the
sequences determined for four
-platein-specific peptides
(Table 1), one of which (f55)
is quite long (31 residues), to devise a PCR strategy. On the basis of the
sequences of
-f55 and of
-f30 (14 residues), six oligonucleotide
primers (termed AP1-AP6) were designed in both forward and reverse
combinations (Table 2) and used
for PCR, with E. aediculatus DNA as the template. A single amplified
fragment of 1025 bp was obtained, using the primer combination AP2 + AP3. This
fragment was cloned and sequenced; within the derived a.a. sequence, peptides
corresponding to f30 and f55 were confirmed (although minor substitutions
occurred towards the end of the long f55 peptide). Additionally, the exact
sequence of peptide
-f42 was found, indicating that this amplified PCR
product corresponded to a portion of an
-platein coding region.
|
In order to obtain the sequence of the entire macronuclear gene encoding
-platein, we utilized a strategy termed RATE-PCR (rapid amplification
of telomere extremities) (Di Guiseppe et al., 2002), based on the organization
of Euplotes macronuclear genes as linear DNA molecules terminating in
telomeres of known sequence. The strategy used as primers two internal
oligonucleotides, termed AP7 and AP8, designed on the basis of the sequence
determined for the amplified fragment, in combination with an oligonucleotide
corresponding to the telomere sequence. Using this strategy, we cloned and
sequenced two fragments that both overlapped the original 1025 bp fragment;
this allowed the reconstruction of an entire macronuclear
-platein-encoding gene. This gene, named the
1-platein gene
(GenBank AY124989), consisted of a coding region of 1611 nucleotides, with
5' and 3' flanking regulatory regions of 87 and 317 nucleotides,
respectively (including the presumed euplotid telomeres). Within the deduced
a.a. sequence, a precise match for the fourth
-platein peptide (f45)
was found.
During the analysis of clones obtained from RATE-PCR, we found a second
clone that only partially overlapped the previously determined 1 coding
sequence. It differed in the 5' flanking noncoding sequence, thus
suggesting the existence of a second
-platein gene. Confirmation of the
C-terminal sequence of a second
-platein gene and details of its
3'-untranslated region were obtained by a RACE-PCR strategy, utilizing
E. aediculatus polyA+ RNA. The complete sequence of the
coding region of this gene, named the
2-platein gene, was determined
(GenBank AY124990). The existence of two
-platein genes was confirmed
by a Southern blot analysis of E. aediculatus macronuclear DNA. Using
the
1-platein coding region as probe, a tight doublet of bands appeared
(data not shown).
Evidence for platein gene expression
Macronuclear gene-sized molecules of hypotrich ciliates are usually
transcribed, and pseudogenes are rarely found. However, to determine whether
both -platein genes are truly expressed, we carried out northern blot
and RT-PCR analyses. Although the northern blot showed only a single band
(data not shown), it is likely that at this resolution two messengers of
similar size would overlap. Indeed, the RT-PCR experiment revealed the
expression of both messengers (Fig.
2A). The difference in their lengths confirms the presence of an
insertion of 40 a.a. in the
1-platein gene with respect to the
2-platein gene (cf. Fig.
3). From the clones produced using the amplified cDNA products,
150 were screened; those corresponding to
1 and
2 cDNAs were
equally represented, suggesting that the transcribed products of the two genes
are also likely to be equally represented.
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Since the original screen that uncovered the ß/-platein gene
sequence was performed on a cDNA expression library using anti-platein
antibodies, it is reasonable to assume that this gene is also expressed in the
cell. However, to demonstrate this directly, northern and RT-PCR analyses were
performed. Hybridization on a northern blot gave a single band at
approximately 1.9-2.0 kb (data not shown). Expression of the gene was also
confirmed by RT-PCR; amplification of reverse-transcribed RNA from vegetative
cells with a ß/
-specific pair of primers gave a major band at the
expected size (approximately 2 kb; Fig.
2B).
Sequence features of the encoded platein proteins
The ß/-platein gene sequence was the first to be obtained. The
predicted protein, of 74.9 kDa, displayed several interesting properties, as
first revealed by analysis of its a.a. composition. Particularly abundant
residues included valine, glutamate, threonine, proline and arginine; these
five a.a. accounted for more than 60% of its 644 residues. The four major
charged residues were notably abundant, in sum almost 30% of the total
protein; negatively charged a.a. (D + E) greatly outnumbered positively
charged (K + R), yielding a predicted pI of 4.88.
Conceptual translation of the 1- and
2-platein gene sequences
revealed encoded proteins (of 536 and 501 a.a., respectively) that were also
predicted to be highly acidic, and similarly rich in V, E, P and T. As shown
in Fig. 3, they are very
similar in overall sequence,
1 having one insert of 40 a.a. not found
in
2, and lacking one 5 a.a. insert found only in
2. In contrast
to their overall highly charged, acidic backbones, the N-termini of the
plateins are by far the most hydrophobic portions of the molecules. When
evaluated by the SignalP V1.1 program
(Nielsen et al., 1997
), the
N-termini of all three sequenced plateins meet the criteria for canonical
starttransfer signal peptides. This program also predicts the most
likely signal cleavage sites, indicated for
1 and
2 in
Fig. 3. The N-terminal peptide
sequence from authentic
-platein has been determined directly. Its
sequence is GEAATPKAAATGS[t][t]A[q]V, where
[x] indicates an uncertain assignment. A corresponding sequence is found
(underlined) in our derived ß/
-platein, beginning with residue 25:
GEAATPKAAATGSTKAPV. This correspondence
provides strong evidence that the predicted signal sequence is indeed cleaved
from the mature platein in vivo. The non-matching residues later in the
respective peptides lend slightly more weight to the suggestion (made above)
that the `ß/
' sequence determined actually represents a
ß-platein gene.
A search for potential phosphorylation sites in the platein sequences was
performed, using the NetPhos 2.0 program
(Blom et al., 1999). Numerous
residues were identified, particularly in ß/
-platein, that show a
strong likelihood of being phosphorylated (output values
0.9); these sites
are highlighted in Fig. 3.
When the predicted -protein sequences were searched against the
BLAST database (Altschul et al.,
1997
), a potential relative was identified: a cytoskeletal protein
from E. gracilis named articulin
(Marrs and Bouck, 1992
).
Similar articulins from the ciliate P. dubius have been identified
and sequenced (Huttenlauch et al.,
1998a
). The primary articulin characteristic is a core of 12-a.a.
repeats, with a VPV... consensus. Consequently, the platein sequences were
scanned, and all three were constructed along a similar plan
(Fig. 3). For example,
1-platein can be arranged with 28 VP-initiated tandem repeats
dodecamers, with some degeneracy (in the form of 8-, 10- or 14-residue units)
and
2 has 24 similar primary repeats. In
ß/
-platein, 316 central residues (nearly half of the total
molecule) can be arranged in 27 such repeats, most of them 12 a.a. in length.
Rather than VPV, most
-platein core repeats in this arrangement
initiate with VPH or VPR, and ß/
-repeats with VPE or VDE (cf.
Table 3).
|
Another characteristic of the known articulin sequences is the presence of
a set of shorter secondary repeat sequences. A search through the
ß/-platein sequence indeed showed additional repeats C-terminally.
These secondary repeats are most easily arranged as 17 proline-initiated
pentamers; included are three exact tandem decapeptides and three exact tandem
pentapeptides (Fig. 3). In the
-plateins, secondary repeats are represented by 15 (
1) or 14
(
2) proline-initiated pentamers, similar in sequence to those of
ß/
-platein. Notably, these secondary repeats in the
-platein isoforms are located on the N-terminal side of the primary
core 12-mer repeats, instead of C-terminally as in ß/
-platein. In
fact, the primary `core' VP-rich dodecamer repeats in both
-plateins
are not at all central, but reside within seven residues of the respective
C-termini.
- and ß/
-plateins co-localize but show differences
in solubility
In the course of studies on the immunofluorescence localization of the
plateins in cells, it became apparent that the pattern of antibody staining
was dependent upon the concentration of the membrane permeant (Triton X-100)
used during cell processing. Therefore, we analyzed the staining pattern of
interphase cells under two conditions of permeabilization, 0.25% and 1% Triton
X-100. After permeabilization with the lower concentration of Triton, the
plates were fully decorated with the affinity-purified serum AP-2 (specific
for -platein), but only partially decorated with the two mAbs PL-3,
specific for the ß/
-plateins, and PL-5, which recognizes all three
plateins on immunoblots (Fig.
4A,B). When the Triton concentration was increased to 1%, the
plates were no longer clearly demarcated with the AP-2 antibody; the cell
surface stained uniformly but less intensely, and many small vesicles were
detected in the cytoplasm (Fig.
4C). The plates were fully stained with the PL-3 antibody under
these more-stringent extraction conditions
(Fig. 4D). These results
suggest that the AP-2 target is located on proteins (
-plateins) that
are at least partly solubilized by the same treatment that retains the PL-3
epitope (presumably on ß/
-plateins), now fully accessible within
the plates. Under these conditions, the PL-5 antibody gave a pattern on
interphase cells (full plate staining) similar to that observed with the PL-3
antibody.
|
The accumulation of plateins in new APs formed during cellular reproduction
was also followed. The pattern of appearance of new plates during pre-division
morphogenesis has been described in detail from silver-stained preparations
(Chatton and Seguela, 1940;
Wise, 1965
;
Ruffolo, 1976
). Briefly, plate
assembly follows a two-step process. New miniature plates first appear in
close association with proliferating basal bodies, both on the ventral and the
dorsal sides; these new APs then gradually enlarge and spread across the cell
surface, while parental plates are resorbed. This process leads to a complete
renewal of the ventral surface, except in the oral area where old plates are
retained and passed to the anterior daughter cell. On the dorsal side, where
basal body duplication begins in the equatorial region of the ciliary rows,
only two-thirds of the APs are initially replaced. Immunofluorescence suggests
that the two platein forms (
vs. ß/
) in these new plates do
not exhibit the same behavior. As in interphase cells, some
-plateins
are partially solubilized: with the AP-2 antibody, the staining of the new
plates even after mild (0.25% Triton) extraction appeared reticulated against
a fluorescent background (Fig.
5A). These plates were not stained at all after 1% Triton
pre-treatment. By contrast, the new plates were fully stained with the PL-3
antibody after both 0.25% (Fig.
5B) and 1% Triton extraction. This indicates that the
ß/
-plateins are less soluble than
-plateins in assembling
plates, as well as in fully formed ones. It appears, however, that the
ß/
-epitopes are more accessible in newly forming plates than they
are in mature APs.
|
![]() |
Discussion |
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Notable unifying features of the articulins are summarized in Table 4, along with features of plateins that distinguish them from previously characterized articulins. The following sections highlight points that deserve special attention.
|
The primary (`core') articulin domain
An a.a. consensus in the tandem 12-mer repeats determined for the ciliate
Pseudomicrothorax (and which also seems to typify the
Euglena articulins) shows alternating V and P residues, with those
residues not conforming strictly to the V or P positions often representing
charged a.a. in an alternating + and consensus arrangement,
respectively (Huttenlauch et al.,
1998a) (Table 3). A
general conservation within this repetitive core domain almost certainly
accounts for the observed crossreactivity of mAbs with different articulin
forms, between similar (Williams,
1991
; Kloetzel et al.,
1992
) and even distantly related species
(Viguès et al., 1987
;
Bricheux et al., 1992
;
Curtenaz et al., 1994
;
Huttenlauch et al.,
1998b
).
While each Euplotes platein shows the hallmark articulin repeat
motif of VP-rich 12-mers (ranging from 24-28 repeats in the three sequenced
molecules), the consensus of these repeats clearly differs from the VPVPV...
motif. The platein consensi are shown in
Table 3, with articulin 1 from
P. dubius for comparison. Each platein type shows a consensus
`fingerprint' that differs for the ß/-platein versus the two
-plateins; each in turn differs in significant ways from the P.
dubius consensus. Notable are the strong preferences for acidic residues
at distinct positions (e.g., glutamate in position 3 of
ß/
-platein, positions 6, 9 and 10 of
-plateins). Thus, the
tendency for positively and negatively charged residues to alternate in the
P. dubius consensus is not followed in the plateins; acidic residues
can show strong preferences for occupying adjoining positions in the platein
consensi. This reflects the overall much higher proportion of acidic residues
in the plateins compared with other articulins (for example, almost a quarter
of the residues in the ß/
-platein core repeat domain are aspartate
or glutamate). The
-plateins in particular are highly acidic, with net
charges within the core domains alone of -50 and -44 for
1 and
2, yielding predicted pIs for those regions of 4.43 and 4.39,
respectively (cf. Table 4). By
comparison, the most acidic of the described articulins, P. dubius
articulin 4, has only a -19 net charge within its even longer primary
domain.
The cortical alveoli, where APs are assembled, have been shown to be
Ca2+ ion reservoirs in some ciliates
(Stelly et al., 1991;
Plattner et al., 1997
;
Plattner and Klauke, 2001
). It
thus seems reasonable to suggest that the abundant acidic residues in the
platein core domain may function in Ca2+ binding, or even that
Ca2+ ions might be included within Euplotes APs as part of
their polymerization process. In another ciliate, Coleps, calcareous
scales have been shown to assemble within the cortical alveoli
(Huttenlauch, 1985
).
The second repetitive articulin domain
In described articulins, a secondary repeat domain is found that is shorter
in the number of repeats and in the length of each repeating unit. In E.
gracilis (Marrs and Bouck,
1992), these repeats number four in each protein, are heptads with
a general consensus of APVT..., and can be located within either the
N-terminal portion of the molecule (articulin 86) or the C-terminal portion
(articulin 80). The P. dubius short repeats are glycine-rich
hexamers, are more numerous (13 and 9 repeats for articulin 1 and 4,
respectively), and are located near the C-termini of both forms
(Huttenlauch et al.,
1998a
).
A second repetitive motif region is also found in Euplotes
plateins; however, the nature of the repeating units is novel. The most
readily discerned repeats can be read as proline-initiated pentamers: 15
repeats in 1-, 14 in
2-, and 17 in ß/
-platein
(Fig. 3). There is no single
consensus, although PAW and PVW are common repeat triplets. Notable is the
absence of glycines, prevalent in the secondary repeat domain of the other
ciliate articulins (those of P. dubius), and the general
proline/tryptophan richness of this region. One striking difference between
the
- and ß/
-plateins is in the overall design of the
molecule; in both
1 and
2, the P-rich pentamer domain is
N-terminal to the primary core of 12-mer repeats, whereas in
ß/
-platein, the pentamer domain is on the opposite side of the
primary core, near the C-terminus.
Anomalous retardation in electrophoretic mobility
The E. gracilis articulins, with apparent molecular masses of 80
and 86 kDa on SDS-PAGE gels, represent proteins whose predicted molecular
masses (from the cloned genes) are about 72 kDa each. Articulins 1 and 4 from
P. dubius migrate more aberrantly; their SDS-PAGE mobilities indicate
proteins of 78-80 kDa, while their derived Mrs are 69.7
and 59.9 kDa, respectively. The mobilities of the plateins in SDS-PAGE are
even more significantly retarded: -plateins migrate with an apparent
Mr of 125 kDa, yet the proteins derived from their cloned
genes predict proteins with Mrs of about 61 kDa (
1)
and 56.3 kDa (
2). While ß/
-plateins migrate at 95-99 kDa by
SDS-PAGE, the derived protein predicts a mass of about 73 kDa. This anomalous
electrophoretic behavior may be related to the high proportion of charged
residues in these proteins. Gumpel and Smith
(Gumpel and Smith, 1992
) found
that an acidic repeat protein from E. gracilis (with an estimated pI
of 3.56) showed similarly retarded gel migration, interpreted to be due to the
high content of acidic residues in the protein. By contrast, the E.
gracilis articulins, with relatively balanced charged residues and
predicted pIs near 8.0, also show SDS-PAGE retardation. Thus, it is possible
that the abundance and regular spacing of proline residues is the significant
feature affecting gel retardation of the articulins. Both charged residues and
prolines might contribute to a persistent secondary structure that is retained
during electrophoresis. Neural-net-based secondary structure prediction
programs (Rost, 1996
) suggest
that the platein molecules exist primarily in an extended form.
Post-translational modifications of the plateins could be an alternative
(or additional) explanation for their anomalous gel migration. One report
(Böhm and Hausmann, 1981)
suggests that APs in E. vannus are coated with a material that reacts
cytochemically with polysaccharide stains. While the protein sequences derived
here from the platein genes reveal no consensi for N-terminal glycosylation,
O-glycosylation prediction programs
(Hansen et al., 1998
)
highlight a large number of potential sites, mostly located N-terminally (see
below). It remains for future biochemical work to determine whether such
glycosylation (or any other post-translational modifications) in fact
occur.
Signal peptides
All three plateins in E. aediculatus are distinguishable quite
clearly from the previously described articulins by their putative N-terminal
signal sequences. Unlike these other articulins (and indeed all other known
cytoskeletal proteins), which typically are assembled free in the cytoplasm,
plateins are polymerized into structural elements (APs) within membrane-bound
cisternae, the cortical alveoli. The N-terminal sequence of E.
aediculatus -platein, determined directly here, matches the
sequence of the derived ß/
-platein gene product, minus its first
24 residues (i.e., starting with residue 25; cf.
Fig. 3). Similarly, the
N-terminal sequences of two platein proteins extracted from E.
eurystomus have been determined (N. Williams, personal communication);
residues 4-11 of the upper platein band of this species are identical to those
same sites in the E. aediculatus
1-platein sequence reported
here, if the signal peptide predicted by the SignalP computer program is first
removed. These results provide strong experimental support for the proposal
that the postulated N-terminal signal sequences of both
- and
ß/
-platein forms are indeed cleaved to yield the mature
proteins.
The presence of signal peptides on the plateins correlates well with their
final intra-alveolar location, and raises questions for future work concerning
the modes of plateins' synthesis, intracellular trafficking and polymerization
into cytoskeletal plates. At this point, it will only be mentioned that many
proteins similarly rich in proline residues have been shown to form strong
`interlocking networks' (Williamson,
1994), which has evident implications for the assembly and
functioning of plateins (and other articulins) as cortical cytoskeletal
elements. The assembly state of such proline-rich protein networks is known in
many cases to be affected by reversible phosphorylation of the proteins
(Williamson, 1994
). Protein
kinases can function within membrane-bound organelles of the secretory pathway
(Drzymala et al., 2000
); since
the plateins predict significant numbers of phosphorylable residues (cf.
Fig. 3), it needs to be
determined whether plateins are in fact phosphoproteins, and if so whether
their phosphorylation state varies as fields of new cortical plates are
assembled during pre-division morphogenesis. Regulation of the assembly state
of another important cytoskeletal element (ciliary rootlets) by reversible
phosphorylation has been shown to occur in Paramecium
(Sperling et al., 1991
).
Platein/articulin domains appear evolutionarily conserved
When used as queries in BLAST homology searches
(Altschul et al., 1997), the
-platein sequences identify the articulins from Euglena, then
Pseudomicrothorax, among the highest scoring matches. However, BLAST
searches using the full ß/
sequence reveal no described articulins
among the first 100 responses; only if the ß/
core alone is
submitted does a known (Euglena) articulin appear, well down the
list. These results indicate that the
-plateins are more-closely
related to `ancestral' articulins, and suggest that the
ß/
-plateins are products of
-platein gene duplications that
have diverged significantly.
With the domain architecture of the plateins somewhat clear
(Fig. 3), and with the
increasing availability of fully sequenced genomes, it has proven instructive
to narrow homology searches by using only selected domains as BLAST queries.
Submitting the major articulin feature of -platein, the VP-rich core
repeats, identifies potential homologs in virtually all taxa, ranging from
bacteria to humans. For example, a predicted VP-rich Drosophila
protein (Adams et al., 2000
)
with a pronounced domain of 12-mer repeats and a likely N-terminal signal
(AAF57876) yields a higher BLAST score than do even the other ciliate (P.
dubius) articulins. Some vertebrate proteins may similarly employ
platein-like domains. One projected human protein (XP_092855) possesses 18
dodecamer repeats (most VP-initiated); another high-scoring protein predicted
from the human genome database (Hs6_7569) contains a core of 25 IP-initiated
12-mer repeats. Both of these human proteins show suggestions of membrane
association (predicted transmembrane helices). Most of the many putative
articulin homologs uncovered are of unknown function or localization; however,
it seems likely that they might assemble to perform cytoskeletal roles, as
demonstrated for the articulins. If true, this would provide another instance
[as in the case of the centrins (Salisbury
et al., 1984
; Chapman et al.,
2000
)] in which the identification and molecular characterization
of new proteins from protists can prove useful in functional genomic studies
of other organisms. As one example, a prokaryotic homolog of articulins has
been found encoded within the recently sequenced genome of Caulobacter
crescentus (Nierman et al.,
2001
). This protein (AAK22660; denoted a `putative articulin')
predicts an N-terminal signal sequence, suggesting that it might function
(structurally) at the plasma membrane or within the periplasmic space.
Performing platein domain homology searches with the secondary P-pentamer
repeat motif results in an entirely different set of responses. Particularly
notable homologies are found among many insect proteins, typically secreted
structural proteins such as those of the chorion or cuticle, or other
secretory products (e.g., Drosophila salivary proteins). One example
is peritrophin-95, an acidic secreted protein with 18 P-initiated pentamers at
its C-terminus, which forms an extracellular mesh (peritrophic matrix) lining
the gut of a larval dipteran (Casu et al.,
1997). Other proteins with prominent domains of proline-initiated
pentameric repeats (often referred to as extensins or proline-rich proteins,
PRPs) are commonly found in plant cell walls (cf.
Hong et al., 1987
;
Muñoz et al., 1998
).
The proline repeats in these wall proteins are presumed to form extended
domains playing roles both structurally (stiffening the extension) and in
binding rapidly and tightly to other proteins (cf.
Williamson, 1994
).
Even the N-termini of all three plateins (internal to the signal sequences)
have an unusual common property; four a.a. (A, T, P, K) make up 80% of these
short (50-60 residue) domains, which are correspondingly very basic (unlike
the primary cores). BLAST searches reveal that sequences with this simple
composition are characteristic of various mucins (heavily O-glycosylated
secretory proteins) (Hanisch,
2001). The NetOGlyc 2.0 program
(Hansen et al., 1998
)
identifies numerous T residues clustered within the N-terminal platein domains
that predict a high likelihood of being O-glycosylated; such clustered sugar
adducts could aid in stiffening this end of the plateins. Since
O-glycosylation occurs in the Golgi apparatus
(Hanisch, 2001
), it would not
be expected for cytoplasmically localized articulins.
Plateins thus can be viewed as composites of a modified (more anionic) articulin core domain, together with other domains differing significantly from those of the previously noted cytoplasmic articulins. The altered molecular architecture of the plateins (including their signal sequences) is most probably related both to their different synthetic/trafficking paths (more like those of secretory proteins) and to the unique intra-alveolar environment within which the final assembled cytoskeletal product is formed.
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