We have isolated a full-length murine clone
corresponding to the rat neuronal p1A75 partial cDNA
(Sutcliffe, J. G., Milner, R. J., Shinnick, T. M., and Bloom, F. E. (1983) Cell 33, 671-682). It
encodes a 185-residue polypeptide that displays 56% identity with p19,
a protein selectively expressed in the Golgi apparatus of neural cells
(Sabéran-Djoneidi, D., Marey-Semper, I., Picart, R., Studler,
J.-M., Tougard, C., Glowinski, J., and Lévi-Strauss, M. (1995)
J. Biol. Chem. 270, 1888-1893). An antibody directed against the recombinant polypeptide allowed us to demonstrate the
existence of the natural 21-kDa protein (p21) in brain and its
prominent juxtanuclear Golgi-like localization in cultured neurons.
Ultrastructural observation of cultured neurons and analysis of
transfected COS cells revealed a specific labeling of the Golgi apparatus, suggesting, as for p19, the presence of a Golgi targeting signal in its primary sequence. Surprisingly, p21, which is much more
strongly expressed in the olfactory epithelium than p19, is also
present in the Golgi complex of spermatocytes and in the flagellar
middle piece of late spermatids.
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INTRODUCTION |
We have previously described a 19-kDa murine protein (p19)
selectively expressed in the Golgi apparatus of neural and
neuroendocrine cells whose human corresponding gene has been localized
in 5q35 (1, 2). The primary sequence of p19 showed a 57% similarity with the translation product of an open reading frame of the rat neuronal p1A75 (3) partial cDNA, which was isolated 14 years ago
and whose human corresponding gene is localized in 4p16 (4). Searches
in protein data bases for other members of this new family have only
revealed that these two proteins share a highly similar short segment
with secretogranin III, which is expressed in intracellular vesicles of
neural cells (1, 5).
The co-localization, in two paralogous chromosomal regions (5q35 and
4p16), of the human p19 and p1A75 genes with other couples of
homologous genes such as, for instance, the D1 and D5 dopamine receptors and the FGFR3 and FGFR4 fibroblast growth factor receptors suggested that these genes originated from the same large gene duplication event, which is thought to be the remnant of an ancient round of tetraploidization (6).
To characterize this new protein family and to study the functional
consequences of a well defined large duplication event, we undertook
the thorough analysis of the protein encoded by the p1A75 cDNA.
This encoded protein is a 21-kDa protein (p21) that is expressed, like
p19, in the Golgi apparatus of neural and neuroendocrine cells.
However, unlike p19, which is absent from the testis and faintly
expressed in the olfactory epithelium, p21 is very strongly expressed
in the olfactory epithelium and in male germ cells.
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EXPERIMENTAL PROCEDURES |
RNA Isolation and Northern Blotting--
Total cellular RNA was
extracted from fresh tissue or cells by the guanidium
thiocyanate/phenol chloroform extraction method (7). Timed pregnant OFA
rats (Iffa-Credo) provided a source of fetal and neonatal brains of
precise gestational or postnatal ages. Other brain structures or
non-neural tissues were dissected from adult Sprague-Dawley (Charles
River) male rats. Dissociated neurons from cerebral hemispheres of
embryonic day 17 rat embryos were plated at high density (6 × 104/cm2) according to Di Porzio et
al. (8) and cultured for 2-4 days; astrocyte cultures were
prepared from the same cerebral areas as described by Denis-Donini
et al. (9).
Total cellular RNA (5 µg/lane) was fractionated on 1.2% agarose gel
containing 3.7% (w/v) formaldehyde, using standard procedures (10).
Gels were blotted onto Hybond N (Amersham Corp.) nylon membranes. The
18 and 28 S ribosomal RNAs observed on the filter by UV light were used
to check that equal amounts of RNA were loaded on each lane.
Hybridization conditions were as follows: 65 °C, 16 h in the
presence of 2 106 cpm/ml of probe in the 5× SSC, 5×
Denhardt's solution, 50 mM sodium phosphate, pH 6.5, and
0.4% SDS. Final washes were done in 0.2 × SSC and 0.1% SDS at
65 °C.
In Situ Hybridization--
Whole heads from 3-day-old
Sprague-Dawley rats were sectioned on a cryostat, and the sections
(10-20 µm thick) were thaw-mounted on silanized glass slides, fixed
with 4% paraformaldehyde, dehydrated, and stocked at
20 °C until
use. Frozen sections were thawed, fixed with paraformaldehyde,
permeabilized with Pronase, and again fixed with paraformaldehyde.
Prehybridization and hybridization were performed at 42 °C
using an oligodeoxynucleotide
(CTCTGCAGCTTCGGTCTCCTGTTCCGACAGCTTCTCTTCTGA) labeled with
[
-33P]dATP and terminal transferase according to
Burgaya et al. (12). Control experiments were performed in
the presence of a 10-fold excess of unlabeled probe. Finally, sections
were dehydrated and exposed to
-Max films (Amersham) for 1-3
weeks.
Characterization of the p21 cDNA--
The cDNA library
was constructed in
gt10, as described previously (13), from
poly(A)+ RNA prepared from cerebral hemispheres of newborn
BALB/c mice. A p21 cDNA probe was amplified by nested RT-PCR from
rat brain cDNA using the four following primers designed using the
published p1A75 sequence (3): first PCR, AATCACTACAACCTGGCCAA and
GGCTTTCTTCCTATCTAGCA; second PCR, ATCACGCGCTCAGTGTCA and
ATCCTCGTGTTCTGCGCA. This cDNA, labeled by random priming (11), was
used as a probe to screen 2 × 105 cDNA clones.
Among 104 positive clones, 15 were analyzed by Southern blot, and the
insert of the largest one was subcloned in the pBluescript plasmid
vector. Both strands of this cDNA (pBS64) were sequenced using the
dideoxynucleotide chain termination technique (14) and the modified T7
polymerase (15) using internal primers.
Recombinant p21 Protein and Antibody Production--
The plasmid
containing the p21 cDNA was amplified with Taq DNA
polymerase (16) using the two following primers: A
(5'-TTCGGATCCATGGTGAAGTTGGGGAATTTC-3') and B
(5'-TAAGGATCCTGCCCGCTTGCTA-3'). These primers were designed to add
BamHI restriction sites on both extremities of the p21 coding sequence. The amplified DNA fragment was restricted using BamHI endonuclease and cloned in the BamHI site
of the pQE-30 (Qiagen) expression vector, which produces a recombinant
protein with a His6 tag on the N terminus. The construction
was sequenced to verify the absence of mutations, and the synthesis of
the recombinant protein was demonstrated by immunoblot using an
antibody directed against a synthetic peptide (HYNLAKQSITRSVSPWMS) that
corresponded to residues 149-166 of p21 and whose sequence was deduced
from the rat cDNA (3). The recombinant p21 protein was produced and
purified using nickel-chelate affinity chromatography (17) in
denaturing conditions according to the protocols provided by Qiagen.
The recombinant protein (0.5 mg) solubilized in 8 M urea was loaded onto a 14% preparative SDS-polyacrylamide gel
electrophoresis. The gel was stained in 0.25 M KCl, and the
band corresponding to the p21 protein was cut and washed in
phosphate-buffered saline. New Zealand White rabbits were immunized
three times subcutaneously with a piece of gel containing approximately
100 µg of protein.
Subcellular Fractionation and Western Blot
Analysis--
Subcellular fractions were prepared from rat postnatal
day 1 brain according to Huttner et al. (18). Briefly,
neonatal rat brains were homogenized in buffered sucrose (320 mM sucrose, 4 mM Hepes, pH 7.4) using a
glass-Teflon homogenizer. This homogenate was centrifuged for 10 min at
800 × g; the pellet was discarded, and the supernatant
was centrifuged for 15 min at 10,200 × g to yield a
pellet (P2) and a supernatant (S2). The S2 fraction was then
centrifuged for 1 h at 165,000 × g to yield a
pellet (P3) fraction and a cytosolic fraction (S3). Equivalent
fractions of each preparation, corresponding to 100 µg of homogenate,
were run on a 14% SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes (Bio-Rad) (19). Carbonate extractability was tested on the P2 fraction as described previously (20). Briefly, pellets were resuspended in a large volume of ice-cold
100 mM Na2CO3 buffer, pH 11.5 (or
phosphate-buffered saline for the control experiment), incubated for
1 h on ice, and centrifuged for 1 h at 200,000 × g. After saturation with 5% nonfat dry milk and
0.05% Tween 20, membranes were incubated with a 1:10,000 dilution of
the rabbit serum followed by a goat anti-rabbit IgG coupled to
peroxidase. Enzymatic activity was revealed using a chemiluminescence
detection kit (Amersham).
Cell Cultures, Transfection, and
Immunofluorescence--
Dissociated neurons from cerebral hemispheres
of embryonic day 17 rat embryos were plated in 35-mm culture dishes at
high density (6 × 104/cm2) in serum-free
medium according to Di Porzio et al. (8) and cultured for 8 days. Neurons were seeded on glass coverslips and fixed with
periodate/lysine/paraformaldehyde (21) for 2 h at room
temperature, permeabilized with 0.005% saponin, and
immunocytochemically stained using the rabbit anti p21 antiserum or
preimmune serum diluted 1:500 and then goat immunoglobulins (IgG)
against rabbit IgG labeled with tetramethylrhodamine (Biosys,
Compiègne, France). For transient transfection experiments, the
BamHI-BamHI restriction fragment used for
prokaryotic expression in the vector pQE-30 (see above) was excised and
inserted in the pcDNA3 eukaryotic expression vector (Invitrogen).
Transfections of COS-7 cells with this construction were performed on
polyornithine-treated glass coverslips using the DOTAP (Boehringer
Mannheim) protocol and reagents. 48 h after transfection, cells
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and stained with anti-p21 antiserum followed by goat anti-rabbit
IgG labeled with fluorescein (Biosys).
Immunohistochemistry of Testis Prints and Isolated Germ
Cells--
Immunocytochemistry was performed on mouse testis prints
and isolated germ cells obtained by seminiferous tubule dilaceration. Prints and cells were fixed for 15 min with 1% formaldehyde in phosphate-buffered saline containing 3% sucrose, treated for 5 min
with Triton X-100, and incubated for 1 h in 5% fat milk in phosphate-buffered saline. Cell preparations were incubated for 40 min
with preimmune serum or anti-p21 antiserum diluted 1:750 and
immunolabeled using biotinylated donkey anti-rabbit immunoglobulins and
streptavidin-biotinylated horseradish peroxidase complexes (Amersham).
Enzymatic activity was revealed using aminoethyl carbazole as a
chromogen, and the cell preparations were counterstained with Harris
hematoxylin and mounted in aqueous medium (Glycergel, Dakopatts).
Stages of mouse spermatogenesis were identified according to Russell
et al. (22).
Immunoperoxidase Electron Microscopy--
The immunoperoxidase
procedure was performed on dissociated neurons using a preembedding
approach, in situ in the Petri dishes, as described
previously (23). Briefly, cells were fixed with periodate/lysine/paraformaldehyde (as described above) and
permeabilized with 0.005% saponin before incubation with the rabbit
anti-p21 antiserum and then with sheep IgG against rabbit IgG labeled
with peroxidase (Institut Pasteur, Paris). After postfixation in 1% glutaraldehyde, detection of peroxidase activity, and postfixation in
1% osmium tetroxide, cells were embedded in situ in Epon
according to Brinkley et al. (24). After observation at the
light microscopic level, selected areas of immunoreactive cells were
sectioned, and ultrathin sections were examined under the electron
microscope without further staining.
 |
RESULTS |
Cloning and Sequence Analysis--
Since the previously described,
1126-base pair-long p1A75 cDNA (3) is shorter than the
1700-base-long mRNA on which it hybridizes on a Northern blot (3),
we decided to isolate a full-length cDNA. To this end, our newborn
mouse (BALB/c) brain cDNA library (13) constructed in the
gt10
phage vector was screened with a probe synthesized by nested reverse
transcription-polymerase chain reaction from rat brain cDNA and
corresponding to nucleotides 136-881 of the p1A75 cDNA (3). Among
15 positive clones analyzed by Southern blot, the clone pBS64 had the
largest cDNA insert. Its length (2083 bases without the poly(A)
tract) corresponded to our estimation of the size of the rat mRNA
whose migration was slightly faster than that of the 18 S rRNA (see
below). The cDNA terminates by a poly(A) tract preceded by a
typical polyadenylation signal (AATAAA) at position 2062 (Fig.
1A). The longest open reading frame encodes a polypeptide of 185 residues with a predicted molecular mass of 21 kDa. This open reading frame is initiated by the first ATG
(position 53) surrounded by a sequence matching very well the proposed
consensus for the initiation of translation (25) (consensus,
CC(A/G)CCATGG; p21cDNA, CAACCATGG).

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Fig. 1.
A, nucleotide sequence of the murine p21
cDNA and translation of its longest open reading frame. The stop
codon (position 608) and polyadenylation signal (position 2062) are
underlined. B, alignment of the p21 and p19 (1)
murine sequences. A double bar indicates an identity, and a
single bar indicates a replacement by an isofunctional amino
acid.
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Comparison of the complete sequence of this polypeptide with that of
p19 (1) confirmed their highly significant similarity (Fig.
1B). If one excepts an insertion of 12 residues in p21
(residues 74-85) (16 for p19 and 19 for p21), these two
polypeptides display 56% identity (96 of 171 identical residues)
(Fig. 1B).
Study of hydropathy of this polypeptide by the method of Kyte and
Doolittle (26) indicated that it is moderately hydrophilic with the
exception of two adjoining stretches, being, respectively, strongly
hydrophilic (residues 47-68) or hydrophobic (residues 83-103) (Fig.
2). The N terminus of the polypeptide
does not fit the outline proposed for signal sequences
(27).
Expression of the p21 mRNA--
The p21 mRNA was further
characterized by Northern blot analysis (Fig.
3, A and B) and
in situ hybridization (Fig.
4). The p21 cDNA probe recognized an
apparently unique mRNA with a migration rate slightly faster than
the 18 S ribosomal RNA (Fig. 3A). Its pattern of
hybridization to RNAs from a variety of rat tissues revealed an absence
of expression in liver, spleen, kidney, and heart and a strong but
variable signal in all brain and spinal cord samples tested (Fig.
3A). The p21 mRNA was also strongly expressed in the
pituitary, in a crude preparation of the olfactory epithelium, and, to
a lesser extent, in the adrenal gland and in the testis (Fig.
3A). Comparison of the p21 hybridization signal in RNA
samples extracted from cultured astrocytes or neurons suggested a
neuronal origin for this RNA, which was absent from the astrocyte culture (Fig. 3A). As shown in Fig. 3B, the p21
mRNA was present as early as embryonic day 14 in cerebral
hemispheres, where its expression increased during embryogenesis. The
maximal abundance of this mRNA was found around embryonic day 20, and its expression faded afterward to reach adult levels at P15 (Fig.
3B).

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Fig. 3.
Expression pattern of p21 mRNA.
A, Northern blot of total RNA samples from various rat
organs, brain structures, or cultured cells using the p21 cDNA
probe selected for the screening of the cDNA library. The migration
of the 18 and 28 S ribosomal RNAs is indicated, and the hybridization
of the same blot with a p19 probe is shown (reproduced from Ref. 1)
(4-day exposure of the autoradiogram). B, Northern blot of
total RNA samples from rat cerebral hemispheres from various embryonic
(E) or postnatal (P) ages (overnight exposure of
the autoradiogram).
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Fig. 4.
In situ hybridization to p21
mRNA. Horizontal section of a 3-day-old rat head. Note the
labeling of the olfactory epithelium and of the retinas.
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Since the olfactory epithelium RNA sample was extracted from a crude
homogenate of the inside of nasal cavities, attempts were made to
further demonstrate that the marked p21 mRNA expression occurred
indeed in the epithelium. We therefore performed an in situ
hybridization experiment on a postnatal day 3 horizontal section of a
rat head. As expected, an intense labeling was observed in the
olfactory epithelium (Fig. 4). Moreover, the brain area and the retinas
were clearly labeled.
Expression of the p21 Protein--
Western blot analysis of p21
expression using rabbit antiserum raised against recombinant p21 showed
the existence of a single band in the brain of the postnatal day 1 rat
(Fig. 5A). The migration level
(21 kDa) of the corresponding protein corresponded exactly to the
calculated molecular mass of p21. p21 was absent from the soluble
cytosolic fraction and was found at comparable levels in the
postnuclear pellet (P2) and the supernatant (S2), in which it was fully
associated with the pellet (P3) obtained after ultracentrifugation (Fig. 5A). A carbonate treatment, which allows
discrimination between integral and peripheral membrane proteins (26),
was also performed on the P2 fraction (Fig. 5B). Fig.
5B shows that p21 is not released from the P2 fraction
following carbonate treatment. Control experiments, performed with an
anti-synapsin antibody indicated that this peripheral protein is, as
expected, completely extracted from the P2 pellet by the carbonate
treatment (Fig. 5B).

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Fig. 5.
Immunoblot detection of p21 in subcellular
fractions of a postnatal day 1 rat brain homogenate. A,
fractions equivalent to 100 µg of homogenate were loaded on each
lane. H, homogenate; S2, postnuclear supernatant;
P2, postnuclear pellet; S3, cytosol; P3, ultracentrifugation pellet. The migration of the
molecular mass markers is indicated. B, carbonate
extractability of p21. The P2 fraction was treated with sodium
carbonate (+) or with phosphate-buffered saline as a control ( ) as
described under "Experimental Procedures." Immunodetection of
synapsin is used as a control for the efficiency of the carbonate
treatment.
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Cellular and Subcellular Localization of p21--
In primary
neuronal cultures from the embryonic striatum and cerebral cortex, the
anti p21 antiserum labeled a prominent juxtanuclear Golgi-like area in
the majority of neurons (Fig. 6). In
contrast, the few contaminating glial cells were devoid of
immunolabeling (not shown). Analysis of the same preparation by
immunoelectron microscopy showed a specific labeling of all the
saccules and of some vesicles in the Golgi zone (Fig.
7A). The perinuclear cistern
was also labeled (Fig. 7C) as well as some dispersed small vacuolar structures and multivesicular bodies in the cell body or in
neurites (Fig. 7B).

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Fig. 6.
Immunofluorescence of cultured rat neurons.
A, the juxtanuclear area of the majority of neurons is
conspicuously labeled with the anti-p21 antiserum. B,
absence of labeling with the preimmune serum. Bars, 10 µm.
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Fig. 7.
Ultrastructural localization of p21 in
cultured neurons. A, the stack of Golgi saccules is labeled
as well as a small vesicle (arrowhead) in the Golgi zone
(G). B, two labeled multivesicular bodies.
C, a conspicuous labeling of the perinuclear cisterna. Bars, 0.5 µm.
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An immunocytochemical analysis of testis prints and isolated germ cells
was performed to identify, within this highly heterogenous tissue, the
p21-expressing cell type. Fig.
8A shows a labeling of the
Golgi complex of primary spermatocytes as early as the pachytene stage.
The Golgi complex was found to be stained at all the subsequent stages
of meiosis (data not shown). In mature spermatids, both the whole
acrosomal region and the flagellar middle piece were labeled (Fig.
8C). At this latter level, the staining was localized in the
flagellar layer surrounding the axial structures (i.e.
axoneme and dense fibers) with a periodical transversal pattern related
to the mitochondrial sheath. No staining of the spermatocytes and
spermatids was observed with the preimmune serum at the same dilution
(Fig. 8, B and D).

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Fig. 8.
Testicular expression of p21.
Immunocytochemical analysis of murine testis prints (A and
B) and isolated germ cells (C and D)
using anti-p21 antiserum (A and C) or preimmune
serum (B and D) is shown. A, the
arrow points to the labeling of the Golgi complex of primary
spermatocytes at the pachytene stage. C, the long
arrow points to the labeling of the flagellar middle piece of
mature spermatids; the short arrow in the inset
points to the labeling of the acrosomal region. Bar, 10 µm.
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Immunocytochemical analysis of transfected COS cells expressing the p21
cDNA revealed a conspicuous immunolabeling of a juxtanuclear zone
(Fig. 9). No specific immunoreactivity
was detected in wild-type COS cells (data not shown).

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Fig. 9.
Immunofluorescence of COS cells transfected
with the p21 cDNA inserted in the pCDNA3 expression
vector. Note the strong juxtanuclear labeling with the anti-p21
antibody.
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 |
DISCUSSION |
In this study, we have characterized the product of the p1A75
cDNA (3), which belongs to the same new family as the p19 protein
(1). This 21-kDa protein (p21) is, like p19, expressed in the Golgi
apparatus of neural cells. However, in contrast to p19, which is absent
from the testis and faintly expressed in the olfactory epithelium, p21
is very strongly expressed in the olfactory epithelium and in male
gametes at the late stages of their differentiation.
The similarity (56% identity) of the primary sequences of murine p19
and p21 indicates that these proteins belong to the same family. The
main differences between these two proteins consist in the presence of
a 12-amino acid-long insertion (TEGVTERFKVSV) in the p21 sequence and
in a complete divergence of their 15 most carboxyl-terminal residues.
Searches in data banks for related sequences yielded a wealth of
overlapping human expressed sequence tags that exhibit a very high
similarity level with either of the two murine sequences. These
overlapping expressed sequence tags allowed the reconstruction of the
primary sequence of human p19 and p21 (data not shown). The very high
similarity of the murine and human p19 and p21 sequences (respectively,
95 and 98% identity for p19 and p21) suggests that these proteins are
submitted to a strong selection pressure. In addition to these
expressed sequence tags, searches in protein data bases revealed that
the highly similar short segment that is shared by p19, p21, and
secretogranin III (5) is also found in a subset of highly related
members of the yeast ABC protein family (28) (Fig.
10).

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Fig. 10.
Alignment of residues 149-166 of p21 with
residues 138-155 of p19 (1) and with residues 136-149 of
secretogranin III (5), residues 475-492 of the CDR1 Candida
albicans multidrug resistance protein (36), and residues 483-500
of the PDR5 Saccharomyces cerevisiae pleiotropic drug
resistance protein (37). Identical or isofunctional amino acids
are boxed.
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Western blot experiments performed with an antiserum made against the
recombinant protein indicated the existence of a single band in the
brain of the postnatal day 1 rat whose migration rate (21 kDa)
corresponded to the calculated molecular mass of p21 (Fig.
5A). This molecular mass differs from the one (25 or 28 kDa)
determined by Sutcliffe et al. (3) for the same polypeptide using hybridization translation or immunoprecipitation experiments, respectively. This difference is probably due to a lack of accuracy in
the determination of the molecular mass rather than to the recognition
of another protein by the antipeptide antiserum used by these authors.
Indeed, the organelle-like pattern observed in their
immunocytochemistry experiments corresponds very well to the Golgi
apparatus localization of p21 (3). This subcellular localization of p21
is closely similar to that of p19. Indeed, similar patterns of
localization were found for these two proteins using either
immunoelectron microscopy or immunofluorescence analysis. The
Golgi-like localization of transfected p21 in COS cells (Fig. 9)
indicated that, like p19 (1), its primary sequence contains a Golgi
targeting signal. Moreover, the presence, in p21 and p19, of a highly
hydrophobic segment and their carbonate-resistant membrane association
indicated that they are both integral membrane proteins. The absence of
a signal peptide in the p21 sequence suggests that, like p19, this
protein could be inserted in the membrane by a C-terminal anchor (1,
29).
The main difference between p19 and p21 seems to reside in their
tissular pattern of expression. p21 mRNA is indeed prominently produced in the olfactory epithelium and to a lesser extent in testis,
two tissues in which p19 expression is, respectively, weak and
undetectable (1). The high expression of p21 in cultured neurons and in
neural and neuroendocrine tissues, on the one hand, and its absence
from all other organs except testis, on the other hand, strongly
suggest a neuronal localization of p21 within the olfactory epithelium.
Very likely, the extremely high hybridization signal observed for p21
mRNA in Northern blot analysis (Fig. 3A) of a crude
olfactory epithelium preparation (which also contains supporting
tissues) probably underestimates the real concentration of p21 mRNA
in the epithelium. Immunohistochemistry experiments (Fig. 8) indicated
that, within the testis, p21 is expressed, as expected, in the Golgi
complex of primary spermatocytes and in the acrosomal region of mature
spermatids. More surprisingly, p21 is also expressed in the flagellar
middle piece of late spermatids, which contains the mitochondrial
sheath.
This surprising localization of a protein in olfactory neurons and in
germinal cells of the testis, two apparently unrelated cell types, has
already been described in the case of a subset of odorant receptors and
their transducing proteins (30-32). These proteins, which are mainly
located in the flagellar middle piece of sperm cells, could be
responsible for the marked augmentation of the respiratory activity
that is associated with chemotaxis (32). The localization of p21 in the
tail middle piece of sperm cells suggests that this protein could also
be involved in this process, reinforcing therefore the hypothesis of a
resemblance, at the molecular level, between olfaction and germ cell
chemotaxis. In addition, the presence of p21 in the acrosomal region,
which originates from Golgi vesicles, can also be related to sperm
chemoattraction. Cohen-Dayag et al. (33) have indeed shown
that the potential to undergo the acrosome reaction (sperm
capacitation) is correlated with sperm chemotactic activity.
Future studies will be necessary to investigate the role of p21 in
olfactory neurons and in sperm cells. In addition to their interest for
the biology of these two cell types, these studies should also help to
understand the functional consequences of a large gene duplication
event, which, in addition to p19 and p21, gave rise to numerous other
couples of genes located, in the human genome, on the telomeric ends of
the long arms of chromosomes 4 and 5 (6). Interestingly, as seems to be
the case for p19 and p21, the other couples of genes suspected to
originate from this large gene duplication event differ mostly by their
tissular pattern of expression. For instance, the dopamine receptors D1 and D5 (34) and the fibroblast growth factor receptors FGFR4 and FGFR3
(35), which exhibit 50-60% similarity within each couple and whose
genes are located in 5q35 (D1 and FGFR4) and 4p16 (D5 and FGFR3), have
retained similar binding specificities and transducing activities but
differ by their expression pattern.
We are grateful to Dr. Ferran Burgaya for
help with the in situ hybridization experiments, to
Françoise Arnos for technical assistance, and to Eric Etienne for
the microscopy pictures.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF035683.