1
Dipartimento di Biologia Evoluzionistica Sperimentale, Via Selmi 3, 40126
Bologna, Italy
2
Istituto Internazionale di Genetica e Biofisica; Via Marconi 10, 80125 Napoli,
Italy
*
Present address: Centro di Oncologia Sperimentale, CNR, Via Pansini 2 Napoli,
Italy
Author for correspondence (e-mail:
gargiulo_g{at}biblio.cib.unibo.it
)
Accepted April 27, 2001
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SUMMARY |
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Key words: Oogenesis, Follicle cells, Eggshell, Vitelline membrane, Endochorion
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INTRODUCTION |
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The analysis of mutations affecting the eggshell has suggested the
structural function of specific proteins. Females homozygous for the
cor36 mutation do not synthesize the early s36 chorion protein and a
defective endochorion layer is formed (Digan et al.,
1979). Homozygous females for
null dec-1 mutations produce eggshells altered in both organization
and stability of the endochorion layer (Bauer and Waring,
1987
). The dec-1 gene
encodes multiple protein products that show distinct localization patterns in
the mature eggshell. Their diverse localizations have suggested that the
different dec-1 derivatives might play different roles in the
assembly and stabilization of the mature eggshell (Nogueron et al.,
2000
). Females homozygous for
the fs(2)QJ42 mutation fail to accumulate the vitelline membrane
protein VM26A.2 and produce eggs with an altered vitelline membrane onto which
the endochorion layer collapses during stage 14, suggesting that the vitelline
membrane has an important role in the stabilization of the outer chorion
layers (Savant and Waring,
1989
; Pascucci et al.,
1996
). Vitelline membrane
defects have been also reported for some alleles of the nudel gene
(Hong and Hashimoto, 1995
;
Hong and Hashimoto, 1996
; Le
Mosy et al., 1998
). This gene
is expressed in follicle cells surrounding the oocyte and encodes a large
mosaic protein with a central serine protease domain involved in the
establishment of the dorso-ventral axis of the Drosophila embryo
(Hong and Hashimoto, 1996
). It
has been demonstrated that nudel mutations that compromise Nudel
protease function also result in the failure of covalent cross-linking of the
vitelline membrane in the laid egg, suggesting an integral role for Nudel
protease in eggshell biogenesis (LeMosy and Hashimoto,
2000
). Certain mutant alleles
of the fs(1)polehole and fs(1)Nasrat genes produce eggs with
leaky vitelline membranes (Ambrosio,
1989
; Casanova and Struhl,
1989
; Degelman et al., 1990).
These genes, not yet cloned, belong to the terminal group genes involved in
the activation of Torso at the poles of the embryo, but nothing is known about
their function in the vitelline membrane formation. A compelling question
arising from these data concerns the involvement of the vitelline membrane in
the localization of the maternal signals required for embryonic axis
determination. The vitelline membrane might form a matrix structure necessary
for the functioning of the cues relevant for embryonic development.
Four Drosophila vitelline membrane protein genes have been cloned
so far: VM26A.1, VM34C, VM26A.2, and VM32E (Higgins et al.,
1984; Mindrinos et al.,
1985
; Burke et al.,
1987
; Popodi et al.,
1988
; Gigliotti et al.,
1989
). Although the other
vitelline membrane genes are expressed from stage 8 to stage 10 of oogenesis,
the VM32E gene, expressed only at stage 10, can be considered a
`late' vitelline membrane gene (Gigliotti et al.,
1989
). Compared with the other
members of the same family, the VM32E gene is under complex temporal
and spatial regulation (Gargiulo et al.,
1991
; Cavaliere et al.,
1997
; Andrenacci et al.,
2000
). This might reflect some
special functions played by the VM32E protein in eggshell formation.
In this report, we describe the distribution and fate of the VM32E protein in the initial and late stages of eggshell assembly. The results clearly show that, during the final stages of oogenesis, some VM32E protein molecules are released from the vitelline membrane and become stably integrated into the endochorion. The VM32E protein is therefore an integral component of both the vitelline and the endochorion layers. The work presented here offers new insights into the process of eggshell assembly, and allows the identification of VM32E protein functional domains required for its integration into the vitelline membrane and its recruitment in the endochorion layer.
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MATERIALS AND METHODS |
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Construction of chimeric VM32E-MYC genes
Standard molecular biology techniques were carried out essentially as
described in Sambrook et al. (Sambrook et al.,
1989). The VM32E-MYC,
1,
2 and
3 chimeric genes were produced by
fusing specific PCR amplification products to DNA fragments obtained by
designed endonuclease digestions of the HindIII/HindIII
VM32E genomic clone (Gigliotti et al.,
1989
). The PCR reactions were
performed using the same clone as template, various internal DNA primers and a
designed synthetic primer (Fig.
1) carrying the coding sequences for the MYC epitope, a
SnaBI restriction site and three stop codons.
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All the chimeric genes contain the minimal VM32E promoter (up to
nucleotide -465), the untranslated 5' end, and the 3' end
including the polyadenylation site of the wild-type VM32E gene. The
VM32E-MYC gene covers the full coding region of the VM32E
gene, whereas the 1,
2 and
3 genes
carry different deletions of the coding sequences, which are:
1, from
amino acid residue 24 to 33;
2, from 50 to 59;
3, from 73 to
116. All the chimeric genes encode proteins with the MYC epitope at the
C-terminal region. The genes were first cloned in pUC19 vector, then sequenced
and subcloned in the XbaI site of the pCaSpeR4 vector (Brand
and Perrimon, 1993
).
Transgenic lines
P-element-mediated transformation was carried out essentially as described
in Spradling and Rubin (Spradling and Rubin,
1982) and Rubin and Spradling
(Rubin and Spradling, 1982
).
Dechorionated embryos from the yw67c23 strain were
injected with the various DNA constructs and helper plasmid
p
25.7wc (Karess and Rubin,
1984
).
In situ hybridization
Whole-mount in situ hybridization with digoxigenin-labeled (Roche) probes
was performed as described by Tautz and Pfeifle (Tautz and Pfeifle,
1989). The 3' end of the
VM32E cDNA (Gigliotti et al.,
1989
) and the DNA encoding the
MYC epitope were used as probes. The egg chambers were viewed with Nomarski
optics on a Zeiss microscope.
Immunofluorescence microscopy
Fixation and antibody staining of hand-dissected ovaries were carried out
as previously described (Gigliotti et al.,
1998). Anti-CVM32E or anti-VMP
antibodies were used at 1/50 dilution and reacted with Cy3-conjugated
anti-rabbit secondary antibody (1/100 dilution). Anti-MYC monoclonal antibody
were used at 1/100 dilution and reacted with Cy3-conjugated anti-mouse
secondary antibody (1/100 dilution). Stained egg chambers mounted in Aquamount
(Polyscience) were analyzed with conventional epifluorescence and with a
Biorad laser confocal microscope attached to a Zeiss Axiophot microscope.
Immunoelectron microscopy
Ovaries were removed in cold insect Ringer's solution and fixed as
described (Pascucci et al.,
1996), dehydrated, and
embedded in the acrylic resin Bioacryl, polymerized at 4°C under UV
irradiation according to Scala et al. (Scala et al.,
1992
). After sectioning with
an ultramicrotome, thin sections were collected on nickel grids, and incubated
overnight at 4°C in the primary antibody diluted (anti-CVM32E at 1:40;
anti-MYC at 1:80) in Tris-buffered saline (TBS) at pH 8.2 containing 0.1%
Triton-X100 and 2% bovine serum albumin (BSA). After repeated washes in TBS,
sections treated with anti-CVM32E antibody were incubated for 1 hour at room
temperature with a goat anti-rabbit IgG; those treated with anti-MYC antibody
were incubated in the same conditions with a goat anti-mouse IgG. In both
cases, IgG were conjugated to 10 nm gold particles and used at 1:40 dilution.
Controls were performed on samples from which the treatment with the first
antibody was omitted. After rinsing in TBS and in distilled water, the
sections were lightly stained in uranyl acetate and lead citrate for
observation with a CM 100 Philips electron microscope. For double labeling,
the detection of the MYC antigen with IgG conjugated to 10 nm gold particles
was followed by the detection of the second antigen, VM32E, with IgG
conjugated to 20 nm gold particles. As control of the quality of our
immunoelectron microscopy procedure, we checked the proper distribution of the
VM26A.2 protein (Pascucci et al.,
1996
) using the anti-VMP
antibody (data not shown).
Western blot analysis
Ovaries and staged egg chambers were quickly collected and placed in cold
Ringer's solution and frozen in liquid nitrogen. Ovaries analyzed in
Fig. 4B were homogenized by
sonication in Laemmli sample buffer (31 mM Tris-HCl, pH 6.8, 2.5% glycerol,
0.5% SDS, 177 mM 2ß-mercaptoethanol, 0.01% bromophenol blue (Laemmli,
1970)), boiled for five
minutes and, following the removal of insoluble material by centrifugation (10
minutes at 15,000 g), the soluble proteins were run on
SDS-PAGE.
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Egg chambers analyzed in Figs 4A,B and 9A were homogenized in ice-cold Ringer's solution with 0.5% Nonidet P-40, 1.3 µg ml-1 pepstatin A, 1.3 µg ml-1 antipain, 3.3 µg ml-1 leupeptin, 2.3 mM PMSF. Following centrifugation (10 minutes at 15,000 g) the supernatant fraction was mixed with an equal volume of 2x Laemmli sample buffer and boiled for five minutes. The pellet phase was washed three times in homogenization buffer, suspended in Laemmli sample buffer and boiled for 1 hour. After boiling, the insoluble residues were removed by centrifugation and the solubilized material was run on SDS-PAGE. Two-hour-old eggs were collected in a wire basket, rinsed with distilled water and, where required, dechorionated with 50% Chlorox. The eggs were homogenized as described here and only the pellet phases were processed and loaded on the gel.
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SDS solubilization of vitelline membrane proteins, shown in Figs 4C, 5B and 9B, was achieved by adding SDS to the homogenization buffer at the concentrations indicated in the text. The samples were incubated at room temperature for 10 minutes. Following centrifugation (10 minutes at 15,000 g) the supernatant fraction was treated as described above. Pellets were washed three times in homogenization buffer without SDS and processed as described above.
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All the samples obtained by the different procedures used were run on 15%
polyacrylamide gels. Protein transfer to nitrocellulose and western blotting
were performed using standard methods (Harlow and Lane,
1998). The chimeric proteins
were detected using anti-MYC monoclonal mouse antibody diluted 1/500; the
VM32E protein was detected by using the anti-CVM32E polyclonal rabbit antibody
(diluted 1/100), whereas the VM26A.2 protein was detected by using the
anti-VMP polyclonal rabbit antibody (diluted 1/100). All the primary
antibodies were detected using horseradish peroxidase conjugated horse
antibody (1/500 dilution) and ABC detection kit (Vector ABC Universal kit no.
pk-6200).
For each set of experiments, the number of ovaries, egg chambers or eggs analyzed were exactly the same and all the material extracted was loaded into the gel. Each gel lane was loaded with four ovaries or 400 egg chambers or eggs (600 for transgenic flies). The experimental procedure used could not cause any loss of material. After the removal of the soluble phase, which was transferred to a new tube and processed for the western blot analysis, the pellet was kept in the original tube in which the egg chambers were homogenized. Furthermore, as an additional control to compare the relative amount of proteins in the various samples, filters were stained after blotting with Ponceau and gels with Coomassie blue. In all the experiments performed, the proteins detected in each lane were of comparable and highly reproducible amount.
Antibodies
The anti-VMP and the anti-CVM32E polyclonal antibodies were generated in
rabbits (Primm) using synthesized peptides located at the N and C termini of
the VM32E protein. The N-terminal peptide was SCPYAAPAPAYSAPAASSG (residues 18
to 36) and the C-terminal peptide was EELRGLGQGIQGQQY (residues 102 to 116).
Anti-MYC mouse monoclonal antibody 9E10 was purchased from Santa Cruz
Biotechnology. Cy3 conjugated anti-rabbit and anti-mouse antibodies were
purchased from Sigma. 10 nm and 20 nm gold particles conjugated anti-rabbit
and anti-mouse antibodies were respectively from Sigma and Chemicon.
All the images were processed in Photoshop 5.0 (Adobe Systems, Mountain View, CA, USA).
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RESULTS |
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Analysis by confocal microscopy showed that, at stage 10B, the VM32E
protein is synthesized in the columnar cells and secreted into the
extracellular space between the follicle cells and the oocyte, where the
vitelline membrane is forming (Fig.
2C,D). No synthesis of the protein was detectable in the anterior
and posterior follicle cells. In these regions, only a faint signal of reacted
antibody was visible (Fig.
2E,G). By stage 11, although the pole follicle cells remain
silent, the VM32E protein was also present at the poles of the oocyte, which
appeared to be uniformly surrounded by it
(Fig. 2F,H). This indicates
that, once secreted, the VM32E protein moves to the poles. In comparison to
this, we analyzed the distribution of the VM26A.2 protein, which has been
reported to be expressed in all follicle cells surrounding the oocyte (Popodi
et al., 1988). To detect this
protein we used a polyclonal antibody (anti-VMP) originally raised against the
N-terminal SCPYAAPAPAYSAPAASSG peptide of the VM32E protein that can also
recognize the VM34C and the VM26A.2 proteins. It reacts most strongly with
VM26A.2 and barely with the other two proteins (data not shown), probably
because a PAYSAPAA peptide is repeated four times in VM26A.2. As shown in
Fig. 2I,L, the signal was
detected in all follicle cells surrounding the oocyte.
The distribution of VM32E was also studied at the ultrastructural level by immunogold electron microscopy on egg-chamber thin sections. At stage 10, the immunogold particles were detected in the secretory vesicles of the follicle cells and appeared to be widely distributed in the vitelline bodies (Fig. 3A), with the exception of those present at the poles, in which a very low density of gold particles was scored (Fig. 3B). At the late stage 10B, the immunogold particles strongly labeled the vitelline membrane (Fig. 3C). At stage 12 (Fig. 3D), immunogold particles were detected in the vitelline membrane and in the forming endochorion pillars. Finally, at stage 14, gold particles labeled both the vitelline membrane and the endochorion (Fig. 3E). The presented data clearly indicate that, at the end of egg chamber development, the VM32E protein is a component not only of the vitelline membrane but also of the endochorion layer.
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Western blot analysis of VM32E protein
We analyzed the VM32E protein during egg chamber development using a
protein extraction procedure that allowed us to follow up its integration into
the eggshell. Staged egg chambers were homogenized and, after centrifugation,
the pellet and supernatant phases were separated and analyzed by western blot.
Proteins recovered in the pellet phase should be those assembled within the
eggshell, whereas proteins in the supernatant phase should be free, or
aggregated in small macromolecular complexes not yet integrated in the
eggshell.
According to the mRNA open reading frame, the VM32E protein should be 116
amino acids long with an expected molecular mass of 12 kDa, but because the
first 17 amino acids follow all the rules for a predicted cleavage site of a
signal peptide (Von Heijne,
1984), the secreted protein
must be shorter and with a molecular mass of 10.4 kDa. A prominent band of
10 kDa, corresponding to the hypothetic mature VM32E protein, was
observed in all the stages analyzed (Fig.
4A). Two additional bands of slightly different size were detected
by the antisera and might represent unknown modifications of the protein. At
stage 10, most of the VM32E protein was detected in the pellet phase,
suggesting its incorporation into the vitelline membrane. VM32E protein was
recovered in the supernatant fraction until stage 12. This might suggest that
the VM32E protein is gradually integrated into the eggshell, because no VM32E
transcript is detectable after stage 11 (Gigliotti et al.,
1989
). By stage 13, no protein
was detected in the supernatant phase and the amount of VM32E protein
extracted from the pellet was lower than at the earlier stages, and even more
so at stage 14. In comparison to this, using the same extraction procedure
described above, we analyzed the vitelline membrane protein VM26A.2
(Fig. 4B). As reported by
Pascucci et al. (Pascucci et al.,
1996
), the VM26A.2 pattern is
quite complex because the protein undergoes proteolytic cleavage in the late
stages of oogenesis. At stage 9, the ongoing synthesis of the VM26A.2 protein
was clearly visible and, by stage 10, most of this protein was recovered from
the pellet phase. At stage 14, the VM26A.2 protein was fully releasable from
the pellet phase. Therefore, the observed reduced level of VM32E protein
detected at stage 14 (Fig. 4A)
might be due to the cross-linking with other endochorion components.
It is thought that vitelline membrane protein cross-linking occurs in two
steps. First, during oogenesis, the vitelline membrane proteins are bound by
disulfide cross-linking, and treatments with reducing agents result in
solubility of the membrane. Second, successive non-disulfide cross-linking,
which renders the vitelline membrane completely insoluble, occurs around the
time of oviposition (Petri et al.,
1979). To determine whether or
not the VM32E protein undergoes disulfide cross-linking, the proteins from
ovaries and stage 14 egg chambers were extracted in presence of 2% SDS, which
permits the release of the non-covalently bound proteins. After
centrifugation, the pellet and supernatant phases were separated, submitted to
SDS-PAGE and analyzed by western blot (see Materials and Methods). To compare
the extraction patterns of VM32E and VM26A.2, we cut the membrane into two
parts after blotting that were separately reacted with the proper antibodies.
As shown in Fig. 4C, the mature
form of the VM26A.2 protein, present only at the terminal stages of oogenesis,
was held in the pellet phase, indicating its cross-linking by disulfide
bridges. The VM32E protein extracted from the whole ovary was mostly
solubilized by the SDS but, at stage 14, this protein was detected only in the
pellet phase. These data clearly indicate that, by the end of oogenesis, the
VM32E protein too is cross-linked by disulfide cross-linking into the
vitelline membrane.
Analysis of VM32E in fs(2)QJ42 mutant
To investigate the interaction occurring among different structural
components at the early stage of vitelline membrane assembly, we have analyzed
the distribution of VM32E in egg chambers obtained from fs(2)QJ42
females that fail to accumulate the VM26A.2 protein and have vitelline
membrane defects (Savant and Waring,
1989). The VM32E protein was
present in both the anterior and posterior regions of vitelline layer already
at stage 10B (Fig. 5A). Then,
in the absence of the VM26A.2 gene product, this protein can move to the poles
as soon as it is secreted from the follicle cells, perhaps because of the
looser structure of the mutant vitelline membrane. However, as assayed by
western blot analysis, the extraction pattern of the VM32E protein was the
same as that of wild-type of fs(2)QJ42 ovaries
(Fig. 5B, the first two lanes),
which indicates that this protein keeps its ability to bind to the other
vitelline membrane components. To ascertain whether or not the increased
mobility of the VM32E protein in fs(2)QJ42 vitelline envelope could
be due to a change of its non-covalent interactions, extraction from ovaries
was performed in the presence of increasing amounts of SDS. As shown in
Fig. 5B, in the mutant, the
VM32E protein was totally released from the vitelline membrane with 0.5% SDS,
whereas 2% of SDS was required in the wild type completely to solubilize this
protein. Although these data do not prove their direct interaction, the VM32E
and VM26A.2 proteins might fit together in the assembling vitelline
membrane.
Dissection of VM32E protein domains
All the putative Drosophila vitelline membrane proteins so far
identified are rich in proline and alanine, and contain a highly conserved
hydrophobic domain (VM domain) of 38 amino acids (Scherer et al.,
1988; Gigliotti et al.,
1989
)
(Fig. 6B). A similar domain,
showing conserved amino acid residues, is also present in the vitelline
membrane proteins 15a-1, 15a-2 and 15a-3 of the mosquito Aedes
aegypti (Lin et al.,
1993
; Edwards,
1996
; Edwards et al.,
1998
). An additional conserved
region of ten amino acids is also present in the VM32E, VM34C and VM26A.2
proteins, but absent from VM26A.1. These homologies might reflect some common
structural features of these proteins relevant to building up the vitelline
membrane. In order to investigate the hypothesis that these protein domains
are required for the integration of VM32E in the eggshell layers, we produced
transgenic flies encoding two differently deleted VM32E proteins
(Fig. 6A) fused at the C
terminus with a MYC epitope of 14 amino acids. As control, we analyzed the
localization and integration of a chimeric protein containing the complete
VM32E coding region fused to the MYC epitope (VM32E-MYC). The expression of
these chimeric genes in the same cell types involved by the native
VM32E gene was driven by the minimal promoter, 5' and 3'
flanking regions of VM32E, and analyzed through in situ hybridization
(data not shown). Because the activity of the minimal promoter used is lower
than that of the wild type (Gargiulo et al.,
1991
), although they are very
similar, the expressed amounts of the different chimeric proteins were lower
than the wild-type VM32E protein (data not shown). As judged by egg
permeability and egg eclosion, no effect on the vitelline membrane protein
structure was produced by the expression of the different chimeric genes (data
not shown). As shown in Fig.
7A,B, the localization of the VM32E-MYC protein at stage 10B was
identical to that of native VM32E and, by stage 11, this chimeric protein was
also localized at the poles (data not shown). Immunoelectron microscopy of
double labeled sections of stage 10B egg chambers showed a wide distribution
of both the MYC-tagged and wild-type VM32E proteins in the vitelline membrane
(Fig. 7C). At the terminal
stages of oogenesis, by using only the anti-MYC antibody, we detected the
VM32E-MYC protein in the vitelline membrane and in the endochorion
(Fig. 7D), as observed for
wild-type VM32E protein. In addition, the VM32E-MYC protein also appeared in
vesicles within the oocyte (Fig.
7E). In these cellular structures, we had also detected the
wild-type VM32E protein (data not shown); the high efficiency and specificity
of the anti-MYC antibody allow us to be more confident in pointing out this
feature.
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In the 1 construct, the deletion removes ten amino acids that are
almost identical in the VM32E, VM34C and VM26A.2 proteins. The removal of this
region did not cause any alteration of the localization of the
1
protein, as assayed by confocal and electron microscopy analysis (data not
shown). In the
2 construct, the deletion removes ten of the 38 amino
acids of the highly conserved hydrophobic VM domain (Figs
6A,8F).
Confocal analysis of egg chambers from flies carrying
2 construct
revealed an altered distribution of the chimeric protein; in fact, the protein
was already present in both anterior and posterior regions of vitelline layer
at stage 10B (Fig. 8A,B). It is
worth noticing that the early mobility of the
2 protein at stage 10
appeared similar to that of the wild-type VM32E protein when the VM26A.2
protein was missing (Fig. 5A).
This result suggests that the VM domain is required for protein-protein
interaction among the various vitelline membrane proteins. Migration of the
2 protein into the endochorion was not affected
(Fig. 8C), indicating that the
integrity of VM domain is not necessary for VM32E movement from the vitelline
membrane to the endochorion.
|
The VM32E protein presents a carboxyl end of 44 amino acids that does not
share significant homology with the other vitelline membrane proteins. This
region might provide the protein with its special feature. Therefore, we
analyzed in transformed flies the localization and integration into the
eggshell of a chimeric VM32E protein, deleted of its carboxyl end (3,
Figs
6A,8F).
Confocal analysis of stage 10B egg chambers showed for the truncated protein
the same distribution pattern as the wild-type VM32E
(Fig. 8D). In stage 11 egg
chambers,
3 protein was also localized at the poles (data not shown).
This suggests that the C-terminal deletion does not alter the proper
localization of the protein at these stages of egg chamber development.
Electron-microscopy analysis of
3 protein distribution in the eggshell
revealed that, at the terminal stages of oogenesis, it was localized only in
the vitelline membrane, which showed a very strong immunogold signal
(Fig. 8E). Therefore, the
C-terminal domain is indispensable for localizing the VM32E protein in the
endochorion. As a control, we analyzed the wild-type VM32E protein in sections
from the same egg chambers and, as expected, it appeared in both the vitelline
and the endochorion layers (data not shown). As shown for the VM32E-MYC
protein, these chimeric proteins were found also in the ooplasm of stage 14
egg chambers (data not shown).
The various VM32E chimeric proteins (Figs
6A,9D)
were analyzed by western blot at stages 10B and 14 of egg-chamber development
(Fig. 9A). The VM32E-MYC
control and the 1 product generated the same pattern as the wild type
VM32E protein (Fig. 9A). The
strong signal of the
2 protein detected in the supernatant phase of
stage 10B egg chambers indicates that a considerable amount of this protein is
not integrated into the membrane. These data agree very well with confocal
analysis showing a high mobility of this protein during stage 10. At stage
10B, the
3 protein showed an extraction pattern similar to the wild
type VM32E protein. The complex distribution pattern of the
3 protein
is probably due to some unknown modifications, because all the major bands
were larger than the predicted one. Also the VM32E-MYC,
1, and
2
proteins might undergo modifications, because their migration on the gel was
slower than expected. Much
3 protein was extracted from stage 14 egg
chambers, probably owing to its inability to localize in the endochorion
layer. It is worth noticing that, at stage 14, the extraction pattern of
3 was identical to that of VM26A.2
(Fig. 4B), which was totally
extracted from the pellet fraction. To assess whether or not the
2 and
3 proteins can form disulfide cross-links, stage 14 egg chambers were
homogenized in presence of 2% SDS and the pellet and supernatant fractions
were analyzed by western blot. As shown in
Fig. 9B, the
2 protein
was fully solubilized by the detergent, indicating that it is not cross-linked
by disulfide bridges. Instead the
3 protein, which contains an intact
VM domain, was recovered in the pellet phase, indicating that it forms
disulfide cross-links.
In laid eggs, the vitelline membrane hardening does not permit the
solubilization of its structural proteins. Therefore, we determined by western
blot whether the deleted 2 and
3 proteins were properly
integrated into the eggshell (Fig.
9C). The wild-type VM32E protein appeared to be tightly
incorporated into the eggshell, as expected. The
3 protein was also
fully insoluble, suggesting that it must be integrated by covalent
cross-links. The analysis of the
2 protein revealed an altered
cross-linking of this protein in the vitelline membrane; the same amount of
2 protein was in fact extracted from whole or dechorionated eggs. These
results indicate that stable integration by cross-linking of the VM32E
protein, and probably of any vitelline membrane protein, is based on the VM
domain. By contrast, the absence of a functional VM domain did not affect the
localization (Fig. 8C) or
integration of the
2 protein in the endochorion layer
(Fig. 9C).
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DISCUSSION |
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The proper assembly of the VM32E protein in the membrane requires the
activity of the VM26A.2 protein. In the fs(2)QJ42 mutant egg
chambers, which lack the VM26A.2 protein, movement to the poles of the VM32E
protein occurs as soon as it is secreted. Therefore, the VM32E-VM26A.2
interaction also appears to regulate the timing of VM32E protein movement to
the poles. The VM domain is necessary for the assembly of the VM32E protein in
the vitelline membrane. We have shown that a deletion of ten amino acids
within this domain impairs the early integration of the VM32E protein into the
vitelline membrane and its cross-linking at the final stages of oogenesis.
Interestingly, this domain is present in all the D. melanogaster
vitelline membrane proteins cloned to date and shares homology with the
vitelline membrane proteins of the mosquito A. aegypti (Scherer et
al., 1988; Gigliotti et al.,
1989
; Lin et al.,
1993
; Edwards,
1996
; Edwards et al.,
1998
). In both species, this
domain contains three precisely spaced cysteine residues, two of which are
ablated by the small deletion analyzed (
2). This strongly suggests that
the VM domain should fulfill a general function in holding together the
various vitelline membrane proteins by disulfide cross-links.
In laid eggs, vitelline membrane hardening is thought to be performed by a
peroxidase-type enzyme that cross-links the tyrosine residues of the different
structural proteins (Petri et al.,
1976; Petri et al.,
1979
). Because the VM32E
protein contains seven tyrosine residues, this protein might also undergo such
cross-linking. Our data indicate that the
2 protein is not tightly
integrated into the vitelline membrane of laid eggs. Because this deletion
does not remove any tyrosine residues, we suggest that the proper vitelline
membrane assembly by disulfide bridges during oogenesis is necessary for any
further covalent protein cross-linking in the egg.
Our data show for the first time that a vitelline membrane component, the
VM32E protein, also participates in the assembly of the endochorion layer.
This implies that VM32E protein molecules are released from the vitelline
membrane and move outwards to the chorion layer. Transient storage of eggshell
components within the vitelline membrane has been reported for some
dec-1 derivatives (Nogueron et al.,
2000) and for the chorion
protein s36, which is produced during early choriogenesis (Pascucci et al.,
1996
; Trougakos and
Margaritis, 1998
). Moreover,
it is not known how these proteins can migrate to the chorion layers nor
particularly whether there are any signal peptide to guide their movement. The
presented results allowed the definition for the first time of an interacting
motif of an eggshell protein involved in its outward movement. Our deletion
analysis indicates that the VM32E C-terminal region is required for the
recruitment of this protein by the endochorion layer. If this region is
deleted, the truncated VM32E protein behaves like the other vitelline membrane
proteins, appearing widely distributed only in the vitelline layer and
becoming cross-linked only in the laid egg. The C-terminal domain might be the
target of an unknown component that will carry the VM32E to the endochorion
layer. Alternatively, this protein domain might allow the interaction of the
VM32E protein with other chorionic components to form protein complexes that
will move to the chorion layer. Based on the presence of nine glutamine
residues in the C-terminal region, cross-linking of the VM32E protein could be
also performed by a transglutaminase-type enzyme, as occurs with the proteins
shaping the cornified cell envelope of mammals, in which glutamyl-lysine
cross-links are formed by transglutaminases (Hohl et al.,
1993
). Even though
transglutaminase activity in egg chamber extracts is still awaiting
documentation, the VM32E protein might be bridged to the s36 and s38 chorion
proteins by cross-links of the glutamine residues in the VM32E carboxyl domain
to the lysine residues of these proteins. It has also been supposed that some
dec-1 derivatives that include the central glutamine-rich repeats
might be cross-linked to other eggshell proteins by this type of cross-linking
(Waring et al., 1990
).
At stage 14, the VM32E also appears in vesicles within the oocyte, and the
same is true for the VM32E chimeric proteins. Besides the endocytotic uptake
of yolk proteins during vitellogenic stages (8-10) (Engelmann,
1979; Schonbaum et al.,
1995
), a late endocytotic
activity at stage 14 has been reported for some dec-1 derivatives
(Nogueron et al., 2000
).
Although the functional significance of the uptake of eggshell components is
not known, our finding that some vesicles loaded with VM32E are contained in
the ooplasm might suggest that, as for the yolk proteins, this protein is
stored to supply nutrients to the developing embryo. A programmed oocyte
uptake of eggshell constituents could be part of the process of eggshell
assembly in order to prevent inappropriate overload of specific proteins in
the different eggshell layers.
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ACKNOWLEDGMENTS |
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