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First published online 5 January 2006
doi: 10.1242/dev.02219
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-like growth factor Gurken
1 Max-Planck-Institut für molekulare Zellbiologie und Genetik,
Pfotenhauerstrasse 108, 01307 Dresden, Germany.
2 Institut für Entwicklungsbiologie, Universität zu Köln,
Gyrhofstraße 17, 50923 Köln, Germany.
Author for correspondence (e-mail:
siegfried.roth{at}uni-koeln.de)
Accepted 23 November 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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growth factor Gurken (Grk) to the oocyte surface.
Here, we show that Cni, but not the second Drosophila Cni homologue
Cni-related (Cnir), binds to the extracellular domain of Grk, and propose that
Cni acts as a cargo receptor recruiting Grk into COPII vesicles. Consequently,
in the absence of Cni function, Grk fails to leave the oocyte ER. Proteolytic
processing of Grk still occurs in cni mutant ovaries, demonstrating
that release of the active growth factor from its transmembrane precursor
occurs earlier during secretory transport than described for the other
Drosophila TGF homologues. Massive overexpression of Grk in a
cni mutant background can overcome the requirement of Grk signalling
for cni activity, confirming that cni is not essential for
the production of the functional Grk ligand. However, the rescued egg chambers
lack dorsoventral polarity. This demonstrates that the generation of
temporally and spatially precisely coordinated Grk signals cannot be achieved
by bulk flow secretion, but instead has to rely on fast and efficient ER
export through cargo receptor-mediated recruitment of Grk into the secretory
pathway.
Key words: Cargo receptor, ER export, Emp24, Axis formation, TGF processing
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). These signals
are mediated by the TGF-like growth factor Gurken
(Neuman-Silberberg and Schüpbach,
1993). Eggs laid by females homozygous for null alleles of
grk are ventralized and lack anteroposterior polarity
(Gonzalez-Reyes et al., 1995;
Roth et al., 1995;
Schüpbach, 1987). Grk
consists of an extracellular growth factor domain containing one EGF repeat, a
transmembrane domain and a short C-terminal cytoplasmic tail, thus resembling
vertebrate TGF (Derynck et al.,
1984) and the other Drosophila TGF family members
Spitz (Rutledge et al., 1992)
and Keren (Reich and Shilo,
2002). The mature, secreted forms of all three Drosophila
TGF homologues are likely to be generated by intramembrane proteases of
the Rhomboid family (Lee et al.,
2001; Urban et al.,
2001; Urban et al.,
2002). Specifically, Grk appears to be cleaved by Rhomboid-2/Brho
(Ghiglione et al., 2002;
Guichard et al., 2000).
Processing of Spitz occurs in the Golgi apparatus, where its protease
Rhomboid1 resides, and is therefore strictly dependent on the prior export of
Spitz from the endoplasmic reticulum (ER), which is in turn mediated by Star
(Lee et al., 2001;
Tsruya et al., 2002;
Urban et al., 2001). By
contrast, we will show here that Grk processing occurs before export from the
oocyte ER.
Generation of the Grk signals depends on the presence of Cornichon [Cni
(Roth et al., 1995)] within
the germline. cni encodes a small hydrophobic protein that is the
founding member of a family of conserved eukaryotic proteins
(Hwang et al., 1999;
Powers and Barlowe, 1998;
Roth et al., 1995). Erv14p,
one of the two S. cerevisiae Cni homologues, was identified as an
integral membrane protein of COPII-coated ER-derived vesicles
(Belden and Barlowe, 1996). The
COPII coat consists of several subunits that are assembled into a
multimolecular coat on the surface of the ER
(Barlowe et al., 1994;
Bednarek et al., 1995) and
serves as an external scaffold organizing the assembly of anterograde
transport vesicles at the ER exit sites
(Bonifacino and Glick, 2004).
Direct or indirect interactions with COPII components can provide an efficient
mechanism for the recruitment of cargo proteins into vesicles leaving the ER
(Barlowe, 2003;
Kuehn and Schekman, 1997).
Erv14p is itself recruited into such vesicles through interactions with the
COPII coat (Powers and Barlowe,
2002). Loss of Erv14p results in a bud site selection defect
caused by inefficient membrane transport of the bud site selection protein
Axl2p (Powers and Barlowe,
1998; Roemer et al.,
1996). In erv14
yeast cells, Axl2p fails to be
sorted into COPII vesicles and accumulates in the ER, while other cargo
molecules are secreted at normal rates. Thus, only a subset of secreted
proteins depends on Erv14p for ER export
(Powers and Barlowe, 1998;
Powers and Barlowe, 2002). In
wild-type oocytes, freshly synthesized Grk protein is efficiently and rapidly
cleared from the large, continuous ER spanning the oocyte. Consistent with Cni
acting as a Grk cargo receptor, reduction in Cni activity causes diffuse
mislocalization of Grk protein within the ER
(Herpers and Rabouille,
2004).
Here, we provide biochemical and genetic evidence for an involvement of Cni in Grk ER export and present data explaining why Cni function is essential for the spatial and temporal specificity of Grk signalling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
grkDC (Schüpbach,
1987), grk2B6, grkHF48
(Neuman-Silberberg and Schüpbach,
1993), shiTS (shi1)
(Grigliatti, 1973) and Df(2L)JS7
(Sekelsky et al., 1995) have
all been described previously.
Immunostaining and electron microscopy
Antibody staining was performed as described
(Peri et al., 1999). The
antisera used were rabbit anti Grk, mouse anti Grk 1D12 (DSHB), mouse anti myc
9E10 (Dianova), rabbit anti myc A-14 (Santa Cruz), mouse anti
Drosophila Golgi 9C1 (Abcam) and mouse anti KDEL-receptor (Abcam).
For the shiTS experiments, freshly dissected ovaries were
incubated for 3 hours at either 25°C or 32°C in Schneider's
Drosophila tissue culture medium (Sigma) before fixation and
staining. Confocal imaging was performed using a Zeiss LSM 510 microscope.
Ultrastructural analysis was carried out as described
(Wilsch-Braeuninger et al.,
1997).
Molecular techniques
cnir and dEmp24 cDNAs were amplified from an ovarian two-hybrid
library (Grosshans et al.,
1994). All Grk transgenes are derived from a Grk genomic rescue
construct containing bases 49777-54827 of P1 DS02110. The region encoding Grk
amino acid 236-294 (transmembrane and cytoplasmic domain) was replaced using
PCR generated restriction sites in domain swaps with the transmembrane or
transmembrane and cytoplasmic domains of Yl (Grk-Yl TM, Grk amino acids 1-235
+ Yl amino acids 1801-1825; Grk-Yl TMC, Grk amino acids 1-235 + Yl amino acids
1801-1984). In three other constructs, the cytoplasmic tail of Grk was
replaced with the cytoplasmic tails of Yl (pGrk-YlCyt, Grk amino acids 1-275 +
Yl amino acids 1826-1984), dEmp24 (pGrk-EmpCyt, Grk amino acids 1-275 + CG3564
amino acids 194-208) or cni (pGrk-CniCyt, Grk amino acids 1-275 + Cni amino
acids 100-145).
The pcni::Cnir plasmid was cloned by replacing the cni-coding
region with a corresponding genomic fragment of cnir in the published
rescue construct (Roth et al.,
1995). The same template was used to introduce a single myc
epitope tag at the Cni C terminus. All constructs were cloned into pCasper4
(Pirrotta, 1988).
For western analysis, ovaries were ground on ice in lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, 2 mM DTT, 1%Triton X-100, 1% SDS 2 mg/ml Aprotinin, 1 mg/ml Leupeptin, 0.5 mg/ml Pepstatin, 10 mM PMSF) and loaded at one to three ovaries per lane. Protein was detected using the A14 and 9E10 anti-myc antisera (Santa Cruz) or the anti-Grk monoclonal 1D12 (DSHB).
Yeast two-hybrid experiments were performed using the system of James et
al. (James et al., 1996). The
control pair Staufen RNA binding domain 5/CG18501 was a gift from Uwe Irion
and Daniel St Johnston. Pulldown experiments were performed by bacterially
expressing the first 57 amino acids of Cni and Cnir or lacZ ORF, respectively,
fused to the maltose binding protein (MBP) of pMal-c2e (NEB). These fusion
proteins were subsequently tested for precipitation from bacterial lysates by
incubation with GST (pGEX2T, Pharmacia) or a GST-Grk fusion (GST and Grk amino
acids 179-245) prebound to Glutathion-Sepharose beads (Pharmacia). The beads
were spun down, washed in PBS with 300 mM NaCl and 1% Triton X-100, and MBP
fusions in the pellet detected by western blots using an anti-MBP monoclonal
antibody (Sigma).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, unlike mutations in grk, loss of
cni function also causes defects in adult somatic tissues. Flies
homozygous for the amorphic allele cniAR55 are subviable
(20% of the expected number hatch), possess rough eyes
(Fig. 1A-C), their
postvertical, interocellar and ocellar bristles are largely missing
(Fig. 1A,B), and the wings have
a truncated vein 2 (Fig.
1E,F).
Transgenes expressing Cni tagged with a single C-terminal myc epitope from
its endogenous promotor rescue the cni germline and somatic defects
(Fig. 1G-I; data not shown).
The tagged Cni protein is detectable in the germline from germarium stages
onwards and becomes enriched within the oocyte during early and middle stages
of oogenesis (Fig. 2A). Cni is
also detectable in the somatic follicular epithelium
(Fig. 2A), in the embryo, and
in male and female somatic tissues (not shown). To address the subcellular
localization of Cni, we turned to the cells of the squamous follicular
epithelium, the large size and flat geometry of which minimizes colocalization
artefacts. Myc-tagged Cni expressed in these cells using the CY2 driver line
is found in small, discrete, dot-like structures that are only partially
overlapping with the ER (Fig.
2B), labelled here using a GFP genetrap of the ER resident protein
Protein Disulfide Isomerase (PDI-GFP)
(Morin et al., 2001) (70% of
all Cni-dots scored in three fields of view associated with PDI-GFP,
n=291). However, whether this represents true colocalization is
difficult to judge as the ER extends widely throughout the cytoplasm. Although
only 8.7% of all Cni-containing structures were also stained with an antibody
against the p120 Golgi protein (Fig.
2C) (n=346 Cni-dots scored in three fields of view), 91%
were positive for staining against the KDEL-Receptor, which is involved in
retrieving KDEL-tagged proteins from the Golgi to the ER
(Fig. 2D) (n=305
Cni-dots scored in three fields of view). As biochemically determined for
Erv14p (Powers and Barlowe,
1998), the subcellular localization of Drosophila Cni is
thus consistent with cycling in and out of the ER. Although almost all
Cni-containing dots were KDEL-receptor positive, the converse is not the case.
Only 20.4% of the KDLR-positive dots contained Cni (n=641).
Drosophila Golgi units have been shown to be biochemically
heterogenous. with different enzyme and cargo content even at the same
cisternal level. Presence in only a subset of retrograde vesicles may
therefore reflect a prior targeting of Cni only towards specific Golgi units
(Yano et al., 2005).
cornichon and cni-related possess partially overlapping functions
The Drosophila genome contains a second cni-like gene (CG17262)
which we named cni-related (cnir). Cni and Cnir share 28.5%
identical and 43.1% similar amino acids, but are each more closely related to
specific vertebrate Cornichons, here exemplified by the human proteins. Cnirel
and Cni4 form a branch distinct from other metazoan Cornichons
(Fig. 3B). Overall structural
properties are conserved between all family members, with a cytoplasmic N
terminus and three transmembrane domains, as determined for Erv14p
(Powers and Barlowe, 2002) and
modelled here for Cni using the TMHMM and TMpred algorithms
(Fig. 3C).
cnir is deleted by Df(2L)JS7
(Sekelsky et al., 1995).
Heterozygosity for the deficiency causes synthetic lethality in an amorphic
cni background (Table
1). In combination with the hypomorphic allele
cniAA12 rare and severely malformed Df(2L)JS7
cniAR55/cniAA12 escapers could be
recovered. The synthetic lethality was completely rescued by introduction of a
transgene driving expression of cnir under control of the
cni promotor and 5' and 3' untranslated regions
(pcni::Cnir), demonstrating that the initial enhancement of the cni
phenotype to lethality was caused by the reduction of the total level of
cni-like genes (P<0.001, Fisher's exact test). The
construct also suppressed the loss of postvertical and ocellar bristles and
the wing venation defects of amorphic
cniAR55/cniAR55 flies
(Fig. 1D,F), but not the rough
eye phenotype and the loss of the interocellar bristles
(Fig. 1D).
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Grk protein is mislocalized to the interior of cni and grkDC oocytes
We re-examined the Grk protein distribution in cni ovaries by
light and immunoelectron microscopy, comparing the results with wild-type and
a grk mutation (grkDC) that produces a
non-secreted Grk protein (Queenan et al.,
1999). In wild-type stage 10 ovaries, Grk is concentrated between
the oocyte nucleus and the adjacent oolemma
(Fig. 4A)
(Neuman-Silberberg and Schüpbach,
1996). Immunoelectron microscopy shows that during vitellogenesis
the oolemma and the apical sides of the follicle cells have enlarged surfaces
covered by microvillous processes to facilitate rapid yolk uptake by the
oocyte (King, 1970)
(Fig. 4D). Grk protein could at
that stage be detected at the oocyte surface, especially on the microvilli.
Grk was also found at the microvilli covering the apical surface of the
follicular epithelium and within follicle cells
(Fig. 4D), confirming Grk
release from the oocyte (Peri et al.,
1999). grkDC is representative of a distinct
class of amorphic grk alleles with amino acid substitutions affecting
a conserved alanine residue within the transmembrane domain
(Queenan et al., 1999). These
mutations apparently block proteolytic release of the extracellular growth
factor domain (see below, Fig.
6C), which has been shown to be necessary for Grk activity
(Ghiglione et al., 2002). Grk
distribution in grkDC oocytes is indistinguishable from
wild type before the onset of vitellogenesis (stage 10A), when the oocyte
increases its endocytosis activity. Grk protein can then also be found in
posterior and ventral parts in homo- or heterozygous grkDC
oocytes, from where it is completely absent in wild-type ovaries
(Fig. 4B). At the
ultrastructural level internalized Grk protein associates with the membranous
cortex of yolk granules in heterozygous grkDC ovaries
(Fig. 4E). Yolk granules are
endosomal derivatives that grow by fusion with endocytic vesicles
internalizing yolk proteins (DiMario and
Mahowald, 1987). Both the timing of the GrkDC
mislocalization and the association of the mutant protein with the cortex of
the yolk granules therefore suggest that the GrkDC protein is
transiently inserted into the plasma membrane, and then reinternalized during
yolk uptake.
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), the
mutant GrkDC protein in wild-type ovaries therefore appears to
reside in a different endomembrane compartment than wild-type Grk protein in
cni mutant egg chambers.
Grk transport to the plasma membrane requires cni function
To confirm a requirement for Cni during Grk transport to the plasma
membrane, we attempted to trap the apparently membrane-tethered
GrkDC protein at the oocyte surface in the presence or absence of
Cni by inactivating a temperature-sensitive allele of the Drosophila
dynamin homologue shibire (shiTS)
(Chen et al., 1991; Grigliatti,
1973; van der Bliek,
1991).
Incubation of dissected shiTS ovaries heterozygous for grkDC at a restrictive temperature (32°C) resulted in the expansion of Grk protein staining around the anterior margin of these oocytes (Fig. 5D). Such an expansion was not observable in control ovaries that contained only wild-type grk (Fig. 5E) or were heterozygous for shiTS (not shown). Incubation at the permissive temperature never resulted in Grk accumulation at the plasma membrane, irrespective of the presence of the grkDC allele (Fig. 5A,B). These observations support our model that grkDC encodes a cleavage-resistant protein that localizes to the plasma membrane prior to its reinternalization.
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Cni acts after the Grk proteolytic processing step
We generated a fully functional (not shown) C-terminally tagged version of
Grk by replacing the intracellular domain, which is dispensable for function
(Queenan et al., 1999), with
five myc epitopes (Grk5myc). When Grk5myc was expressed from the endogenous
promotor, only a short fragment corresponding in size to the expected
C-terminal cleavage remnant (23 kDa) could be detected in western blots by
antisera directed against the C-terminal epitope tags
(Fig. 6A). Thus, at near
endogenous expression levels Grk protein is quantitatively present in the
processed form. As the same band was observed in extracts from flies mutant
for cni (Fig. 6A), Grk
processing does not require Cni function.
In ovary lysates from flies overexpressing Grk5myc in the germline under
control of the maternal -Tubulin Gal4 or nanos driver lines both the
cleavage remnant and a longer protein species (ca. 70 kDa) could readily be
detected (Fig. 6B and data not
shown). The 70 kDa band corresponds to an uncleaved precursor form, as it can
be detected both by a monoclonal antibody directed against the extracellular
growth factor domain (Fig. 6B,
right panel) as well as by antibodies against the intracellular, C-terminal
epitope tag (Fig. 6B, left
panel). Formation of the C-terminal Grk cleavage remnant is independent of
cni also under overexpression conditions
(Fig. 6B, left panel). However,
in the absence of Cni, another species accumulates (48 kDa), which is
recognized by the extracellular but not the C-terminal antisera, and therefore
appears to be the N-terminal cleavage product corresponding to the mature
growth factor. Supporting this interpretation, a band of this size has
previously been identified as the active secreted Grk ligand in cell culture
experiments (Ghiglione et al.,
2002).
Together, these experiments suggest that cni is not required for proteolytic processing of Grk, and that in wild type, the generation, secretion and eventual degradation of the mature growth factor is a rapid process with low steady state levels of the protein. Although the cleavage process is saturable when Grk is overexpressed in wild type, causing accumulation of the uncleaved precursor, the N-terminal cleavage product is detected only after overexpression in a cni mutant background.
|
). Thus, in the absence of Cni, proteolytic cleavage of the
Grk precursor occurs normally, but the extracellular/lumenal fragment that in
wild type forms the mature, secreted growth factor accumulates in the ER and
retains its pre-Golgi glycosylation signature.
Proteolytic processing of Grk therefore differs from that of Spitz which
has to be exported from the ER to be processed by Rhomboid1 within the Golgi
(Lee et al., 2001;
Urban et al., 2001;
Urban et al., 2002). By
contrast, Grk is processed at the ER level, and requires Cni function for
efficient export of the mature ligand. These in vivo observations are
consistent with tissue culture data showing that the presumptive Grk protease
Rhomboid2 (Guichard et al.,
2000) is able to process Grk retained within the ER
(Ghiglione et al., 2002).
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). Unlike
Grk, the Drosophila vitellogenin receptor Yolkless (Yl) is
efficiently transported to the plasma membrane of cni mutant oocytes
as seen from the formation of yolk vesicles in these cells
(Fig. 4D)
(DiMario and Mahowald, 1987).
We therefore generated transgenic flies expressing domain swap constructs from
the endogenous grk promotor, in which either the Grk intracellular
domain, the transmembrane domain, or both, were replaced with the
corresponding fragments of Yl. Although Yl itself is not proteolytically
released, it possesses, like Grk, an alanine residue at the position mutated
in the cleavage resistant allele grkDC (Ala245Val). All
three constructs were able to rescue the oogenesis phenotype of grk
mutant flies (Fig. 7D; data not
shown). As Grk activity depends on prior proteolytic release of the
extracellular growth factor (Ghiglione et
al., 2002), these domain swaps do not interfere with the
processing of Grk despite the reported substrate selectivity of Rhomboid type
proteases (Urban and Freeman,
2003). By contrast, signalling by all three fusion proteins still
depends on Cni function, as neither construct was able to suppress the
cni oogenesis phenotype (Fig.
7E; data not shown). The region crucial for the role of Cni in Grk
secretion shown above must therefore lie within the extracellular domain of
Grk.
Cni interacts with the membrane proximal part of the Grk extracellular domain
We tested for potential corresponding protein interactions using a yeast
two-hybrid system selecting for adenine and histidine autotrophy
(James et al., 1996) with bait
constructs containing different portions of the Grk extracellular domain
extending to the beginning of the transmembrane domain (amino acids 74-245,
179-245, 215-245). Introduction of a prey construct containing the Cni ORF
into this background conferred the ability to grow under stringent selection
conditions, indicating an interaction between the two proteins
(Table 2). Thus, the membrane
proximal 31 residues of the Grk extracellular domain are sufficient to mediate
binding to Cni in a yeast two-hybrid assay. Interaction was also observed with
a prey construct encoding only the first 57 amino acids of Cni. By contrast,
neither of the Grk bait constructs nor the CG18501 control were able to bind
to a prey construct containing Cnir. These two hybrid data were confirmed by
pull-down experiments, where a GST fusion protein containing amino-acids 197
to 245 of Grk could specifically co-purify a MBP fusion construct containing
the N-terminal 57 amino acids of Cni, but not one with the corresponding Cnir
domain or a lacZ control (Fig.
6E). This difference between Cni and Cnir in their ability to bind
Grk may underlie the strict requirement for Cni during Grk secretion, even
though the two Drosophila Cni-like proteins exhibit redundancy in
other contexts.
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). Exit of Emp24p and associated cargo from the ER is in
part mediated by binding of Emp24p to components of the COPII coat through
diaromatic amino acid pairs in the C-terminal cytoplasmic tail. We replaced
the intracellular domain of Grk with the short cytoplasmic tail of one of the
D. melanogaster Emp24 homologues (CG3564 amino acids 194-208). A
transgene expressing this fusion protein from the endogenous promotor
(pGrk-EmpCyt) fully rescued the loss of grk, indicating that the
transgene produced normal amounts of active Grk ligand (not shown).
Interestingly, it also restored some Grk signalling activity in the absence of
cni (Fig. 7F). Eggs
laid by homozygous cni females containing one copy of pGrk-EmpCyt had
normal anteroposterior polarity. Some eggs also possessed recognizable dorsal
appendage material, indicating low to intermediate levels of Grk signalling in
these egg chambers (Fig. 7F).
Thus, fusing Grk to a domain known to mediate selective recruitment into COPII
vesicles partially alleviates its dependence on Cni. Similar results were achieved using an analogous transgene replacing the Grk intracellular domains with a Cni fragment consisting of the C terminus after the second predicted transmembrane domain (Cni amino acids 100-145, pGrk-CniCyt). The transgene fully rescued the loss of grk (data not shown) and restored some signalling activity in the absence of cni. Eggs laid by cni mutant females carrying one copy of this transgene had normal anteroposterior polarity and showed slight and variable rescue of the dorsoventral axis (Fig. 7G). The C-terminal domains of Cni-like and Emp24-like proteins may therefore be functionally equivalent.
If Cni were only functioning as a cargo receptor for ER export of Grk,
massive overexpression of Grk should result in bulk flow ER to Golgi transport
and might thus overcome the requirement for cni. To test this
assumption, we analyzed the egg phenotypes produced by those grk
overexpression lines that were used for the western blots described above.
Expression of grk with the help of the maternal -Tubulin Gal4
driver leads to a strong increase in the amount of Grk protein in stage 9 egg
chambers when compared with endogenous Grk levels
(Fig. 8A,C,E). When
overexpressed in a wild-type background, the bulk of grk mRNA is
still transported to the vicinity of the nucleus (data not shown). Grk protein
remains asymmetrically distributed, although the region with high Grk protein
levels within the oocyte is more expanded when compared with wild type
(Fig. 8A,C). This might be due
to the saturation of the mechanisms normally responsible for retention of the
protein near its site of translation and the subsequent secretion through a
few local ER exit sites (Herpers et al., 2004). The resulting egg chambers
maintain DV polarity although they are severely dorsalized. The operculum, the
dorsal-most chorion structure that is specified in follicle cells receiving
maximal Grk levels, is expanded while the dorsal appendages normally specified
at more lateral positions experiencing slightly lower Grk signalling are
shifted to the ventral side of the egg
(Fig. 8B,C).
Overexpression of Grk in a cni mutant background results in uniform high levels of Grk protein within the oocyte (Fig. 8E). Interestingly, the resulting eggs possess variable amounts of dorsal appendage material (Fig. 8F,G), indicating restoration Grk signalling, albeit to lower levels than in the presence of Cni. However, the eggs lack DV and frequently even AP polarity, as can be seen by the patchy induction of dorsal appendage material around the entire egg circumference (Fig. 8F,G) and the presence of a posterior micropyle (Fig. 8G), respectively.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Bonifacino and Glick, 2004;
Kuehn and Schekman, 1997). Cni
partially colocalizes with the ER resident protein marker PDI-GFP, associates
with the KDEL-receptor (a protein involved in retrieval of escaped ER
proteins) but is largely excluded from the Golgi. This suggests that Cni, like
its yeast homologue Erv14p (Powers and
Barlowe, 1998; Powers and
Barlowe, 2002), has a pre-Golgi localization.
|
),
these data show that the requirement for Cni in Grk export lies at the level
of ER export. Our two hybrid data and Grk domain swap experiments suggest that
Cni binds Grk through an interaction between its lumenal hydrophilic loop and
the membrane-proximal extracellular spacer between the transmembrane and EGF
domains of Grk. This interaction is consistent with the membrane topology
proposed by Powers and Barlowe (Powers and
Barlowe, 2002) for Erv14p, and suggests that Cni is the cargo
receptor for Grk ER export. The inability of Cnir to bind to Grk in the same
assay also correlates with its failure to suppress the cni oogenesis
phenotype.
Processing of Spitz by Rhomboid1 depends on prior export of the
transmembrane precursor from the ER (Lee
et al., 2001; Tsruya et al.,
2002; Urban et al.,
2001). However, Spitz, as well as Grk, can be processed by
Rhomboid2 and Rhomboid3 while still within the ER
(Ghiglione et al., 2002;
Urban et al., 2002). In the
absence of Cni, Grk is effectively processed in the oocyte although it cannot
leave the ER. Thus, during oogenesis Grk processing must occur within the ER,
suggesting that the presumptive Grk protease Rhomboid2
(Ghiglione et al., 2002;
Guichard et al., 2000) either
resides in, or cycles through, the oocyte ER.
|
). By
contrast, our data suggest that Cni serves to specifically ensure efficient
export of Grk after cleavage in the oocyte ER. We propose that Grk interacts
with Cni prior to its proteolytic processing, and that the proteins remain
associated at least until the mature growth factor is recruited into an
outgoing vesicle (Fig. 9A). By
contrast, soluble Grk protein lacking a membrane anchor would diffuse away
into the lumen after synthesis, precluding recruitment by Cni at the ER
membrane, explaining why truncated Grk protein lacking a transmembrane domain
is not secreted from the oocyte and corresponding grk alleles are
nonfunctional (Queenan et al.,
1999). The same fate would await Grk released from its membrane
anchor through proteolytic processing in a cni mutant oocyte
(Fig. 9B).
Constructs expressing Grk fused to the cytoplasmic parts of either Cni or
dEmp24p are partly able to restore Grk signalling in the absence of Cni, but
not to wild-type levels. We suggest that the heterologous cytoplasmic tails,
which contain the respective domains shown in yeast to mediate the COPII
interactions (Schimmoller et al.,
1995; Powers and Barlowe,
2002), are rapidly recruiting unprocessed Grk fusion proteins
towards prospective vesicle budding sites. Because the ER exit motives are
separated from the growth factor part during the processing step, most of the
processed protein will in the absence of Cni still escape into the ER lumen,
explaining the low rescue efficiency of the fusion proteins. However,
proteolytic cleavage would preferentially occur in the vicinity of the
outgoing vesicles, locally increasing the concentration of the soluble mature
growth factor (Fig. 9C). This
appears to be sufficient to ensure inclusion of some processed Grk into the
outgoing vesicles in the absence of Cni, but cannot reconstitute wild-type
rates of Grk signalling.
Conversely, the hypomorphic mutation cniAA12 truncates
Cni after the first two putative membrane-spanning domains
(Roth et al., 1995). It
therefore deletes the second, cytoplasmic loop shown to mediate COPII
interaction in Erv14p (Powers and Barlowe,
2002), but still possesses the first, lumenal loop binding to Grk.
The truncated CniAA12 protein may therefore remain able to keep
processed Grk at the ER membrane. This would limit diffusion of processed Grk
to the two dimensions of the ER membrane, rather than the three dimensions of
the lumen, thereby enriching it to some degree in vesicles leaving the oocyte
ER (Fig. 9D). However, to
achieve the full rate of Grk secretion further cargo concentration into
outgoing vesicles through interaction of the Grk-Cni complexes with the COPII
coat would be required. Consistently, cniAA12 is clearly a
hypomorphic allele with readily detectable remaining Grk signalling activity
(Fig. 7B), but in mutant
oocytes, Grk protein is diffusely mislocalized within the large, continuous ER
and can no longer be found concentrated at ER exit sites
(Herpers and Rabouille,
2004).
Grk is translated from a localized mRNA and becomes translocated into a
giant ER spanning the entire oocyte and containing around 1000 active exit
sites. Nevertheless, in the presence of its cargo receptor Cni, Grk is
exclusively secreted through a few of these sites and their associated Golgi
stacks at the dorsal anterior corner where the grk mRNA is found
(Herpers and Rabouille, 2004),
giving rise to a spatially tightly confined signal to the neighbouring
follicle cells. Concentration-driven bulk flow export from the ER can support
secretion if cargo proteins are synthesized at sufficient rates to allow their
accumulation within the ER
(Martinez-Menarguez et al.,
1999). Correspondingly, we have shown that massive overexpression
of Grk in the oocyte can in principle restore signalling in the absence of Cni
function, most likely via bulk flow ER export. However, in comparison to the
wild-type situation the spatial and temporal precision of the Grk signals is
lost, with severe consequences for the subsequent steps of pattern
formation.
Cni is also required independently from Grk in somatic tissues, where it
appears to act redundantly with Cnir. The reduced viability and life span of
flies lacking cni function and the synthetic lethality when the gene
dose of cnir is reduced in a cni mutant background indicate
a more general cellular function of the Cni proteins. Erv14p, the cni
homologue from S. cerevisiae, is involved in recruiting the golgin
Rud3p to the cis-Golgi stacks (Gillingham
et al., 2004). Besides its function in Axl2p recruitment, Erv14p
therefore may play a more general role in establishing cis-Golgi identity. It
will be interesting to find out whether Cni proteins in general might have a
more fundamental cell biological function, e.g. in establishing Golgi
polarity, that may so far have been masked by redundancy and more easily
detected phenotypes caused by their roles as cargo receptors.
|
ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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