InteractiveFly: GeneBrief

Cornichon: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Drosophila Cornichon (Cni) is the founding member of a conserved protein family that also includes Erv14p, an integral component of the COPII-coated vesicles that mediate cargo export from the yeast endoplasmic reticulum (ER). During Drosophila oogenesis, Cni is required for transport of the TGFalpha growth factor Gurken (Grk) to the oocyte surface. Cni, but not the second Drosophila Cni homologue Cni-related (Cnir), binds to the extracellular domain of Grk, and it is proposed 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 TGFalpha 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 (Bökel, 2006).

During Drosophila oogenesis, signals from the oocyte to the overlying follicular epithelium govern the polarization of the maturing egg and the establishment of the future embryonic body axes (Roth, 2003). These signals are mediated by the TGFalpha-like growth factor Gurken. Eggs laid by females homozygous for null alleles of grk are ventralized and lack anteroposterior polarity. 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 TGFalpha and the other Drosophila TGFalpha family members Spitz and Keren. The mature, secreted forms of all three Drosophila TGFalpha homologues are likely to be generated by intramembrane proteases of the Rhomboid family. Specifically, Grk appears to be cleaved by Rhomboid-2/Brho. 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. By contrast, it is shown in this study that Grk processing occurs before export from the oocyte ER (Bökel, 2006).

Generation of the Grk signals depends on the presence of Cornichon [Cni (Roth, 1995)] within the germline. cni encodes a small hydrophobic protein that is the founding member of a family of conserved eukaryotic proteins (Hwang, 1999; Powers, 1998; Roth, 1995). Erv14p, one of the two S. cerevisiae Cni homologues, was identified as an integral membrane protein of COPII-coated ER-derived vesicles. The COPII coat consists of several subunits that are assembled into a multimolecular coat on the surface of the ER and serves as an external scaffold organizing the assembly of anterograde transport vesicles at the ER exit sites (Bonifacino, 2004). Direct or indirect interactions with COPII components (Barlowe, 2003; Kuehn, 1997) can provide an efficient mechanism for the recruitment of cargo proteins into vesicles leaving the ER (Bökel, 2006).

Erv14p is itself recruited into such vesicles through interactions with the COPII coat (Powers, 2002). Loss of Erv14p results in a bud site selection defect caused by inefficient membrane transport of the bud site selection protein Axl2p (Powers, 1998; Roemer, 1996). In erv14Delta 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, 1998; Powers, 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 (Herpers, 2004) activity causes diffuse mislocalization of Grk protein within the ER (Bökel, 2006).

Biochemical and genetic evidence is provided for an involvement of Cni in Grk ER export and data is presented explaining why Cni function is essential for the spatial and temporal specificity of Grk signalling (Bökel, 2006).

Recruitment within the ER is essential for the efficient ER exit of many different proteins. Cargo proteins may be concentrated into outgoing COPII-coated vesicles at the ER exit sites either by binding directly to vesicle or coat components, or through indirect recruitment using additional adaptors (Barlowe, 2003; Bonifacino, 2004; Kuehn, 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, 1998; Powers, 2002), has a pre-Golgi localization (Bökel, 2006).

Using the cleavage-resistant Grk^DC protein, it has been demonstrated that loss of cni blocks Grk transport to the oocyte plasma membrane, and in the absence of Cni overexpressed Grk protein accumulates within the oocyte and retains an ER-type glycosylation. Together with the accumulation of Grk within the ER of hypomorphic cni mutant oocytes (Herpers, 2004), these data show that the requirement for Cni in Grk export lies at the level of ER export. 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 (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 (Bökel, 2006).

Processing of Spitz by Rhomboid1 depends on prior export of the transmembrane precursor from the ER. However, Spitz, as well as Grk, can be processed by Rhomboid2 and Rhomboid3 while still within the ER. 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 either resides in, or cycles through, the oocyte ER (Bökel, 2006).

Processed Spitz generated before export of the precursor to the Golgi is specifically retained in the ER. By contrast, the data suggest that Cni serves to specifically ensure efficient export of Grk after cleavage in the oocyte ER. It is proposed 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. 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, 1999). The same fate awaits Grk released from its membrane anchor through proteolytic processing in a cni mutant oocyte (Bökel, 2006).

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. It is suggested that the heterologous cytoplasmic tails, which contain the respective domains shown in yeast to mediate the COPII interactions (Schimmoller, 1995; Powers, 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. 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 (Bökel, 2006).

Conversely, the hypomorphic mutation cni^AA12 truncates Cni after the first two putative membrane-spanning domains (Roth, 1995). It therefore deletes the second, cytoplasmic loop shown to mediate COPII interaction in Erv14p (Powers, 2002), but still possesses the first, lumenal loop binding to Grk. The truncated Cni^AA12 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. 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, cni^AA12 is clearly a hypomorphic allele with readily detectable remaining Grk signalling activity, but in mutant oocytes, Grk protein is diffusely mislocalized within the large, continuous ER (Herpers, 2004) and can no longer be found concentrated at ER exit sites (Bökel, 2006).

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, 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, 1999). Correspondingly, 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 (Bökel, 2006).

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, 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 (Bökel, 2006).

REGULATION

Cni interacts with the membrane proximal part of the Grk extracellular domain

Cni interacts with the membrane proximal part of the Grk extracellular domain. Tests were performed for potential corresponding protein interactions using a yeast two-hybrid system selecting for adenine and histidine autotrophy 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. 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. 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 (Bökel, 2006).

Yeast Emp24p is a cargo receptor cycling between the ER and the intermediate compartment (Schimmoller, 1995). 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. The intracellular domain of Grk was replaced 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 rescues the loss of grk, indicating that the transgene produced normal amounts of active Grk ligand. Interestingly, it also restores some Grk signalling activity in the absence of cni. Eggs laid by homozygous cni females containing one copy of pGrk-EmpCyt have normal anteroposterior polarity. Some eggs also possessed recognizable dorsal appendage material, indicating low to intermediate levels of Grk signalling in these egg chambers. Thus, fusing Grk to a domain known to mediate selective recruitment into COPII vesicles partially alleviates its dependence on Cni (Bökel, 2006).

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 rescues the loss of grk and restores some signalling activity in the absence of cni. Eggs laid by cni mutant females carrying one copy of this transgene have normal anteroposterior polarity and show slight and variable rescue of the dorsoventral axis. The C-terminal domains of Cni-like and Emp24-like proteins may therefore be functionally equivalent (Bökel, 2006).

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, the egg phenotypes produced by grk overexpression lines were analyzed. Expression of grk with the help of the maternal alpha-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. When overexpressed in a wild-type background, the bulk of grk mRNA is still transported to the vicinity of the nucleus. Grk protein remains asymmetrically distributed, although the region with high Grk protein levels within the oocyte is more expanded when compared with wild type. 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. 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 (Bökel, 2006).

Overexpression of Grk in a cni mutant background results in uniform high levels of Grk protein within the oocyte. Interestingly, the resulting eggs possess variable amounts of dorsal appendage material, 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 and the presence of a posterior micropyle, respectively (Bökel, 2006).

These observations show that the requirement for Cni can be overcome by Grk overexpression. Thus, cni function is not essential for the formation of an active ligand per se, but the Cni-mediated increase in the efficiency of Grk secretion is a prerequisite for the precise temporal and spatial control of Grk signalling (Bökel, 2006).

DEVELOPMENTAL BIOLOGY

Transgenes expressing Cni tagged with a single C-terminal myc epitope from its endogenous promotor rescue the cni germline and somatic defects. 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. Cni is also detectable in the somatic follicular epithelium, in the embryo, and in male and female somatic tissues . To address the subcellular localization of Cni, cells of the squamous follicular epithelium, the large size and flat geometry of which minimizes colocalization artifacts, were examined. 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, labelled using a GFP genetrap of the ER resident protein Protein Disulfide Isomerase (PDI-GFP) (70% of all Cni-dots scored in three fields of view associated with PDI-GFP). 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, 91% were positive for staining against the KDEL-Receptor, which is involved in retrieval of escaped ER proteins, acting by retrieving KDEL-tagged proteins from the Golgi to the ER. As biochemically determined for mammalian Erv14p (Powers, 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. 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 towards specific Golgi units (Yano, 2005; Bökel, 2006).

EFFECTS OF MUTATION Grk transport to the plasma membrane requires cni function

Owing to a failure in Grk signalling during oogenesis, eggs produced by cni mutant females lack both anteroposterior (AP) and dorsoventral (DV) polarity (Roth, 1995). However, unlike mutations in grk, loss of cni function also causes defects in adult somatic tissues. Flies homozygous for the amorphic allele cni^AR55 are subviable (20% of the expected number hatch); they possess rough eyes, their postvertical, interocellar and ocellar bristles are largely missing, and their wings have a truncated vein 2 (Bökel, 2006).

The second cni-like gene of Drosophila cni-related (cnir) is deleted by Df(2L)JS7. Heterozygosity for the deficiency causes synthetic lethality in an amorphic cni background. In combination with the hypomorphic allele cni^AA12 rare and severely malformed Df(2L)JS7 cni^AR55/cni^AA12 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. The construct also suppresses the loss of postvertical and ocellar bristles and the wing venation defects of amorphic cni^AR55/cni^AR55 flies, but not the rough eye phenotype and the loss of the interocellar bristles (Bökel, 2006).

In contrast to rescue constructs expressing native or myc-epitope tagged cni from either the cni or bicoid promotors, analogous constructs containing cnir are unable to compensate for the loss of cni during oogenesis. Cnir is therefore able to substitute for Cni in some contexts, whereas other processes, most notably Grk signalling by the oocyte, specifically require Cni function (Bökel, 2006).

The Grk protein distribution was examined in cni ovaries by light and immunoelectron microscopy, comparing the results with wild-type and a grk mutation (grk^DC) that produces a non-secreted Grk protein (Queenan, 1999). In wild-type stage 10 ovaries, Grk is concentrated between the oocyte nucleus and the adjacent oolemma (the plasma membrane of the oocyte). 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. Grk protein at that stage can be detected at the oocyte surface, especially on the microvilli. Grk is also found at the microvilli covering the apical surface of the follicular epithelium and within follicle cells, confirming Grk release from the oocyte. grk^DC is representative of a distinct class of amorphic grk alleles with amino acid substitutions affecting a conserved alanine residue within the transmembrane domain. These mutations apparently block proteolytic release of the extracellular growth factor domain, which has been shown to be necessary for Grk activity. Grk distribution in grk^DC 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 homozygous or heterozygous grk^DC oocytes, from where it is completely absent in wild-type ovaries. At the ultrastructural level internalized Grk protein associates with the membranous cortex of yolk granules in heterozygous grk^DC ovaries. Yolk granules are endosomal derivatives that grow by fusion with endocytic vesicles internalizing yolk proteins. Both the timing of the Grk^DC mislocalization and the association of the mutant protein with the cortex of the yolk granules therefore suggest that the Grk^DC protein is transiently inserted into the plasma membrane, and then reinternalized during yolk uptake (Bökel, 2006).

By comparison, Grk is more smoothly distributed within cni mutant oocytes and seems to diffuse away from a source near the nucleus. At the ultrastructural level, the comparison with grk^DC and wild-type ovaries shows that Grk protein in cni oocytes is neither clustered at the cortex of yolk granules nor enriched at the plasma membrane adjacent to the oocyte nucleus. In contrast to earlier suggestions, the mutant Grk^DC protein in wild-type ovaries therefore appears to reside in a different endomembrane compartment than wild-type Grk protein in cni mutant egg chambers (Bökel, 2006).

To confirm a requirement for Cni during Grk transport to the plasma membrane, attempts were made to trap the apparently membrane-tethered Grk^DC protein at the oocyte surface in the presence or absence of Cni by inactivating a temperature-sensitive allele of the Drosophila dynamin homologue shibire (shi^TS). Incubation of dissected shi^TS ovaries heterozygous for grk^DC at a restrictive temperature (32°C) resulted in the expansion of Grk protein staining around the anterior margin of these oocytes. This observation supports the model that grk^DC encodes a cleavage-resistant protein that localizes to the plasma membrane prior to its reinternalization (Bökel, 2006).

By contrast, Grk^DC protein can not be trapped at the plasma membrane of oocytes from shi^TS/shi^TS; grk^DC Df(2L)H60/cni^AR55 females. Both at permissive and restrictive incubation temperatures for shi^TS, all Grk protein remains diffusely distributed throughout the oocyte, in a pattern indistinguishable from the distribution of wild-type Grk in homozygous cni ovaries. Thus, Cni is required for the transport of mutant Grk^DC protein and, by extension, wild-type Grk to the oocyte plasma membrane (Bökel, 2006).

A fully functional C-terminally tagged version of Grk was generated by replacing the intracellular domain, which is dispensable for function, 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. Thus, at near endogenous expression levels Grk protein is quantitatively present in the processed form. Since the same band was observed in extracts from flies mutant for cni, Grk processing does not require Cni function (Bökel, 2006).

In ovary lysates from flies overexpressing Grk5myc in the germline under control of the maternal alpha-Tubulin Gal4 or nanos driver lines both the cleavage remnant and a longer protein species (ca. 70 kDa) can readily be detected. The 70 kDa band corresponds to an uncleaved precursor form, since it can be detected both by a monoclonal antibody directed against the extracellular growth factor domain as well as by antibodies against the intracellular, C-terminal epitope tag. Formation of the C-terminal Grk cleavage remnant is independent of cni also under overexpression conditions. 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 (Bökel, 2006).

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 (Bökel, 2006).

It was next asked where processing of Grk occurs along the secretory pathway. A C-terminally tagged version of Grk was generated that resembles Grk5myc, but in addition carries the point mutation that induces the A to V amino acid exchange found in grk^DC (GrkDC5myc). In contrast to the functional cleavable Grk5myc protein, GrkDC5myc expressed from the endogenous promotor accumulates in the ovary as a high molecular weight form (70 kDa) corresponding in size to the precursor band found in the overexpression situation. This supports the notion that the grk^DC point mutation prevents proteolytic cleavage of the Grk precursor. In lysates from cni heterozygous control ovaries, the GrkDC5myc signal is smeared out into several high molecular weight bands, which is indicative of glycosylation of the protein in the Golgi. Strikingly, this smearing of the GrkDC5myc band does not occur in the absence of Cni. Therefore the glycosylation state of the N-terminal Grk5myc cleavage product accumulating in cni mutant ovaries was checked. The molecular weight of this fragment could be decreased both by treatment with Endoglycosidase H (EndoH) and Protein N-glycosidase F (PNGaseF). Although PNGaseF is capable of removing all N-linked sugar modifications, EndoH sensitivity is characteristic for the high mannose type modifications added already in the ER, which will subsequently be trimmed and replaced by the final sugar modifications upon entry into the Golgi. 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 (Bökel, 2006).

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. 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 is able to process Grk retained within the ER (Bökel, 2006).

Secretion of Grk depends both on the presence of Cni protein and the Grk transmembrane domain, and it has therefore been suggested that an interaction within the membrane might be responsible for the role of Cni in Grk secretion. 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. Therefore transgenic flies were generated 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 grk^DC (Ala245Val). All three constructs were able to rescue the oogenesis phenotype of grk mutant flies. Since Grk activity depends on prior proteolytic release of the extracellular growth factor, these domain swaps do not interfere with the processing of Grk despite the reported substrate selectivity of Rhomboid type proteases. By contrast, signalling by all three fusion proteins still depends on Cni function; neither construct is able to suppress the cni oogenesis phenotype. The region crucial for the role of Cni in Grk secretion must therefore lie within the extracellular domain of Grk (Bökel, 2006).

mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in Gurken transport in Drosophila oocytes

The anteroposterior and dorsoventral axes of the future embryo are specified within Drosophila oocytes by localizing gurken mRNA, which targets the secreted Gurken transforming growth factor-alpha synthesis and transport to the same site. A key question is whether gurken mRNA is targeted to a specialized exocytic pathway to achieve the polar deposition of the protein. This study shows, by (immuno)electron microscopy that the exocytic pathway in stage 9-10 Drosophila oocytes comprises a thousand evenly distributed transitional endoplasmic reticulum (tER)-Golgi units. Using Drosophila mutants, it was shown that it is the localization of gurken mRNA coupled to efficient sorting of Gurken out of the ER that determines which of the numerous equivalent tER-Golgi units are used for the protein transport and processing. The choice of tER-Golgi units by mRNA localization makes them independent of each other and represents a nonconventional way by which the oocyte implements polarized deposition of transmembrane/secreted proteins. It is proposed that this pretranslational mechanism could be a general way for targeted secretion in polarized cells, such as neurons (Herpers, 2004).

To understand how Gurken, as a transmembrane protein, achieves its polar distribution, the organization of the exocytic pathway in Drosophila oocytes was elucidated. The exocytic pathway is similar to that found in other Drosophila cells observed so far. Namely, it contains a continuous ER that pervades the entire ooplasm, from which a multitude of tER-Golgi units arise. In the oocyte, as in S2 cells, the tER-Golgi units comprise an ER exit site (positive for dSec23p), closely apposed to a Golgi apparatus, either under the form of a cluster of vesicles and tubules, or a Golgi stack, both marked by the Golgi marker, dCOG5. S2 cells contain ~20 of these units, whereas the number is much greater in oocytes (~1000) but with an equivalent density (S2 cells have about a 60-100 times smaller volume than a stage 9 oocyte) (Herpers, 2004).

One way to explain Gurken deposition at the dorsal/anterior (D/A) corner is to argue for a concentration of the tER-Golgi units at this corner. It has been shown that cell migration in wound healing is accompanied by the redistribution and concentration of the cellular Golgi complex to the part of the cell facing the injury, thus sustaining a polarized secretion that helps in the healing. This study shows, using immunofluorescence and electron microscopy, that the thousand tER-Golgi units in the Drosophila oocyte are evenly distributed throughout the ooplasm. This concentration is therefore unlikely to be the underlying mechanism for Gurken polarity (Herpers, 2004).

Another way to explain that Gurken is only synthesized and transported in the tER-Golgi units localized at the D/A corner is to argue that they have a unique composition in regard to the three known proteins involved in its movement through the exocytic pathway: Star, Cornichon, and Brother of Rhomboid (Brho). Perhaps these three proteins only reside in the tER-Golgi units at the D/A corner, therefore rendering them, and only them, competent for Gurken transport. Star is thought to act as an ER chaperone helping in the exit from the ER. Cornichon is a potential Gurken receptor at the ER exit sites. Brho is a specific endoprotease located in the Golgi apparatus that cleaves Gurken just after its transmembrane domain, thus generating the lumenal active ligand of Torpedo, and a C-terminal membrane bound fragment whose fate is undetermined (Herpers, 2004).

Several lines of evidence, however, suggest that these proteins are not restricted to the tER-Golgi units of the D/A corner. (1) cornichon and brho mRNAs do not have a polarized localization, but rather occupy the entire oocyte. This suggests that the two proteins are expressed ubiquitously, as is Star protein in stage 6-10 oocytes. (2) In an S2 cell assay, Gurken is only cleaved and secreted when cells are transfected with both Brho and Star. When transfected with Brho alone, Gurken is cleaved but not secreted, and with Star alone, Gurken is neither processed nor secreted. Because in both squid and K10 mutants Gurken protein is transported to all tER-Golgi units along the anterior side and at the ventral/anterior corner. Gurken protein is found in the space between the oocyte and the nurse cells: this suggests that at least Star and Brho are present in all tER-Golgi units, including those away from the D/A corner, and they act in the processing and transport of Gurken. Similarly, Star, Cornichon, and Brho are also likely localized to the tER-Golgi units at the posterior pole, in WT stage 6-7 and stage 9-10 merlin oocytes where Gurken protein is synthesized, transported and processed, so Gurken signals to the posterior follicle cells. (3) It was recently published that in stage 10 germ line clones of the sec5 exocyst complex subunit, Gurken protein is synthesized in the middle and at the posterior pole of the Drosophila oocyte. This experiment, and those described in this study, shows that all tER-Golgi units potentially have the capacity of transporting and processing Gurken protein. Together, the tER-Golgi units at the D/A corner do not seem to contain a different set of transport and processing proteins from the others (Herpers, 2004).

This study shows, by using K10, squid, and merlin mutants, that what dictates the use of these numerous, seemingly identical and evenly distributed tER-Golgi units, is the restricted localization of gurken mRNA. This also could be the case for other transmembrane/secreted proteins (Herpers, 2004).

gurken mRNA is localized in a restricted manner at the D/A corner, where it is then anchored. The anchoring mechanism is not yet clear and is the subject of intense research, but it could be envisaged that an efficient recruitment of local tER-Golgi units would be achieved by anchoring the mRNA directly to the membrane. The ER has been suggested as such an anchor. Whatever the anchoring mechanism and wherever it is localized, gurken RNA diffuses locally, binds to ribosomes, is translated, and recognized by the signal recognition particle that targets it to the most proximal ER membrane, where the protein is synthesized and subsequently transported through the most adjacent tER-Golgi units (Herpers, 2004).

Additional mechanisms also ensure that local synthesis is followed by local polarized delivery at the D/A intercellular space, where the activity of Gurken is necessary but also needs to be restricted for proper oocyte development. In eukaryotic cells, the ER has been shown to be continuous throughout the entire cytoplasm. FRAP experiments on PDI-GFP-expressing oocytes as well as the use of a strong allelic combination of Cornichon have shown that the ER comprises a single lumen throughout the oocyte, including at the D/A corner. Partial diffusion of the newly synthesized Gurken in the membrane of the ER is therefore expected. Such a diffusion over long distance (>0.5 mm) within the ER has been shown for soluble proteins, such as the light and heavy chains of the immunoglobulins in frog oocytes. However, the maximum distance over which intracellular Gurken is found is 20 µm (Herpers, 2004).

Therefore, the diffusion of Gurken is likely to be prevented by efficient sorting mechanisms. Such mechanisms rely primarily on the transmembrane domain of Gurken and Cornichon. Gurken lacking its transmembrane domain diffuses in the ER in a very similar way as WT Gurken does in a strong cornichon mutant, except for the concentration observed at the D/A corner. This could be explained by the difference in diffusion between a transmembrane protein and a lumenal fragment. Nevertheless, this phenocopy suggests that Gurken binds Cornichon through its transmembrane domain. This interaction could mediate the efficient packaging of transmembrane Gurken in COPII transport vesicles. Cornichon presents homology to Erv14p, which is involved, in yeast, in the exit of the plasma membrane Axl2 transmembrane protein from the ER in COPII-coated vesicles. The interaction between Erv14p and Axl2p has been suggested to act via a novel mechanism that might be mediated by interactions of transmembrane segments. The binding of Gurken to Cornichon might rely on a similar mechanism, although it is not clear why a transmembrane cargo protein would need an extra transmembrane chaperone for its sorting and incorporation into COPII buds (Herpers, 2004).

This is particularly intriguing because efficient export from the ER also could be mediated by motifs found in the cytoplasmic domain of Gurken. In type I transmembrane proteins such as ERGIC53 and Emp46, a doublet of phenylalanine and leucine, respectively, is important for the exit from the ER. Both doublets are found in Gurken cytoplasmic domain (aa251 sfpvLLmlss lyvlfaavfm lrnvpdyrrk qqqlhlh kqr FFvrc). The removal of the cytoplasmic domain of Gurken does not seem, though, to affect to a great extent the efficient exit of the truncated protein from the ER, perhaps because it can still interact with Cornichon. The role of the cytoplasmic domain could perhaps be unraveled in a weak cornichon mutant background (Herpers, 2004).

Brefeldin A (BFA) treatment of the WT egg chambers was expected to lead to full retention/retrieval of Gurken in the ER followed by its diffusion. However, many oocytes remained seemingly unaffected, suggesting that the binding of Gurken to its sorting receptor Cornichon locked Gurken at tER sites. When the drug treatment was performed in a weak allele of cornichon, Gurken could be observed diffusing in the ER, suggesting that the locking mechanism was impaired. This diffusion, however, was not as extensive as the diffusion observed in the strong cornichon mutant under nontreated conditions. This partial diffusion could represent that of the complex Cni^CF5/Gurken (two transmembrane proteins) instead of Gurken alone. Why, under BFA treatment, Cni^CF5/Gurken complex diffuses more readily than Cornichon/Gurken is still not understood and further work is needed to elucidate the molecular details of the BFA effect in oocytes (Herpers, 2004).

Further work is needed to find out whether the polar synthesis and deposition relied upon by other transmembrane proteins stands alone as a pretranslational mechanism (through mRNA localization), or is coupled to efficient protein sorting events from the ER (Herpers, 2004).

All tER-Golgi units are able to work in synchrony for the transport of transmembrane proteins, of which the RNAs are not localized, such as Yolkless. However, a subset of tER-Golgi units can be recruited to perform the specific task of transporting a given transmembrane/secreted protein. This suggests that the different tER-Golgi units within a single cell can function in an uncoupled/nonsynchronous/independent manner, even though the ER is continuous. The restricted localization of transcripts is a necessary cue for imposing this uncoupling, as has been suggested for muscle heterokaryons and hybrid myotubes, although it is not clear in these systems whether the ER is continuous (Herpers, 2004).

This study has exemplified the functional uncoupling of the tER-Golgi units in Drosophila oocytes, and it is proposed that a similar mechanism also could take place for other types of highly polarized cells, such as neurons. This is suggested by a series of observations, showing that RNA encoding transmembrane proteins specific for the dendrites are translated in the dendrites themselves, and not exclusively in the cell body. It is also suggested by the immunofluorescence labeling of Golgi markers such as galactosyltransferase and GM130, in a dotty pattern along the dendrites, suggesting that perhaps Golgi-like structures could underlie this labeling. It is therefore possible that the mechanism identified here also occurs in neurons. mRNA encoding transmembrane/secreted proteins specific for the dendrites could be localized in these specialized domains and use dendritic Golgi-outposts to induce the local synthesis and transport of the proteins they encode. Whether in mammalian cells, the multiple ER exit sites and the dozens of Golgi stacks making up the Golgi ribbon also could function in an uncoupled manner and respond to a restricted mRNA localization remains to be elucidated (Herpers, 2004).

REFERENCES

Search PubMed for articles about Drosophila Cornichon

Barlowe, C. (2003). Signals for COPII-dependent export from the ER: what's the ticket out? Trends Cell Biol. 13: 295-300. 12791295

Bökel, C., Dass, S., Wilsch-Bräuninger, M. and Roth, S. (2006). Drosophila Cornichon acts as cargo receptor for ER export of the TGF-like growth factor Gurken. Development 133: 459-470. 16396907

Bonifacino, J. S. and Glick, B. S. (2004). The mechanisms of vesicle budding and fusion. Cell 116: 153-166. 14744428

Gillingham, A. K., Tong, A. H., Boone, C. and Munro, S. (2004). The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi. J. Cell Biol. 167: 281-292. 15504911

Herpers, B, and Rabouille, C. (2004) mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in Gurken transport in Drosophila oocytes. Mol. Biol. Cell 15(12): 5306-17. 15385627

Hwang, S. Y., Oh, B., Zhang, Z., Miller, W., Solter, D. and Knowles, B. B. (1999). The mouse cornichon gene family. Dev. Genes Evol. 209: 120-125. 10022955

Kuehn, M. J. and Schekman, R. (1997). COPII and secretory cargo capture into transport vesicles. Curr. Opin. Cell Biol. 9: 477-483. 9261052

Martinez-Menarguez, J. A., Geuze, H. J., Slot, J. W. and Klumpermann, J. (1999). Vesicular tubular clusters betweenn the ER and golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles. Cell 98: 81-90. 10412983

Powers, J. and Barlowe, C. (1998). Transport of axl2p depends on erv14p, an ER-vesicle protein related to the Drosophila cornichon gene product. J. Cell Biol. 142: 1209-1222. 9732282

Powers, J. and Barlowe, C. (2002). Erv14p directs a transmembrane secretory protein into COPII-coated transport vesicles. Mol. Biol. Cell 13: 880-891. 11907269

Queenan, A. M., Barcelo, G., Van Buskirk, C. and Schüpbach, T. (1999). The transmembrane region of Gurken is not required for biological activity, but is necessary for transport to the oocyte membrane in Drosophila. Mech. Dev. 89: 35-42. 10559478

Roemer, T., Madden, K., Chang, J. and Snyder, M. (1996). Selection of axial growth sites in yeast requires Axl2p, a novel plasma membrane glycoprotein. Genes Dev. 10: 777-793. 8846915

Roth, S., Neuman-Silberberg, F. S., Barcelo, G. and Schupbach, T. (1995). Cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in Drosophila. Cell 81: 967-978. 7540118

Roth, S. (2003). The origin of dorsoventral polarity in Drosophila. Philos. Trans. R. Soc. Lond. B Biol. Sci. 358: 1317-1329. 14511478

Schimmoller, F., Singer-Kruger, B., Schroder, S., Kruger, U., Barlowe, C. and Riezman, H. (1995). The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. EMBO J. 14: 1329-1339. 7729411

Yano, H., Yamamoto-Hino, M., Abe, M., Kuwahara, R., Haraguchi, S., Kusaka, I., Awano, W., Kinoshita-Toyoda, A., Toyoda, H. and Goto, S. (2005). Distinct functional units of the Golgi complex in Drosophila cells. Proc. Natl. Acad. Sci. 102: 13467-13472. 16174741

date revised: 10 April 2006

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