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).

GENE STRUCTURE

cDNA clone length - 844

Bases in 5' UTR - 92

Exons - 4

Bases in 3' UTR - 317

PROTEIN STRUCTURE

Amino Acids - 144

Structural Domains

The Drosophila genome contains a second cni-like gene (CG17262) named in this study 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, exemplified by the human proteins. Cnirel and Cni4 form a branch distinct from other metazoan Cornichons. Overall structural properties are conserved between all family members, with a cytoplasmic N terminus and three transmembrane domains, as determined for Erv14p (Powers, 2002) and modelled for Cni using the TMHMM and TMpred algorithms (Bökel, 2006).

EVOLUTIONARY HOMOLOGS

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER-vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not efficiently delivered to the cell surface. Axl2p is required for selection of axial growth sites and normally localizes to nascent bud tips or the mother bud neck. In erv14Delta strains, Axl2p accumulates in the ER while other secretory proteins are transported at wild-type rates. It is proposed that Erv14p is required for the export of specific secretory cargo from the ER. The polarity defect of erv14Delta yeast cells is reminiscent of cornichon mutants, in which egg chambers fail to establish proper asymmetry during early stages of oogenesis. These results suggest an unforeseen conservation in mechanisms producing cell polarity shared between yeast and Drosophila (Powers, 1998).

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are introduced into this loop, an Erv14p-Axl2p complex accumulates in the endoplasmic reticulum, suggesting that Erv14p links Axl2p to the COPII coat. Based on these results and further genetic experiments, it is proposed Erv14p coordinates COPII vesicle formation with incorporation of specific secretory cargo (Powers, 2002)

Rud3p is a coiled-coil protein of the yeast cis-Golgi. Rud3p is localized to the Golgi via a COOH-terminal domain that is distantly related to the GRIP domain that recruits several coiled-coil proteins to the trans-Golgi by binding the small Arf-like GTPase Arl1p. In contrast, Rud3p binds to the GTPase Arf1p via this COOH-terminal 'GRIP-related Arf-binding' (GRAB) domain. Deletion of RUD3 is lethal in the absence of the Golgi GTPase Ypt6p, and a screen of other mutants showing a similar genetic interaction revealed that Golgi targeting of Rud3p also requires Erv14p, a cargo receptor that cycles between the endoplasmic reticulum and Golgi. The one human protein with a GRAB domain, GMAP-210 (CEV14/Trip11/Trip230), is known to be on the cis-Golgi, but the COOH-terminal region that contains the GRAB domain has been reported to bind to centrosomes and gamma-tubulin. In contrast, it is found that this region binds to the Golgi in a GRAB domain-dependent manner, suggesting that GMAP-210 may not link the Golgi to gamma-tubulin and centrosomes (Gillingham, 2004).

date revised: 10 April 2006

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