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First published online 5 January 2006
doi: 10.1242/dev.02219

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Drosophila Cornichon acts as cargo receptor for ER export of the TGF-like growth factor Gurken

Christian Bökel¹, Sajith Dass^2,*, Michaela Wilsch-Bräuninger¹ and Siegfried Roth^2,

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.

View larger version (118K): [in a new window]   Fig. 1. cni-related and cni show partial redundancy in the soma but not the germline. (A) Wild-type head. Ocellar (arrowhead), interocellar (black arrow), and post-vertical bristles (white arrow) are marked. (B) cniAR55/cniAR55 head. Ocellar (arrowhead), interocellar (black arrow) and post-vertical bristles (white arrow) are missing and the eyes are rough. (C) cniAR55/cniAR55 rough eye phenotype with fusion of ommatidia (white arrow), specification of ommatidia with aberrant numbers of cone cells (white arrowhead), and missing (black arrow) or ectopic (black arrowhead) interommatidial bristles. (D) The pcni::Cnir transgene (cnir ORF under cni control) rescues ocellar (arrowhead) and post-vertical (white arrow), but not intraocellar bristles (black arrow) or the rough eye phenotype. (E) Homozygous cniAR55/cniAR55 wing. Vein 2 is truncated before it reaches the margin (arrow). (F) One copy of the pcni::Cnir transgene suppresses the cni wing phenotype (arrow). (G) Wild-type egg with two large dorsal respiratory appendages and an anterior micropyle. (H) Egg from a cniAR55/cniAR55 mother. Owing to the loss of Grk signalling, the egg is ventralized and both ends differentiate into anterior structures. (I) Egg from a cniAR55/cniAR55 female carrying a pCnimyc transgene. Establishment of the anteroposterior axis is completely rescued and that of the dorsoventral axis largely rescued. (J) Egg from a cniAR55/cniAR55; pcni::Cnir female. Expression of cnir under control of cni promotor and UTR sequences fails to rescue Grk signalling.

View larger version (94K): [in a new window]   Fig. 2. Cni protein localization. (A) Myc-tagged Cni (Cni-myc) expressed from the endogenous promotor accumulates in the oocyte during early and middle oogenesis (white arrows). At stage 10, strong expression in the nurse cells indicates maternal loading (arrowhead). The protein is also detectable in the somatic follicular epithelium (black arrows). Right panel, magnification of boxed area. (B) C-terminally myc-tagged Cni protein (Cni-myc, red) expressed in the squamous follicle epithelium forms discrete punctate structures, that are often associated (arrows) but not necessarily tightly colocalized with the large reticulate ER of these cells labelled with PDI-GFP (green). (C) Cni-myc (red) does in most cases (arrows) not colocalize with the punctate and disperse Golgi apparatus of the same cells, marked by the p120 Golgi protein (blue). (D) Cni-myc containing structures (red) are largely co-stained (arrows) with an antibody against the KDEL receptor (blue). This protein cycles between ER and Golgi. The total pool of KDEL-receptor positive structures exceeds that co-staining with Cni-myc. (B-D) DNA (DAPI) is shown in light blue. Scale bars: 10 µm.

View larger version (56K): [in a new window]   Fig. 3. Cornichon belongs to a conserved protein family. (A) Drosophila Cni and Cni-related, and their human homologues HsCni (gb| AAD20960.1) and HsCni4 (gb| NP_054903.1) share 21.5% identical (dark grey) and 43.9% similar (light grey) amino acids. Conservation is much higher between pairs of homologues: 66.7% identitical/85.4% similar in the case of Cni and HsCni, and 51.2% identitical/81.8% similar for Cnirel and HsCni4 (residues identical between homologues in medium grey). Conserved residues are distributed along the entire length of the proteins, predicted transmembrane domains are underlined. (B) Rootless phylogenetic tree for the fly, human and yeast Cni family members. Cnirel and human Cni4 form a distinct branch. Tree constructed by neighbour-joining, bootstrap support>98% for all nodes. (C) Model of the membrane topology predicted for Cni using TMHMM (www.cbs.dtu.dk/services/TMHMM) and TMpred (www.ch.embnet.org), with the N terminus facing the cytoplasm. The three transmembrane domains are labelled in light grey, while the Grk-binding domain (dark grey) maps to the N terminus, including the first lumenal loop. Black residues denote the conserved diaromatic motive.

View larger version (99K): [in a new window]   Fig. 4. Grk protein distribution in wild-type, grkDC and cni mutant egg chambers. (A) Stage 10 wild-type egg chamber. Grk protein is concentrated at the anterodorsal corner of the oocyte in the vicinity of the nucleus. Some Grk can be seen in the follicle cells receiving the Grk signal. (B) Stage 10 grkDC/grkDC egg chamber. Grk protein is mislocalized to the interior of the oocyte. The staining appears patchy and granular, with high protein concentration near the nucleus. Grk is not detectable in the follicular epithelium. (C) Stage 10 cniAR55/Df(2L)H60 egg chamber. Grk protein is distributed in a gradient with a high point near the posteriorly mislocalized oocyte nucleus and in a cortical ring at the anterior end of the oocyte adjacent to the nurse cells. There is no detectable Grk uptake into the follicular epithelium. (D) Immunoelectron microscopy of the anterodorsal corner of a stage 10 wild-type egg chamber. Grk immunogold staining (small black dots) can be detected in the oocyte (ooc) near the nucleus (nuc, arrows) and at the plasma membrane facing both follicle cells (fc) and nurse cells (nc). Grk is also present at the microvillous processes of the follicle cell surface (arrowheads) and within the follicle cells. Grk is absent from the cortex of yolk granules (yg). (E) Immunoelectron microscopy of the ventral posterior part of a heterozygous stage 10 grkDC/CyO oocyte. Most Grk staining is associated with the cortex of growing yolk granules (arrows). (F) Immunoelectron micrograph of a stage 10 cniAR55/Df(2L)H60 egg chamber. Grk staining is found scattered throughout the oocyte (arrows) but does not accumulate at yolk granules or the oolemma.

View larger version (116K): [in a new window]   Fig. 5. Trapping of GrkDC protein at the plasma membrane through inactivation of the Drosophila Dynamin homologue Shibire requires Cni function. (A) shiTS; grkDC/CyO egg chamber incubated at the permissive temperature (25°C). Grk staining at the membrane is limited to the vicinity of the nucleus. Ovaries preincubated in Schneider's medium exhibit higher than usual background, irrespective of their genotype. (B) shiTS; cniAR55/CyO egg chamber incubated at 25°C. Grk is distributed in the wild-type pattern. (C) shiTS; cniAR55/grkDC Df(2L)H60 egg chamber incubated at 25°C. Grk is diffusely distributed throughout the oocyte. (D) Incubation at 32°C blocks endocytosis in shiTS; grkDC /CyO oocytes. Membrane tethered GrkDC protein accumulates at the oolemma. (E) Blocking endocytosis in shiTS; cniAR55/CyO ovaries has no effect on the wild-type Grk protein not anchored to the oocyte plasma membrane. (F) shiTS; cniAR55/grkDC Df(2L)H60 egg chamber incubated at the restrictive temperature. In the absence of cni, GrkDC protein cannot be trapped at the plasma membrane but remains mislocalized to the interior of the oocyte.

View larger version (44K): [in a new window]   Fig. 6. Western blots with epitope tagged Grk protein and GST pull-downs. (A) Grk tagged C-terminally with five myc tags (Grk5myc) under endogenous control. Only the C-terminal cleavage residue but not a full-length precursor can be detected using the 9E10 anti-myc monoclonal. No differences are visible between homozygous cniAR55 mutant ovaries and heterozygous sibling controls. (B) Grk5myc overexpressed in wild-type (+) and homozygous cniAR55 (cni) ovaries using the TubGal4VP16 driver. The 9E10 anti-myc monoclonal reveals two bands. Although the lower band corresponds in size to the expected C-terminal cleavage residue, the upper band, corresponding to an uncleaved precursor, is larger than predicted from the protein sequence, indicative of glycosylation. In addition to the precursor band, the 1D12 anti-Grk monoclonal reveals a shorter band corresponding in size to the released N-terminal growth factor fragment in the mutant but not the wild-type lane. (C) A protein species corresponding in size to the uncleaved Grk precursor accumulates in lysates expressing a Grk5myc construct carrying the grkDC point mutation from the endogenous promotor (GrkDC5myc). This does not occur in ovaries expressing the functional Grk5myc version. Smearing of the GrkDC5myc band into several high molecular weight species indicative of Golgi glycosylation only occurs in the presence of Cni. (D) The N-terminal growth factor fragment accumulating in cni mutant ovaries upon Grk overexpression is sensitive to EndoH and PNGaseF, indicating ER-type high Mannose glycosylation. (E) Grk and Cni interact directly. MBP fusion proteins with lacZ or the N-terminal 57 amino acids of Cni or Cnir were pulled down using GST or GST-Grk (GST fused to Grk amino acids 179-245) prebound to beads. Probing the pellets with anti-MBP antibody reveals a specific interaction between the juxtamembrane domain of Grk and the Cni N terminus.

View larger version (60K): [in a new window]   Fig. 7. Fusions of Grk to the cytoplasmic tails of Yl, dEmp24 or Cni. (A) Wild-type egg. The anterior pole bears a micropyle, and the dorsal side carries two respiratory appendages. (B) Egg from a cniAA12/cniAR55 female. The egg is ventralized (reduction of dorsal appendages), but the anterior and posterior structures differentiate correctly. (C) Egg from a cniAR55/cniAR55 female. The egg is completely ventralized and both ends differentiate into anterior structures (micropyle), indicating complete loss of Grk function. (D) Egg from a grkHF48/grk2B6 female carrying a transgene replacing the Grk transmembrane and cytoplasmic domains with the corresponding domains of Yl (grk-Yl TMC). The transgene is able to rescue the grk oogenesis phenotype. (E) The grk-Yl TMC transgene shows no function in an amorphic cni background. (F) Egg from a cniAR55/cniAR55 female expressing the Grk extracellular and transmembrane domains fused to the dEmp24 cytoplasmic domain (grk-Emp24 Cyt). The egg is partially ventralized, but the anteroposterior axis is correctly specified. (G) Egg from a cniAR55/cniAR55 female expressing the Grk extracellular and transmembrane domains fused to the presumptive cytoplasmic domain of Cni (grk-Cni Cyt). The anteroposterior axis is correctly polarized and the dorsal appendages are partially rescued.

View larger version (94K): [in a new window]   Fig. 8. Gal4-mediated Grk overexpression in the germline can overcome the secretion block in cni ovaries. (A) Wild-type egg chamber. Endogenous Grk (red) is tightly localized to the dorsal anterior corner of the oocyte. Cells are outlined with Phalloidin (green). (B) Wild-type egg. The dorsalmost chorion structure is the operculum (op). Dorsal appendages (da) are derived from more lateral positions. (C,D) Grk overexpression in a wild-type background using the maternal tubulin:Gal4-VP16 driver. (C) Despite a massive increase in the amount of Grk protein, there is still a Grk gradient within the oocyte with highest levels close to the oocyte nucleus. (D) The eggs maintain DV polarity although they have an expanded operculum (op) and the dorsal appendages (da) are shifted to the ventral side. (E-G) Overexpression of Grk in a cniAR55/Df(2R)H60 background using the maternal tubulin:Gal4-VP16 driver line. (E) High amounts of Grk protein are evenly distributed within the oocyte. (F) The eggs lack DV polarity, but the presence of dorsal appendage material (da) and the suppression of a posterior micropyle indicate low levels of Grk signalling. (G) Despite formation of dorsal appendage material (da), 30% of the eggs lack anteroposterior polarity as can be seen from the presence of a posterior micropyle (m). This indicates a lack of temporal control of Grk signalling.

View larger version (22K): [in a new window]   Fig. 9. Schematic representation of Cni function. (A) Wild-type situation. Grk protein binds to Cni after processing in the ER and the released growth factor is recruited into COPII coated vesicles by Cni. (B) In the absence of Cni, processing still occurs but the released growth factor is lost into the ER lumen. (C) Loss of Cni can be partially overcome by fusion of Grk with a COPII targeting motive from Cni or dEmp24. Processing will preferentially occur near ER exit sites, locally increasing the concentration of the soluble growth factor. (D) The truncated CniAA12 protein can keep processed Grk at the membrane, but fails to efficiently recruit it into outgoing vesicles.