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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.004200

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Cornichon regulates transport and secretion of TGF-related proteins in metazoan cells

Department of Cell and Tissue Biology, Program in Cell Biology, University of California at San Francisco, San Francisco, CA 94143-0512, USA

View larger version (48K): [in this window] [in a new window]   Fig. 1. Polypeptide sequences and predicted transmembrane topology for human, Drosophila and yeast cornichon proteins. (A) Alignment of the amino acid sequences CNIH, CNIH2, CNIH3 and CNIH4. The identity of CNIH with each of the other paralogs is: 105/159 (66%) with CNIH2; 109/160 (68%) with CNIH3; 45/118 (38%) with CNIH4. (B) Alignment of CNIH, Drosophila Cni (dCni) and Erv14. The identity of CNIH with each ortholog is: 96/144 (66%) with dCni and 45/137 (32%) with Erv14. Predicted transmembrane domains are underlined. The conserved `Cni domain' is boxed. Identical residues are in red.

View larger version (106K): [in this window] [in a new window]   Fig. 2. Cornichon localizes in the ER and partially in the Golgi, and colocalizes with TGF ligands. (A,B) Colocalization of HA-tagged CNIH with endogenous calnexin, a marker of the ER, and GM130, a cis-Golgi marker, in HeLa cells. HA-CNIH is only expressed in transfected cells, whereas calnexin and GM130 are expressed in all cells. Bar, 10 µm. (C) Detection of endogenous CNIH in HeLa cells and colocalization with calnexin. Bar, 10 µm. (D) Partial colocalization of CNIH and TGF in transfected HeLa cells. (E) Partial colocalization of SEC23 (Sec23) with CNIH in HeLa cells. Bar 2 µm. (F-H) Myc-tagged Drosophila Cni (dCni-M) in transfected S2 cells colocalizes with the ER marker KDEL-GFP (F), partially colocalizes with the Golgi marker p120 (G), and colocalizes with ectopically expressed Grk. Bar, 10 µm (H). (I,J) Immunoelectron microscopy detection of dCni-M in S2 cells. (I) Anti-Myc antibody (10-nm gold particles) detected dCni-Myc (red arrowheads) on buds from the ER, and adjacent vesicular and tubular membranes. Brackets indicate clusters of vesicles and tubules of the ER. (J) Double immunodetection of dCni and Sec23 (dSec-23p), a COPII vesicle marker. Myc-tagged dCni (red arrowheads) and Sec23 in COPII vesicles (black arrowheads) were visualized using 10 nm and 15 nm gold particles, respectively. Bar, 200 nm.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Cornichon interacts with TGF family members. (A) CNIH interacts with TGF. Left: lysates of HeLa cells expressing TGF and/or Myc-tagged CNIH subjected to immunoprecipitation with anti-Myc, and immunoblotting with indicated antisera. Right: total cell lysates subjected to immunoblotting. TGF co-precitated with CNIH-Myc only when coexpressed and not when separately expressed and cell lysates were mixed (lane 5). CNIH did not interact with SEC61 (Sec61), an ER resident protein, or transferrin receptor (Tf-R). (B) CNIH interacts with amphiregulin (AR). Left: lysates of HeLa cells expressing Myc-tagged AR and/or Flag-tagged CNIH, subjected to immunoprecipitation with anti-Flag, and immunoblotting with anti-Myc. Right: total cell lysates subjected to immunoblotting to detect AR. Immature AR is marked. (C) Drosophila Cni interacts with Grk. S2 cells were transfected or not transfected to express Myc-tagged dCni (dCni-Myc) and treated or not with dsRNAi targeting dCni. Lysates were processed for Grk immunoprecipitation and immunoblotting with anti-Myc or anti-Grk antibodies. dCni-Myc co-precipitated with Grk, only when Grk and dCni-Myc were coexpressed in the absence of dsRNAi targeting Cni. dsRNAi targeting Cni did not affect Grk expression. (D) The EGF core of transmembrane TGF is required for interaction with CNIH. HeLa cells were transfected to express HA-tagged CNIH and full-length TGF, TGFC or TGFE. Cell lysates were subjected to anti-HA immunoprecipitation, and immunoblotting to detect CNIH-associated TGF. TGF and TGFC were detected using an anti-TGF antibody Ab-1, and TGFE was detected using anti-Myc. Lysates of non-transfected cells served as controls. Dividing vertical white lines indicate parts from the same gel (A,D).

View larger version (51K): [in this window] [in a new window]   Fig. 4. CNIH interacts with proteins of the secretory pathway. (A) CNIH interacts with GM130. HeLa cells, transfected to express Myc-tagged GM130 with or without Flag-CNIH, were subjected to anti-Flag immunoprecipitation and immunoblotting with anti-Myc, or conversely with anti-Myc followed by anti-Flag immunoblotting. (B) HeLa cells, transfected to express Flag-tagged GRASP55, with or without TGF or Myc-tagged CNIH, were subjected to anti-Flag immunoprecipitation, followed by immunoblotting for TGF or Myc-CNIH. GRASP55 immunoprecipitated with CNIH in the presence of TGF, but not in its absence. *Bands corresponding to a remaining GRASP55 immunoblot signal prior to tubulin immunoblotting. All samples were prepared and loaded in duplicate.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Effects of CNIH on TGF maturation. (A) CNIH increased the immature TGF form. CHO cells were transfected to express TGF in the presence of increasing CNIH-Myc levels. Upper panel: expression of TGF in cell lysates, showing an increase in immature TGF (form 1), relative to the mature forms 2 and 3. Lower panels: increased TGF levels co-precipitated with CNIH-Myc. (B) Effect of CNIH on endogenous TGF in HCA-7 cells. Cells were transfected to express CNIH-Myc (20 and 100 ng plasmid), and co-precipitation of endogenous TGF with CNIH was examined. (C) Effect of CNIH-HA on TGF localization in transfected CHO cells. In the absence of HA-CNIH, TGF had a diffuse distribution. CNIH expression conferred TGF accumulation in the ER, where CNIH is localized. Bar, 20 µm. (D) The Semi-quantitative image analysis of TGF immunostaining (given as relative TGF intensity units) shows three times more accumulation of intracellular TGF signal in comparison with cells not transfected with CNIH. Error bars represent s.e.m.; *t-test, P<0.05. (E) Comparison of full-length CNIH and CD1 deletion mutant; TM, transmembrane. Transfected cells expressed full-length or CD1 mutant CNIH-Myc and TGF. Cell lysates were processed for anti-Myc immunoprecipitation and immunoblotting with anti-TGF. Dividing vertical lines indicate parts from the same gel (B).

View larger version (36K): [in this window] [in a new window]   Fig. 6. Increased CNIH expression decreases the cell surface level and secretion of TGF. (A) CNIH decreases cell surface TGF in transfected HCA-7 cells, assessed by cell surface protein biotinylation, followed by anti-TGF immunoprecipitation and streptavidin blotting. Duplicate samples were analyzed. Lower panel: total TGF in cell lysates, demonstrating increased relative level of immature TGF (form 1) in the presence of CNIH. (B) CNIH does not affect the cell surface expression of gp130, assessed by cell surface protein biotinylation, followed by avidin precipitation and anti-gp130 blotting. (C) CNIH significantly reduces the level of soluble TGF. Secretion of AP-TGF, measured using an alkaline phosphate assay, in the medium of HeLa cells with or without CNIH. PMA was added to activate shedding. Error bars represent s.e.m.; *t-test, P<0.05. (D) CNIH does not affect the subcellular distribution of TACE. Upper panel: cell surface protein biotinylation followed by avidin precipitation and anti-TACE western blot. Lower panel: immunoflorescence staining of HeLa cells. Transfected CNIH-HA is expressed only in transfected cells, whereas endogenous hTACE is expressed in all cells. Bar, 20 µm. (E) Effect of CNIH on N-glycosylation of TGF. HeLa cells were transfected to express TGF with or without CNIH. Cell lysates were treated with N-glycosidase F, and analyzed by SDS-PAGE and western blotting for TGF. Increased mobility after treatment indicates N-glycosylation prior to treatment. CNIH increases the level of immature TGF (form 1) without affecting its N-glycosylation. Dividing vertical white lines indicate parts from the same gel (A).

View larger version (45K): [in this window] [in a new window]   Fig. 7. Role of Drosophila Cni (dCni) in Grk processing and secretion. (A) S2 cells were transfected to express Grk, Star, B-Rho and/or a secreted form of Grk (secGrk). Grk expression was visualized by immunoprecipitation and/or blotting with an antibody against the Grk ectodomain. (B) dCni enhanced the level of non-processed Grk. S2 cells were transfected to express Myc-Grk, Star, B-Rho and/or dCni-Flag. Myc-Grk in cell lysates and Grk in the media were visualized using anti-Grk ectodomain antibody. (C) Silencing of dCni in S2 cells causes accumulation of processed Grk (pGrk) in the presence of Star and B-Rho, and reduces Grk secretion. Top panel: downregulation of endogenous dCni mRNA expression by dsRNA-dCni, as assessed by RT-PCR. Lower panel: S2 cells were transfected to express Grk, B-Rho and/or Star, and treated with dsRNA-dCni. Grk expression was visualized, as in B. Accumulation of pGrk with dsRNA-Cni treatment was 2.7 fold and secretion of secGrk was 0.5 fold. Treatment with a lysosome inhibitor (chloroquine; 0.5 mM) does not affect pGrk relative accumulation and secretion. (D) Effect of dsRNA-Star on Grk processing and secretion. Top panel: downregulation of endogenous Star mRNA by dsRNA-Star, assessed by RT-PCR. Lower panels: S2 cells were transfected to express Grk, B-Rho, Star and/or dCni-Myc, and treated with dsRNA targeting Star. Grk was visualized using anti-Grk ectodomain antibody. (E) Silencing of dCni does not affect the general secretory pathway in cells transfected with secretable GFP (Grk-signal peptide-GFP). Dividing vertical white lines indicate parts from the same gel (A,E).

View larger version (29K): [in this window] [in a new window]   Fig. 8. Schematic representation of the model of cornichon action in transport of TGF family proteins. In both Drosophila and vertebrates, cornichon interacts in the ER with TGF family proteins, such as Grk in Drosophila and TGF in mammals, and controls and is required for their transport from the ER into the Golgi. In Drosophila, Star and Rhomboid control the transport and cleavage of Grk to the cell surface. The late endosomal compartment that may be important for the activities of Rhomboid and Star is not shown. In vertebrates, no roles for Rhomboid or Star homologs in the transport or cleavage of TGF family proteins have been characterized. Instead, the transmembrane metalloprotease TACE is known to cleave TGF to release the soluble ectodomain, and this may primarily occur late during transport and at the cell surface. Other ADAM proteases may control the ectodomain release of other TGF family proteins. The precise localization of proteins and timing of cleavage of TGF remains to be better characterized and, as shown, is not meant to be definitive.

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