Consortin is a disordered integral membrane protein. (A) Structure of mouse consortin short and long isoforms, showing the position of the predicted transmembrane segment (blue bar, residues 652–669 in the long isoform) and of the DDSDLL sequence (green, residues 557–562 in the long isoform) for interaction with the monomeric GGA (consensus DXXLL) and tetrameric AP-1, AP-2 and AP-3 adaptors [consensus (D/E)XXXL(L/I)]. The regions for binding to Cx26 (residues 521–711 in the long isoform) and Cx30 (residues 503–631 in the long isoform), deduced from the overlap of consortin yeast two-hybrid clones interacting with the connexin baits, are indicated. (B) Prediction of disordered regions in consortin by the IUPred algorithm, showing the positions of the transmembrane domain and connexin-interaction region (residues 521–631). The short C-terminal tail of consortin is well structured and likely to form a globular domain. In contrast, the putatively cytosolic, N-terminal segment of consortin, which includes the connexin-interacting region, is highly disordered. This structural arrangement is conserved in all full-length consortin orthologs known.

Subcellular localization of consortin in the Golgi apparatus. (A) Detection of consortin by an affinity-purified polyclonal antibody in lysates of untransfected HeLa cells (HeLa) or HeLa cells transiently transfected with a GFP-tagged human consortin construct (HeLa/consortin-GFP). Exogenously expressed consortin-GFP and endogenously expressed consortin are indicated by arrows. The lower molecular mass bands detected by the antibody (asterisks) may correspond to isoforms generated by translation started from a hypothetical internal ribosomal entry site or by post-translational processing. (B, C) Confocal microscopy images of HeLa cells stained for endogenous consortin (green) and the Golgi apparatus markers (red) GM130 (B) and p58 (C). (D) Modification of the distribution of consortin (green) in HeLa cells transiently transfected with the dominant negative Q71L mutant of human ARF1 (red). (E, F) Modification of the distribution of consortin in HeLa cells treated with Golgi-disrupting agents nocodazole (15 µm for 2 h) (E) and brefeldin A (5 µm for 45 min) (F); cells were stained for consortin (green) and for the Golgi markers (red) GM130 (E) or TGN48 (F). Bars, 5 µm.

Consortin interacts with the TGN adaptors GGA1 and GGA2. (A) GST-pulldown assay of in vitro synthesized GGA1 with the GST-consortin chimera. (B) Co-localization of consortin (green) with GGA1-myc (red), in transfected HeLa cells synthesizing this chimeric protein. (C) GST-pulldown assay of in vitro synthesized GGA2 with GST-consortin. (D) Co-localization of consortin (green) with endogenous GGA2 (red) in HeLa cells. Bars, 10 µm.

Consortin silencing alters the plasma membrane targeting of connexins 32, 43 and 45. (A) Immunoblot of consortin in lysates of HeLa-RNA-NT (stably expressing a non-silencing control shRNA) and HeLa-shRNA15 cells (stably expressing a consortin-targeting shRNA), after 72 h of siRNA-mediated consortin knockdown. Ten microgram total cellular protein extract were loaded per lane and β-actin was used as loading control. Maximum consortin silencing was obtained when combining shRNA stable knockdown with transfection of either CNST-826 or CNST-841 siRNAs. (B) Effect of consorting silencing on the number of connexin immunolabeled plaques in connexin-expressing HeLa-RNA-NT and HeLa-shRNA15 cells after 72 h of siRNA-mediated consortin knockdown. In all cases, differences between control and consortin-silenced cells were statistically significant (Supplementary Material, Table S1). (C–E) Effects of consortin knockdown on connexin distribution in HeLa/RNA-NT and HeLa-shRNA15 cells expressing Cx32 (C), Cx43 (D) or Cx45-eGFP (E) after 72 h of siRNA transfection. Cells were stained for the appropriate connexin (green) and the Golgi marker GM130 (red), and counterstained with DAPI (blue). Cell contours are drawn as white lines. Arrowheads indicate immunostained connexin plaques at intercellular contacts. Note the absence of plaques and the intracellular connexin accumulation (asterisks) in cells undergoing consortin silencing. Bars, 5 µm.

The intracellular trafficking of gap junction plaques is affected by consortin redistribution. (A) Immunogold labeling of Cx43 in a typical gap junction plaque at the membrane interface of two adjacent HeLa-RNA NT cells stably expressing Cx43. (B) In HeLa-shRNA15 cells stably expressing Cx43, many annular gap junction plaques are found within the cytoplasm. (C) In the same cells, much lower levels of Cx43 are detected in Golgi-related vesicles (arrow) and tubular structures (arrowheads). (D, E) Several of the annular, Cx43-containing gap junctions found in the cytoplasm of HeLa-shRNA15 cells show a partial splitting of the two interacting membranes (D, arrow) or lie within lysosomal structures (E). Bar, 170 nm.

Consortin interacts with connexins both in vitro and in vivo. (A) Pulldown experiments of different in vitro synthesized and radioactively labeled connexins (Cx26, Cx30, Cx31, Cx32, Cx43 and Cx45) by a batch-purified GST-consortin (short isoform) chimera. For each pulldown assay, three lanes are shown, corresponding to the relevant in vitro translation reaction (input), a control assay with batch-purified GST (GST), and the assay with the appropriate GST chimera (GST-consortin). Bands corresponding to putative connexin monomers and hexamers are indicated by arrows and arrowheads, respectively. Note that the hexamer band is consistently enriched in the GST-consortin pulldown lane, compared with the input lane. (B) In vitro synthesized, radioactively-labeled Cx26 and Cx30 were pulled down by batch-purified GST-consortin-ΔCter synthesized in Escherichia coli, which indicates that connexin binding does not occur through hydrophobic interactions with the putative transmembrane domain of consortin. (C) Pulldown assay of Cx26 from detergent-free lysates of HeLa-Cx26 cells with GST-consortin (short isoform) and GST-consortin-ΔCter chimeras synthesized in E. coli. A band corresponding to putative Cx26 hexamers, detected with an anti-Cx26 antibody, is observed in the GST-consortin-ΔCter lane. In contrast, no band was detected in the GST-consortin lane, indicating that membrane-inserted Cx26 does not interact with the short isoform of consortin. This may be due to a blockade by the transmembrane segment and C-terminal tail of non-membrane-inserted consortin, which are not part of the minimal connexin-interaction region of consortin. (D) Immunoprecipitation of Cx43 from extracts of HeLa cells transiently transfected with expression plasmid pNTCx43. Extracts were immunoprecipitated with an irrelevant monoclonal antibody (anti-myc), anti-Cx43 monoclonal antibody P2D12, or a mixture of anti-Cx43 monoclonal antibodies IF1 and CT1. Bands corresponding to Cx43 and the short isoform of consortin are detected by immunoblotting in the immunoprecipitates obtained with the anti-Cx43 antibodies, but not in those obtained with the irrelevant antibody, indicating that consortin and Cx43 co-precipitate and may interact in vivo. (E) Escherichia coli-synthesized, batch-purified, GST-consortin and GST-consortin-ΔCter do not pulldown in vitro synthesized pannexin 1 (Panx1).

Endogenous consortin localizes to the Golgi apparatus and the cell membrane. (A) Immunogold labeling of HeLa cells reveals most consortin in tubulo-vesicular structures (some pointed by arrowheads) of the TGN, and rarely in the cisternae of Golgi stacks (double arrowhead). (B) Consortin is also immunolabeled in tubulo-vesicular structures (white arrowheads) at a distance from the TGN (black arrowhead), as well as at the plasma membrane (arrows), mostly at microvilli. (C–E) Consortin-containing tubulo-vesicular structures (arrowheads) are observed throughout the cytoplasm (C), close to the plasma membrane (D) and fusing with it (E). Bars, 300 nm (A, B) and 200 nm (C–E).

Mutant NSDAA-consortin alters the intracellular traffic of connexins. (A) Pulldown of Cx26, Cx30, Cx43 and Cx45 by GST-NSDAA-consortin. Bands corresponding to the monomeric form of the indicated connexin and its putative hexamer are indicated by arrows and arrowheads, respectively. (B) In HeLa cells expressing either Cx45-eGFP or Cx26, co-expression of wild-type consortin has no effect on the cell surface display of either connexin, which formed plaques at intercellular contacts consistent with the presence of gap junctions (arrowheads). (C) In contrast, expression of the NSDAA-consortin mutant caused the intracellular accumulation (asterisks) of both Cx45-eGFP and Cx26. Thus, NSDAA-consortin behaves as a dominant-negative mutant. White lines outline cell contours, as observed in differential interference contrast (DIC) confocal images. Bars, 5 µm.

The distribution of Cx43 in transfected HeLa cells changes with that of consortin. (A) Immunofluorescence labeling of consortin shows a loose distribution throughout most of the cytoplasm in HeLa-Cx43 and HeLa-RNA NT-Cx43 cells. In contrast, consortin is mostly restricted to the perinuclear Golgi apparatus in HeLa-shRNA 15-Cx43 cells (right panel). (B) Immunostaining of Cx43 detects this connexin at small plaques along the membrane interfaces of HeLa-Cx43 and HeLa-RNA NT-Cx43 cells. In HeLa-shRNA 15-Cx43 cells, Cx43 forms larger plaques at the cell surface and also appears as discrete spots within the cytoplasm (arrowheads). Bar, 10 µm.