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doi: 10.1242/10.1242/jcs.00440

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A tyrosine-based sorting signal is involved in connexin43 stability and gap junction turnover

Marc A. Thomas^1,2,3, Nathalie Zosso¹, Isabelle Scerri², Nicolas Demaurex³, Marc Chanson^2,*, and Olivier Staub^1,*,

¹ Institute of Pharmacology and Toxicology, University of Lausanne, 1005 Lausanne, Switzerland
² Department of Pediatrics, University Hospitals, 1211 Geneva, Switzerland
³ Department of Physiology, University of Geneva, 1211 Geneva, Switzerland

View larger version (40K): [in a new window]   Fig. 1. Mutation of tyrosine 286 to alanine affects Cx43 steady-state levels. (A) The amino acid sequence of Cx43 spanning residues 280-291. The putative PY-motif and tyrosine-based sorting signal are indicated and their consensus sequences displayed. (B) Transient expression of human Cx43 constructs in SKHep1. Cells were transfected with cDNA plasmids encoding either Cx43-WT, Cx43 with a substitution of tyrosine to alanine at position 286 (Cx43-Y286A) or neither (untransfected). Following expression (48 hours), western blot analysis was performed on cellular lysates using either an anti-Cx43 antibody (top panel) or a ß-actin antibody (bottom panel) to control protein loading. Cx43 was detected in its nonphosphorylated (NP) and phosphorylated (P1, P2) forms (indicated by arrows) and was determined by treating cellular lysates with CIP before western blot analysis.

View larger version (30K): [in a new window]   Fig. 2. The tyrosine-based sorting signal, adjacent to the PY-motif, is the major element affecting Cx43 levels. (A) Transient expression of Cx43 mutant constructs in SKHep1 cells. Mutant Cx43 plasmids were developed by exchanging proline with leucine at position 283 (Cx43-P283L), glycine with alanine at position 285 (Cx43-G285A), and valine with aspartate at 289 (Cx43-V289D). Cells were transfected with these plasmids, as well as Cx43-WT and Cx43-Y286A, cellular lysates prepared (48 hours after transfection) and western blot analysis performed using an anti-Cx43 antibody (top panel) or a ß-actin antibody (bottom panel) to control protein loading. (B) Quantitation of transfected Cx43 transiently expressed in SKHep1. Experiments were performed as in A, and the levels of total Cx43 and ß-actin detected by western blot fluorography quantified on a molecular imager FX. The Cx43:actin ratio value was determined for each construct and displayed as mean±s.e.m. (n=4 separate experiments). Asterisks indicate differences at P<0.01 vs control as determined by the Student's t-test.

View larger version (36K): [in a new window]   Fig. 3. Tyrosine 286 is involved in Cx43 stability. (A) Pulse-chase analysis was performed on stably transfected SKHep1 cells expressing either Cx43-WT or Cx43-Y286A, by pulsing for 60 minutes with [35S]-methionine and then chasing for 0, 2, 4 or 6 hour periods. Cells were then lysed, Cx43 immunoprecipitated overnight, and SDS-PAGE and fluorography performed. (B) Pulse-chase experiments were performed as in A, and the level of immunoprecipitated [35S]-labelled Cx43 was quantified from fluorographs using a molecular imager FX. A plot of the mean±s.e.m. percentage of pulse-labelled Cx43 remaining after 0, 2, 4 and 6 hours of chase from four independent experiments fitted using a mono-exponential decay function is shown. ANOVA statistical analysis indicates that the difference in the degradation curves between Cx43-WT and Cx43-Y286A is highly significant (P<0.0001).

View larger version (54K): [in a new window]   Fig. 4. Differential responses of wild-type Cx43 and Cx43-Y286A to both proteasomal and lysosomal inhibitors. (A) The stable cell lines, Cx43-WT and Cx43-Y286A, were treated for 3 hours with the proteasome inhibitor lactacystin (10 µM) or the lysosomal inhibitors leupeptin (10 µM) or NH4Cl (10 mM). Western blot analysis was then performed on the cellular lysates using either an anti-Cx43 (top panel) or a ß-actin (bottom panel) antibody. Steady-state levels of Cx43-Y286A were consistently greater than Cx43-WT, therefore reduced fluorography was performed for comparative analysis. (B) Quantitation of Cx43 protein levels in response to pharmacological agents. Experiments were performed as in A, and the amounts of Cx43-NP (left panel) and Cx43-P1,P2 (right panel) detected by western blot fluorography were quantified using a molecular imager FX. Mean ± s.e.m. values of normalised Cx43-WT (grey columns) and Cx43-Y286A (black columns) expression levels are shown (n=3 independent experiments). Asterisks represent P<0.01 versus control determined using the Student's t-test.

View larger version (44K): [in a new window]   Fig. 5. The lysosomal inhibitor, NH4Cl, affects wild-type, but less efficiently, mutant (Y286A) Cx43 turnover. (A) Pulse-chase analysis was performed on either Cx43-WT cells or Cx43-Y286A cells by pulsing for 60 minutes with [35S]-methionine, and then chasing for 0, 3 or 6 hour periods in the absence (control) or presence of the lysosomal inhibitor, NH4Cl. (B) Pulse-chase experiments were performed as in A, and the level of immunoprecipitated [35S]-labelled Cx43 was quantified from fluorographs using a molecular imager FX. A plot of the mean±s.e.m. percentage of pulse-labelled Cx43 remaining after 0, 3 and 6 hours of chase (n=4 independent experiments), fitted using a mono-exponential decay function is shown. ANOVA statistical analysis reveals that the difference in the degradation curves between Cx43-WT (control and NH4Cl) is highly significant (P<0.0001), in contrast to the Cx43-Y286A (control and NH4Cl), which is significant (P=0.0303).

View larger version (58K): [in a new window]   Fig. 6. Differential localisation of immunoreactive Cx43-WT compared with Cx43-Y286A in response to pharmacological agents. Cells expressing either wild-type Cx43 (A) or Y286A-mutated Cx43 (B) were incubated for 3 hours in new medium either alone (control) or supplemented with 10 µM lactacystin (Lactacystin), 10 mM NH4Cl (NH4Cl) or 2 µg/ml Brefeldin A (BFA). The cells were then fixed, immunostained with anti-Cx43 antibodies and visualised using fluorescence microscopy. Staining at appositional membranes is indicated by arrows.

View larger version (19K): [in a new window]   Fig. 7. NH4Cl and BFA, but not lactacystin, affect wild-type, but not mutant (Y286A) Cx43-dependent gap junctional communication. (A) SKHep1 cells stably expressing Cx43-WT or Cx43-Y286A were incubated for 3 hours either in new medium alone (control) or new medium supplemented with 10 µM lactacystin (Lactacystin), 10 mM NH4Cl (NH4Cl) or 2 µg/ml Brefeldin A (BFA). Intercellular communication was then assessed by microinjecting individual cells within a cluster, and recording three minutes later the number of lucifer yellow (LY)-labelled cells by fluorescent microscopy, the results of which are expressed as mean±s.e.m. The number of injections is displayed above the column and asterisks indicate differences at P<0.01 versus control as determined by the Student's t-test. (B) Distribution of junctional conductance values (open circles) evaluated in Cx43-WT and Cx43-Y286A cell pairs monitored under the dual patch-clamp technique. The mean±s.e.m. junctional conductance value calculated for each distribution is indicated by the black triangle and error bars. No significant difference (P=0.064) between the two cell clones was detected using the Student's t-test for unpaired data.