Depletion of ERp29 by siRNA. ROS (A–E) or HeLa/Cx43 (F–J) cells were either untreated or transfected with control or ERp29 siRNA. After 48 h, cells were harvested, homogenized, and postnuclear supernatants were resolved by reducing SDS-PAGE, transferred to Immobilon membranes, and immunoblotted for ERp29 (A and F), PDI (B and G), ERp57 (C and H), BiP (D and I), and Cx43 (E and J). As expected, Cx43 migrated as a doublet, reflecting multiple phosphorylation states of the protein. Parallel samples were immunoblotted to normalize for total protein and calculated as mean ± SE. *p < 0.05 (n = 5), significantly less than control treated cells.

Model for ERp29/Cx43 interactions. ERp29 binding to one or both of the EL domains helps stabilize monomeric Cx43. On exit from the ER to the Golgi apparatus, ERp29 dissociation allows Cx43 oligomerization, presumably by allowing an conformational shift in Cx43. Here, the hexamers are represented by two of the six connexins in the oligomeric complex. The possibility that other protein cofactors may also be part of the ERp29–connexin complex is indicated by the gray circles. In the absence of ERp29, Cx43 prematurely oligomerizes, which increases retention in the Golgi apparatus, increases the potential for Cx43 misfolding/degradation and decreases gap junction formation.

Intracellular Cx43 localized predominantly to the Golgi apparatus in ERp29-depleted cells. ROS cells were transfected with either ERp29 siRNA (A–I) or with control siRNA (J–R) and further incubated for 48 h. The cells were then fixed and immunostained for Cx43 (A, D, G, J, M, and P) and an ER marker (PDI) (B and K), cis-Golgi marker (GM130) (E and K), or early endosome-associated protein 1 (EEA1) (H and Q). Merged images are in C, F, I, L, O, and R. In cells treated with ERp29 siRNA, Cx43 was retained in an intracellular compartment (arrowheads) that colocalized predominantly with GM130 (G–I). To a lesser extent, Cx43 also colocalized with PDI, in cells treated with ERp29 siRNA. (C) Colocalization between Cx43 and EEA1 was limited (I). Cells treated with control siRNA showed less intracellular Cx43. Bar, 10 μm.

Truncated ERp29 inhibits Cx43-mediated gap junctional communication. (A–F) HeLa/Cx43 cells were transfected with either the N-terminal half of EGFP-ERp29 (EGFP-ERp29-N; A–C) or EGFP-ERp29 (D–F) and then fixed, permeabilized, and immunolabeled. Cells expressing EGFP-ERp29 showed accumulation of Cx43 inside the cells (arrows). In contrast, untransfected cells and cells transfected with EGFP-ERp29 had Cx43 localized to gap junctions (arrowheads). Bar, 10 μm. (G–L) Cells transfected with either EGFP-ERp29-N (G–I) or EGFP-ERp29 (J–L) were imaged, and gap junctional communication was assessed by measuring the intercellular transfer of microinjected Alexa568. The microinjected cell is denoted by asterisk (*). Data show the average ± SE of at least 15 microinjections, performed on two independent preparations each. Cells expressing EGFP-ERp29-N showed decreased dye transfer compared with cells expressing EGFP alone (*p < 0.05) and EGFP-ERp29 (#p < 0.05).

ERp29 depletion interferes with gap junctional communication. ROS (A–D) or HeLa/Cx43 cells (E–J) were transfected with ERp29 siRNA (A, B, and E–G) or control siRNA (C, D, and H–J) and then further incubated for 48 h. For HeLa/Cx43 cells, the siRNA was labeled with Cy3 (F and I). Gap junctional communication was assessed by measuring the intercellular transfer of microinjected calcein. The injected cell is denoted by asterisk (*). Note that diffusion of microinjected dye was uniform for control cells; however, it was uneven for cells treated with ERp29, where arrowheads denote uncoupled cells (B and G). Bar, 50 μm. K. For each condition, cell coupling was scored as the number of calcein labeled cells per microinjection. Data show the average ± SE of 15–20 microinjections, performed on two independent preparations each. Cells treated with ERp29 siRNA showed decreased dye transfer compared with untreated and control siRNA-treated cells (*p < 0.01).

ERp29 forms a complex with Cx43. (A) HeLa/Cx43 cells were either untreated (−) or incubated for 5 h with 6 μg/ml BFA (+). The cells were then harvested, solubilized in 0.1% Triton X-100, and magnetically immunopurified with mouse anti-Cx43 and anti-mouse IgG BioMags, resolved by SDS-PAGE, and immunoblotted with rabbit anti-ERp29 or rabbit anti-Cx43. ERp29 preferentially coimmunopurified with Cx43 isolated from BFA-treated cells, suggesting an enhanced interaction when Cx43 is retained in the ER. (B and C) Immunolabeled HeLa shows retention of Cx43-HKKSL in the ER (B), whereas cells expressing wild-type Cx43 show localization to gap junctions (C). Bar, 10 μm. (D) Cells expressing different connexin constructs were homogenized, processed, and immunopurified as described above. ERp29 preferentially coimmunopurified with constructs that were predominantly monomeric (Cx43-HKKSL and Cx32/43/32-HKKSL), but not Cx32-HKKSL, which is oligomerized (Maza et al., 2005). Control samples processed in the absence of mouse anti-Cx43 showed no interaction with ERp29. (E–H) HeLa/Cx32 cells were either untreated (E) or transfected with ERp29 siRNA (F) or with control siRNA (G) and further incubated for 48 h. The cells were then fixed and immunostained for Cx32, which was quantified as number of plaques per cell (H).

ERp29 depletion interferes with Cx43 trafficking to the plasma membrane. ROS (A–C), HeLa/Cx43 (D–F) or HeLa/cldn-4 (G–I) cells were either untreated (A, D, and G), transfected with ERp29 siRNA (B, E, and H) or with control siRNA (C, F, and I), and further incubated for 48 h. The cells were then fixed and immunostained for either Cx43 (A–F) or claudin-4 (G–I). Both untreated and control cells showed Cx43 present at cell–cell interfaces with a distribution consistent with gap junction plaque formation (arrows). However, 48 h after treatment with ERp29 siRNA, surface labeling of Cx43 was diminished and instead accumulated in the perinuclear region of the cell (arrowheads). In contrast to Cx43, a tight junction protein, claudin-4, remained plasma membrane localized (arrows), even in cells treated with ERp29 siRNA. Bar, 10 μm. (J) Gap junction plaques at intercellular junctions were quantified by image analysis for ROS and HeLa/Cx43 cells that were either untreated (un), treated with control siRNA (con), or two different target-specific ERp29 siRNAs specific for rat ERp29 (r1, r2) or human ERp29 (h1, h2). ERp29 depletion decreased the average number of gap junction plaques/cell (*p < 0.05 vs. control-treated cells).

ERp29 depletion promotes early oligomerization of Cx43. (A and B) Blue native analysis of HeLa/Cx43, HeLa/Cx32 or NIH 3T3 cells that were either untreated (−) or incubated for 5 h with 6 μg/ml BFA (+), harvested, solubilized in 0.1% Triton X-100, resolved by blue native gel electrophoresis, and then transferred to immobilon membranes. Cx32 and Cx43 were visualized by immunoblot. (B) Note that BFA significantly increased the percentage of monomeric Cx43 (*p < 0.05; n = 3), in contrast to Cx32, which is not sensitive to BFA. (C–E) ROS cells were either untreated, or treated with control siRNA or ERp29 siRNA for 48 h and then harvested and further analyzed by blue native gel electrophoresis and immunoblot for Cx43 and ERp29. Note the ERp29 band that comigrated with monomeric Cx43. (D) Line scans from representative gels (a–c) in C show that ERp29 depletion decreased the amount of monomeric Cx43 (arrowhead), which was also quantified in E (*p < 0.05; n = 3). (F–H) HeLa/Cx43 cells were treated with control or ERp29 siRNA for 48 h and then further incubated for 5 h in either the absence (−) or presence (+) of BFA, and the oligomerization state of Cx43 was analyzed by blue native gel electrophoresis. (G) Line scans for samples treated with BFA (a and b) in F show that in cells in which ERp29 was depleted, the ability of BFA to promote the retention of monomeric Cx43 was diminished (H) (*p < 0.05; n = 3) compared with control cells, suggesting a role for ERp29 in limiting Cx43 oligomerization in the ER.

ERp29 prevents lindane-induced decreases in Cx43 expression, assembly, and gap junctional communication. (A–C) ROS cells were treated with 0 (A), 50 (B), or 100 μM lindane (C) for 16 h and then fixed, permeabilized, and immunolabeled for Cx43. With increasing lindane treatment, there was an increase in the relative amount of Cx43 localized to the perinuclear region of the cell (arrowheads) and a decrease in punctate gap junctional labeling. Bar, 10 μm. (D and E) ROS cells were treated with 0, 50, or 100 μM lindane for 16 h and then harvested and analyzed by immunoblot for ERp29 (D) and Cx43 (E) expression, normalized to actin expression. Asterisk (*), significantly less than control treated cells (p < 0.05; n = 7). (F–H) ROS cells were transfected with EGFP-ERp29 2 d before treatment with lindane as described above. Overexpression of EGFP-ERp29 partially prevented the decrease in Cx43 expression at 100 μM lindane (G) compared with untransfected cells (E) (#p < 0.01, n = 4). (I–N) Untransfected, control ROS cells immunolabeled for Cx43 (I) and ERp29 (L) show Cx43 localized to gap junction plaques at the cell surface (arrowheads). Transfection of ROS cells with EGFP-ERp29 before lindane treatment retained gap junction-localized Cx43 (K) in cells overexpressing EGFP-ERp29 (N). Bar, 10 μm. (O–U) Untransfected, control ROS cells show high levels of intercellular communication, as assessed by measuring the intercellular transfer of microinjected calcein (O). The injected cell is denoted by asterisk (*). Untransfected ROS cells treated for 16h with 100 μM lindane showed a decrease in gap junctional communication (P); however, cells transfected with EGFP-ERp29 before lindane treatment retained gap junctional communication (Q). Bar, 10 μm. (U) For each condition, cell coupling was scored as the number of calcein labeled cells per microinjection. Data show the average ± SE of at least 24 microinjections performed on three independent preparations each. Cells that were either untransfected or transfected with EGFP alone showed a significant decrease in gap junctional communication, compared with untreated controls (*p < 0.01). By contrast, ROS cells transfected with EGFP-ERp29 were resistant to Lindane and had increased gap junctional communication, as compared with control cells transfected with EGFP alone (#p < 0.02).