Altered trafficking of mutant connexin32

Abstract

We examined the cellular localization of nine different connexin32 (Cx32) mutants associated with X-linked Charcot-Marie-Tooth disease (CMTX) in communication-incompetent mammalian cells. Cx32 mRNA was made, but little or no protein was detected in one class of mutants. In another class of mutants, Cx32 protein was detectable in the cytoplasm and at the cell surface, where it appeared as plaques and punctate staining. Cx32 immunoreactivity in a third class of mutants was restricted to the cytoplasm, where it often colocalized with the Golgi apparatus. Our studies suggest that CMTX mutations have a predominant effect on the trafficking of Cx32 protein, resulting in a potentially toxic cytoplasmic accumulation of Cx32 in these cells. These results and evidence of cytoplasmic accumulation of other mutated myelin proteins suggest that diseases affecting myelinating cells may share a common pathophysiology.

Fig. 2.

Analysis of Cx32 expression in PC12J cells stably transfected with normal and mutated Cx32 cDNAs. A, As negative controls for B, immunoblots of the parental cells (P; lane 1) and two clones transfected with the vector alone (V; lanes 2–3, clones pREP9.6.1 and pREP9.6.4) were hybridized with a polyclonal antiserum (Lola) that recognizes an epitope in the cytoplasmic loop of Cx32. The bands seen across alllanes in A and B, including a high molecular weight band (asterisk), are proteins nonspecifically detected by Lola. B, Immunoblots of three clones transfected with 175fs(lanes 1–3, clones 5117.6.1, 5117.4.9, and 5117.4.3) were hybridized with Lola. C, Immunoblots of three clones expressing various amounts of R220Stop protein (lanes 1–3), the parental cells (P;lane 4), and two clones transfected with the vector alone (V; lanes 5–6) were performed with a mouse monoclonal antibody against the cytoplasmic loop (M12.13). An arrow indicates truncated Cx32. The two bands seen across all lanes (asterisks) are proteins nonspecifically detected by the M12.13 antibody.D, Immunoblots of rat liver (+;lane 1) and representative clones transfected with wild-type Cx32 (WT; lane 2, clone 517.4.7), R15Q (lanes 3–4, clones 15.23 and 15.57), V63I (lanes 5–6, clones 63.36 and 63.55), R142W (lanes 7–8, clones 116.41 and 116.38), E186K (lanes 9–10, clones 412.5 and 414.10), and E208K(lanes 11–12, clones 208.47 and 208.49) were performed with a mouse monoclonal antibody that also recognizes the C terminal (7C6.C7). Dashes indicate the positions of molecular weight markers of 46 and 30 kDa, respectively. E, Northern blots of four clones transfected with 175fs(lanes 1–4, 5117.2.3, 5117.4.9, 5117.2.10, and 5117.4.3), one clone expressing wild-type Cx32 (WT;lane 5, 517.4.7), and the parental cells (P; lane 6) were performed using a full-length, wild-type human Cx32 cDNA probe. The Cx32 transcripts are of the expected size (∼1.3 kb). The same blots were stripped and rehybridized with a full-length rat GAPDH cDNA probe to assess the relative quantity of RNA in each lane. Thedashes indicate the positions of 18S rRNA (∼2.4 kb). For immunoblots, the single and double arrowheads represent the positions of the monomeric and dimeric forms of rat liver Cx32 on the same gels. The migration of rat liver Cx32 is slower because the homogenate was prepared in a different lysis buffer relative to that used for the cell lysates (see Materials and Methods).

Fig. 1.

Diagram of Cx32, with the mutations studied in this paper indicated by arrows. Asterisks indicate conserved cysteine residues.

Fig. 3.

Localization of wild-type Cx32 and CMTX mutants in PC12J cells by indirect immunofluorescence using scanning laser confocal microscopy. Immunocytochemistry was performed with 7C6.C7 for all clones but R220Stop, for which M12.13 was used. A, Untransfected cells, PC12J. B,Wild-type Cx32, clone 517.4.7. C,R15Q, clone 15.38. D,V63I, clone 63.55. E,V139M, nonclonal cells. F,R220Stop, clone 220.37. G,R220Stop, clone 220.37. H,G12S, nonclonal cells. I,R142W, clone 116.38. J,E208K, clone 208.37. The magnification ofJ is higher to show greater cytoplasmic detail. Note that the diffuse staining seen in clone 208.37 is the predominant pattern observed in all clones expressing E208K. Scale bars, 10 μm.

Fig. 4.

Localization of Cx32 mutantsE186K and E208K in PC12J cells by indirect immunofluorescence using scanning laser confocal microscopy.A–C, Cells expressing E186K (clone 414.10) were double-stained with the mouse monoclonal antibody 7C6.C7 against Cx32 (A; rhodamine) and a polyclonal antiserum against rat α-mannosidase II (MnII) (B; fluorescein).A and B are superimposed inC. D–F, Cells expressingE208K (clone 208.49) were double-stained with the polyclonal antiserum B₁J against Cx32(D; rhodamine) and the monoclonal antibody 10A8 againstMG160 (E; fluorescein). Dand E are superimposed in F. The characteristically punctate staining of E186K obtained with 7C6.C7 (see Fig. 5; data not shown) is difficult to see because of high background staining contributed by the α-mannosidase II antibody; arrowheads are used to indicateCx32 immunoreactivity in the cytoplasm of these cells. Note the absence of Cx32 at the cell surface in these two mutants. Scale bars, 10 μm.

Fig. 5.

Effect of BFA on intracellular localization of E186K in clone 414.10. Immunocytochemistry was performed on cells treated with ethanol or 15 μg/ml BFA for 1 hr, and the cells were visualized with scanning laser confocal microscopy. A, Cx32 (antibody 7C6.C7); ethanol; 520×. B, Cx32; BFA; 760×. C, MG160 (antibody 10A8); ethanol; 760×.D, MG160; BFA; 760×. Arrows indicate the compact Cx32 or Golgi staining in control cells. Regardless of BFA concentration (data not shown), residual bright MG160 immunoreactivity on one side of the nucleus (arrowheads) was always observed in BFA-treated cells, including wild-type Cx32-expressing clones and vector-alone clones.