Cysteine residues in the C-terminal tail of connexin32 regulate its trafficking.

Gap junctions (GJs) are formed by the assembly of constituent transmembrane proteins called connexins (Cxs). Aberrations in this assembly of Cxs are observed in several genetic diseases as well as in cancers. Hence it becomes imperative to understand the molecular mechanisms underlying such assembly defect. The polarized cells in the epithelia express Connexin32 (Cx32). The C-terminal tail (CT) of Cx32 orchestrates several aspects of GJ dynamics, function and growth. The study here was aimed at determining if post-translational modifications, specifically, palmitoylation of cysteine residues, present in the CT of Cx32, has any effect on GJ assembly. The CT of Cx32 was found to harbor three cysteine residues, which are likely to be modified by palmitoylation. The study here has revealed for the first time that Cx32 is palmitoylated at cysteine 217 (C217) in cell line derived from prostate tumors. However, it was found that mutating C217 to alanine affected neither the trafficking nor the ability of Cx32 to assemble into GJs. Intriguingly, it was discovered that mutating cysteine 280 and 283, only in combination, blocked the trafficking of Cx32 from the trans-Golgi network to the cell surface. The mutants showed reduced stability due to enhanced lysosomal degradation. Overall, the findings reveal the importance of the two C-terminal cysteine residues of Cx32 in regulating its trafficking and stability and hence its ability to assemble into GJs.