Leucine-Zipper Mediated Intermolecular Interaction between MG53 is Essential for Cellular Membrane Repair

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We recently found that MG53, a muscle-specific TRIM family protein, functions as a sensor of oxidation to nucleate the assembly of cell membrane repair machinery (Cai et al, 2009 Nature Cell Biology). Our data showed that disulfide formation mediated by Cys242 is critical for MG53-mediated translocation of intracellular vesicles toward the injury sites. Here we test the hypothesis that leucine-zipper motifs in the coiled-coil domain of MG53 constitute an additional mechanism that can facilitate oligomeric formation of MG53 for assembly of the cell membrane repair machinery. Chemical cross-linking studies show that the coiled-coil domain of MG53, which contains the two putative leucine zipper motifs LZ1 (L176/L183/L190/V197) and LZ2 (L205/L212/L219/L226), is involved in formation of MG53 dimers. While mutation of LZ2 does not affect oligomeric interaction of MG53, replacement of 3 leucine residues to alanines in LZ1 leads to compromised oligomeric formation of MG53. Moreover, double mutation of LZ1 and LZ2 completely disrupts MG53 oligomeric formation, even under non-reducing conditions. Live cell imaging revealed that the movement of GFP-tagged MG53 mutants, GFP-LZ1 and GFP-LZ2, in response to mechanical damage of the cell membrane is significantly reduced relative to the wild type GFP-MG53 construct. Furthermore, the GFP-LZ12 double mutant is completely ineffective in translocation toward the injury sites, and cannot repair acute damage to cell membranes. Our data show that leucine-zipper mediated oligomer formation is essential for cell membrane repair. LZ1 likely constitutes a critical motif for disulfide cross-link between Cys242, during assembly of the membrane repair machinery.