GJA1-20k, an internally translated isoform of Connexin 43, is an actin capping protein

Previously, we identified that GJA1-20k, an internally translated isoform of Connexin 43, mediates an actin-dependent protective form of mitochondrial fission (Shimura, Nuebel et al. 2021). We found that when GJA1-20k is present, bands of actin surround mitochondria at locations enriched with GJA1-20k, inducing mitochondrial fission which generates less oxygen free radicals, protecting hearts subjected to ischemia-reperfusion injury. Here, we report that GJA1-20k is a direct actin binding protein and thereby identify the mechanism by which GJA1-20k is able to recruit and stabilize actin filaments around the mitochondria. Surprisingly, GJA1-20k functions as a canonical actin capping protein, producing both truncated actin puncta and stabilized actin filaments. GJA1-20k contains an RPEL-like actin binding motif, and we confirm with both computational modeling and biochemistry, that this domain is crucial for actin capping. The actin capping functionality of GJA1-20k adds GJA1-20k to the family of proteins that regulate actin dynamics. As a stress responsive protein, GJA1-20k can help explain cytoskeletal dependent responses to cellular stress, from delivery of channels to affecting mitochondrial size and function.