Neural Regeneration Laboratory, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
J Proteome Res. 2013 Jun 7;12(6):2597-610. doi: 10.1021/pr301166p. Epub 2013 Apr 26.
Connexins are the structural subunits of gap junctions and act as protein platforms for signaling complexes. Little is known about tissue-specific connexin signaling nexuses, given significant challenges associated with affinity-purifying endogenous channel complexes to the level required for interaction analyses. Here, we used multiple subcellular fractionation techniques to isolate connexin32-enriched membrane microdomains from murine liver. We show, for the first time, that connexin32 localizes to both the plasma membrane and inner mitochondrial membrane of hepatocytes. Using a combination of immunoprecipitation-high throughput mass spectrometry, reciprocal co-IP, and subcellular fractionation methodologies, we report a novel interactome validated using null mutant controls. Eighteen connexin32 interacting proteins were identified. The majority represent resident mitochondrial proteins, a minority represent plasma membrane, endoplasmic reticulum, or cytoplasmic partners. In particular, connexin32 interacts with connexin26 and the mitochondrial protein, sideroflexin-1, at the plasma membrane. Connexin32 interaction enhances connexin26 stability. Converging bioinformatic, biochemical, and confocal analyses support a role for connexin32 in transiently tethering mitochondria to connexin32-enriched plasma membrane microdomains through interaction with proteins in the outer mitochondrial membrane, including sideroflexin-1. Complex formation increases the pool of sideroflexin-1 that is present at the plasma membrane. Together, these data identify a novel plasma membrane/mitochondrial signaling nexus in the connexin32 interactome.
间隙连接蛋白是间隙连接的结构亚基，作为信号复合物的蛋白质平台发挥作用。由于与亲和力纯化内源性通道复合物相关的重大挑战，以至于我们对组织特异性连接蛋白信号连接体知之甚少，这些挑战需要达到进行相互作用分析的水平。在这里，我们使用多种亚细胞分级分离技术从鼠肝中分离富含连接蛋白 32 的膜微区。我们首次表明，连接蛋白 32 定位于肝细胞的质膜和内线粒体膜。我们使用免疫沉淀-高通量质谱、相互免疫沉淀和亚细胞分级分离方法的组合，报告了使用缺失突变对照物验证的新相互作用组。鉴定出 18 个与连接蛋白 32 相互作用的蛋白质。大多数代表驻留的线粒体蛋白，少数代表质膜、内质网或细胞质伴侣。特别是，连接蛋白 32 在质膜上与连接蛋白 26 和线粒体蛋白 sideroflexin-1 相互作用。连接蛋白 32 的相互作用增强了连接蛋白 26 的稳定性。趋同的生物信息学、生化和共聚焦分析支持连接蛋白 32 通过与外线粒体膜中的蛋白质（包括 sideroflexin-1）相互作用，在瞬时将线粒体固定到富含连接蛋白 32 的质膜微区的作用。复合物形成增加了存在于质膜上的 sideroflexin-1 池。总之，这些数据确定了连接蛋白 32 相互作用组中一个新的质膜/线粒体信号连接体。
