RESUMO
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.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Conexinas/metabolismo , Hepatócitos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Transdução de Sinais , Animais , Proteínas de Transporte de Cátions/genética , Fracionamento Celular , Membrana Celular/química , Conexina 26 , Conexinas/genética , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica , Hepatócitos/citologia , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/química , Proteínas Mitocondriais/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteína beta-1 de Junções ComunicantesRESUMO
Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes.