title: The liver connexin32 interactome is a novel plasma membrane-mitochondrial signaling nexus.
abstract: 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.
authors.affiliation: Neural Regeneration Laboratory, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Fowler
authors.forename: Mark
authors.initials: M
authors.lastname: Akins
authors.forename: Hu
authors.initials: H
authors.lastname: Zhou
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
authors.forename: Steffany A L
authors.initials: SA
authors.lastname: Bennett
mesh_terms: D000818:Animals; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism; D005786:Gene Expression Regulation; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008930:Mitochondria, Liver / Q000737:chemistry / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D015398:Signal Transduction; D000097002:Gap Junction beta-1 Protein
chemical_list: D027682:Cation Transport Proteins; D000072259:Connexin 26; D017630:Connexins; D024101:Mitochondrial Proteins; D000097002:Gap Junction beta-1 Protein; C424902:Sfxn1 protein, mouse