The liver connexin32 interactome is a novel plasma membrane-mitochondrial signaling nexus

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cation Transport Proteins / genetics
  • Cation Transport Proteins / metabolism*
  • Cell Fractionation
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism*
  • Connexin 26
  • Connexins / genetics
  • Connexins / metabolism*
  • Gap Junctions / metabolism
  • Gene Expression Regulation
  • Hepatocytes / cytology
  • Hepatocytes / metabolism*
  • Liver / cytology
  • Liver / metabolism
  • Mice
  • Mice, Knockout
  • Mitochondria, Liver / chemistry
  • Mitochondria, Liver / metabolism*
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*
  • Protein Binding
  • Protein Interaction Mapping
  • Signal Transduction*


  • Cation Transport Proteins
  • Connexins
  • Mitochondrial Proteins
  • Sfxn1 protein, mouse
  • connexin 32
  • Connexin 26