In Vivo Identification of the Outer Membrane Protein OmcA−MtrC Interaction Network in Shewanella oneidensis MR-1 Cells Using Novel Hydrophobic Chemical Cross-Linkers

Abstract

Outer membrane (OM) cytochromes OmcA (SO1779) and MtrC (SO1778) are the integral components of electron transfer used by Shewanella oneidensis for anaerobic respiration of metal (hydr)oxides. Here the OmcA-MtrC interaction was identified in vivo using a novel hydrophobic chemical cross-linker (MRN) combined with immunoprecipitation techniques. In addition, identification of other OM proteins from the cross-linked complexes allows first visualization of the OmcA-MtrC interaction network. Further experiments on omcA and mtrC mutant cells showed OmcA plays a central role in the network interaction. For comparison, two commercial cross-linkers were also used in parallel, and both resulted in fewer OM protein identifications, indicating the superior properties of MRN for identification of membrane protein interactions. Finally, comparison experiments of in vivo cross-linking and cell lysate cross-linking resulted in significantly different protein interaction data, demonstrating the importance of in vivo cross-linking for study of protein-protein interactions in cells.