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 proteinprotein interactions in cells.

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

“…Despite their great potential, current XL-MS studies that have aimed to identify cross-linked peptides have been mostly limited to in vitro cross-linking experiments, with few successfully identifying protein interaction interfaces in living cells (24,25). This is largely because XL-MS studies remain challenging due to the inherent difficulty in the effective MS detection and accurate identification of cross-linked peptides, as well as in unambiguous assignment of cross-linked residues.…”

Protein Interactions, Post-translational Modifications and Topologies in Human Cells

“…The use of XL-MS to study protein structures and interactions directly from living cells was first demonstrated on the bacterial system Shewanella oneidensis, where new details on the membrane protein electron transport machinery used during anaerobic respiration were discovered (14). More recently XL-MS has been applied to E. coli cell lysates (15,16) as well as intact E. coli cells (17,18).…”

Antibody Recognition Force Microscopy Shows that Outer Membrane Cytochromes OmcA and MtrC Are Expressed on the Exterior Surface of Shewanella oneidensis MR-1

“…Prior to conducting the Ig-RFM experiments, the specificity of each polyclonal antibody (i.e., anti-OmcA and anti-MtrC) for OmcA or MtrC was verified by Western blot analysis as described previously (24,28). Proteins were resolved by both denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE).…”

“…Anti-OmcA bound exclusively to OmcA, anti-MtrC bound exclusively to MtrC, and neither antibody showed crossreactivity with the other cytochrome. Antibody specificities of anti-OmcA and anti-MtrC were also validated by immunoblot analysis of S. oneidensis whole-cell lysate (28).…”