Protein Interactomes Identify Distinct Pathways for Streptococcus mutans YidC1 and YidC2 Membrane Protein Insertases

Virulence properties of cariogenic Streptococcus mutans depend on integral membrane proteins. Bacterial protein trafficking involves the co-translational signal recognition particle (SRP) pathway components Ffh and FtsY, the SecY translocon, and membrane-localized YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway. In addition, S. mutans has two yidC paralogs. The{Delta} yidC2 phenotype largely parallels that of{Delta} ffh and{Delta} ftsY while the{Delta} yidC1 phenotype is less severe. This study defined YidC1 and YidC2 interactomes to identify their respective functions alone and in concert with the SRP, ribosome, and/or Sec translocon. A chemical cross-linking approach was employed, whereby whole cell lysates were treated with formaldehyde followed by Western blotting using anti-Ffh, FtsY, YidC1 or YidC2 antibodies and mass spectrometry (MS) analysis of gel-shifted bands. Cross-linked lysates from WT and{Delta} yidC2 strains were also reacted with anti-YidC2 antibodies coupled to magnetic Dynabeads, with co-captured proteins identified by MS. Additionally, C-terminal tails of YidC1 and YidC2 were engineered as glutathione-S-transferase fusion proteins and subjected to 2D Difference Gel Electrophoresis and MS analysis after being reacted with non-cross-linked lysates. Results indicate that YidC2 works in concert with the SRP-pathway, while YidC1 works in concert with the SecY translocon independently of the SRP. In addition, YidC1 and/or YidC2 can act alone in the insertion of a limited number of small integral membrane proteins. The YidC2-SRP and YidC1/SecY pathways appear to function as part of an integrated machinery that couples translation and transport with cell division, as well as transcription and DNA replication. ImportanceStreptococcus mutans is a prevalent oral pathogen and causative agent of tooth decay. Many proteins that enable this bacterium to thrive in its environmental niche, and cause disease, are embedded in its cytoplasmic membrane. The machinery that transports proteins into bacterial membranes differs between Gram-negative and Gram-positive organisms. One important difference is the presence of multiple YidC paralogs in Gram-positive bacteria. Characterization of a proteins interactome can help define its physiological role. Herein, we characterized the interactomes of S. mutans YidC1 and YidC2. Results indicate that YidC1 and YidC2 have individualized functions in separate membrane insertion pathways, and suggest putative substrates of the respective pathways. Furthermore, S. mutans membrane transport proteins appear as part of a larger network of proteins involved in replication, transcription, translation, and cell division/cell shape. This information contributes to our understanding of protein transport in Gram-positive bacteria in general, and informs our understanding of S. mutans pathogenesis.