Project Summary. Streptococccus mutans is a ubiquitous oral bacterium and prominent etiologic agent of human dental caries. Certain strains can also cause bacterial endocarditis and invade human coronary epithelial cells. US health care costs associated with dental decay alone are over $64 billion annually. This project addresses membrane protein insertion in S. mutans. A semi-permeable lipid bilayer membrane encloses all cells and determines cellular function by dictating what can cross by virtue of proteins embedded within differing lipid milieus. S. mutans shares features common to other bacterial membrane protein insertion systems, and those of eukaryotic endoplasmic reticulum and organelles, but also exhibits unique properties. S. mutans is becoming an established model for studying protein transport in Gram+ bacteria. All known regulatory circuits and multiple virulence attributes of S. mutans stem from its membrane protein composition. Biological membranes are ~50% protein by mass and membrane proteins represent most known drug targets. Thus understanding S. mutans protein transport and insertion pathways will facilitate targeted therapy against this and related pathogens. This project focuses on co-translational protein transport including YidC insertases (bacteria, mitochondria, and chloroplasts), and the signal recognition particle (SRP) pathway conserved in all living cells. We identified respective S. mutans YidC1, YidC2, and SRP substrates and identified four pathway frameworks: canonical SRP pathway, autonomous YidC pathway, coordinated YidC2-SRP pathway, and an SRP-independent pathway in which YidC1 interacts with the SecYEG translocon and an uncharacterized protein called Jag. We also characterized the cardiolipin-rich lipidome of the S. mutans membrane and showed the influence of lipid composition on membrane partitioning of specific components of the transport machinery. In this renewal application we will identify interactions of protein components of the transport machinery with anionic lipids using Martini22 coarse-grain molecular dynamic simulation in conjunction with circular dichroism, solution NMR, and biolayer interferometry (SA1). We will also evaluate protein-protein interactions in the context of varying lipid milieus by molecular dynamic simulation, blue-native polyacrylamide gel electrophoresis, and will utilize solid state NMR to evaluate protein-protein contacts and structures of protein pairs and oligomers. Findings will be validated using insertion of epitope-tagged substrates in vivo, and in vitro transcription-translation-insertion assays with customized proteoliposomes (SA2). Lastly, membrane- localized Jag is a predicted RNA binding protein (RBP). Mitochondrial membrane-localized RBPs have recently been shown to chaperone specific mRNAs for coupled translation-insertion. jag and rnpA encoding ribonuclease P are in the same operon as yidC1. Therefore we will also evaluate the roles of Jag and RnpA in post-tra...