ABSTRACT Tuberculosis is one of the leading causes of death due to infectious disease despite the availability of anti- tubercular drugs. The cell envelope of Mycobacterium tuberculosis (Mtb) is notable for the abundance of mycolic acids (MAs), which are essential to mycobacterial viability. The mycobacterial cell envelope is extremely hydrophobic, contributes to virulence and antibiotic resistance. Yet, exactly how glycoconjugates and other species-specific lipids are transported across the inner membrane for cell envelope biosynthesis is incompletely understood. Recent work shows that the Mycobacterial membrane protein Large (MmpL) transporters export lipids synthesized in the mycobacterial cytoplasm for incorporation into the cell envelope. These transporters are therefore important for bacterial viability and virulence. MmpL3, the focus of this proposal, is essential and required for transport of trehalose monomycolate (TMM), the precursor of trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP). The exact mechanism of MmpL3 export remains elusive. RND family proteins typically possess adaptor proteins that assist in substrate transport. As presented in our preliminary data and recent publication, we identified several lipoproteins that interact with MmpL3 and MmpL11. We characterized an Mtb lpqN mutant and demonstrated that LpqN has a lipid binding pocket and interacts with the Ag85 mycolyl transferases. Based on these data, we propose a model where mycobacterial lipoproteins form a complex with MmpL protein to promote lipid secretion and localization. Our hypothesis is that MmpL3 interacts with lipoproteins that facilitate export of TMM and its incorporation into the cell envelope. The proposed study will combine biochemical and genetic approaches to identify proteins that complex with MmpL3 to facilitate substrate transport. .