MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

Significance The unique architecture of the mycobacterial cell wall plays a predominant role inMycobacterium tuberculosis pathogenesis. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs) across the cell membrane for cell-wall biosynthesis. Here we report a crystal structure ofMycobacterium smegmatis MmpL3 that indicates a plausible pathway for TMM translocation. Native mass spectrometry suggests that the monomeric MmpL3 transporter is able to specifically bind TMM and phosphatidylethanolamine lipids. Overall our data suggest a mechanism for TMM transport involved in mycobacterial cell-wall biogenesis.