Evidence for the mycobacterial Mce4 transporter being a multiprotein complex.

Mycobacteria possess Mce transporters that import lipids and are thought to function analogously to ATP-binding cassette (ABC) transporters. However, whereas ABC transporters import substrates using a single solute binding protein (SBP) to deliver a substrate to permease proteins in the membrane, mycobacterial Mce transporters have a potential for six SBPs (MceA-F), working with a pair of permeases (YrbEA-B), a cytoplasmic ATPase (MceG), as well as multiple Mce Associated Membrane (Mam) and Orphaned-Mam (Omam) proteins to transport lipids. In this study we used the model mycobacteria, Mycobacterium smegmatis, to study the requirement of individual Mce, Mam, and Omam proteins in Mce4 transport of cholesterol. All the Mce4 and Mam4 proteins we investigated were required for cholesterol uptake. However, not all Omam proteins, which are encoded by genes outside of mce loci, proved to contribute to cholesterol import. OmamA and OmamB were required for cholesterol import, while OmamC,D,E, and F were not. In the absence of any single Mce4, Mam4, or Omam that we tested, the abundance of Mce4A and Mce4E declined. This relationship between the levels of Mce4A and 4E and these additional proteins suggests a network of interactions that assemble and/or stabilize a multiprotein Mce4 transporter complex. Further support for Mce transporters being multiprotein complexes was obtained by immunoprecipitation-mass spectrometry where we identified every single Mce, YrbE, MceG, Mam, and Omam protein with a role in cholesterol transport as associating with Mce4A. This study represents the first time any of these Mce4 transporter proteins were shown to associate.Importance:How lipids travel between membranes of diderm bacteria is a challenging mechanistic question because lipids, which are hydrophobic molecules, must traverse a hydrophilic periplasm. This question is even more complex for mycobacteria, which have a unique cell envelope that is highly impermeable to molecules. A growing body of knowledge identifies Mce transporters as lipids importers for mycobacteria. Here, using protein stability experiments and immunoprecipitation-mass spectrometry we provide evidence for mycobacterial Mce transporters existing as multiprotein complexes.