The cytoplasmic C-terminal domain of the MmpL11 lipid transporter is required for interaction with its co-cistronic partner MSMEG_0240 in Mycobacterium smegmatis

MmpL proteins play an important role in the various mechanisms associated with mycobacterial virulence. Identification of interacting protein partners required for a detailed understanding of their role remains hampered because of their large size (> 100 kDa) and the presence of twelve transmembrane domains by classical methods. In this study, we used two independent biotin proximity labelling assays (APEX2 and BioID) to define the proxisome of MmpL11 in M. smegmatis. Indeed, these techniques are performed directly in the organism of interest, allowing the detection of potentially transient or weak interactions in multiprotein complexes and preserving the subcellular structures and the presence of cofactors or post-translational modifications that can also impact protein-protein interactions. BioID leads to the biotinylation of lysine residues, whereas APEX2 leads to the biotinylation of mainly tyrosine residues; they have also been shown to have different effective labelling radii. On one hand, an interaction was detected between the cytoplasmic C-terminal domain of MmpL11 and MSMEG_0240, a protein of unknown function, using BioID. This interaction was confirmed using both MmpL11 and MSMEG_0240 as fusions with BirA and was corroborated by AlphaFold3 prediction. On the other hand, APEX2 failed to detect an interaction between MmpL11 and MSMEG_0240, probably due to the absence of accessible tyrosines. However, both approaches identified MSMEG_0940 as an additional interactant with MmpL11 that also depends on the C-terminal domain. Overall, this study demonstrates that APEX2 and BioID as complementary tools for defining the proxisome of mycobacterial proteins.