Such a robust method to rigorously define IMD proteins will benefit future investigations to decipher the synthesis, maintenance, and functions of this membrane domain and help delineate a more general mechanism of subcellular protein localization in mycobacteria. Using a combination of fluorescence microscopy, proteomics, and subcellular fractionation, we identified three new proteins associated with the IMD. Here, we describe fluorescent protein colocalization as an alternative, independent approach. However, proteomics on its own is unlikely to detect every IMD-associated protein because of technical and biological limitations. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis. IMPORTANCE The intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Taken together, this new colocalization strategy is effective in verifying the IMD association of proteins found by proteomic analyses while facilitating the discovery of additional IMD-associated proteins. Of the nine remaining IMD candidate proteins, three were confirmed by biochemical methods to be associated with the IMD. Twenty of these had previously been suggested to localize to the IMD based on proteomic data. We identified 29 fusion proteins that showed polar fluorescence patterns characteristic of IMD proteins. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains, each producing a Dendra2-FLAG-tagged recombinant protein. Furthermore, some IMD-associated proteins may have escaped proteomic identification and remain to be identified. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a few of these have been verified by independent experimental methods. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized biosynthesis of envelope components. The IMD is concentrated in the polar regions of growing cells and becomes less polarized under nongrowing conditions. Mycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD).
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