Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins in Mycobacterium smegmatis

Abstract

Mycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD). The IMD is concentrated in the polar regions of growing cells and becomes less polarized under non-growing conditions. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized envelope biosynthesis. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a handful of these have been verified by other experimental methods. Furthermore, we speculate that some IMD-associated proteins may have escaped proteomic identification and remain to be identified. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains each expressing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed fluorescence patterns similar to those of IMD proteins and, consistent with this co-localization, we had previously identified 20 of these using a proteomics approach. Of the nine remaining IMD candidate proteins, three were confirmed to be associated with the IMD while some others appear to be lipid droplet-associated. Taken together, our newly devised strategy is effective in verifying the IMD association of proteins found by proteomic analyses, while facilitating the discovery of additional IMD-associated proteins. ImportanceThe intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis, that likely contribute to the function of the IMD. How can we find more IMD-associated proteins that escaped proteomic detection? Here, as an alternative approach, fluorescence microscope images of 523 proteins were screened to identify IMD-associated proteins. We confirmed the IMD association of previously identified proteins and discovered three additional proteins associated with the IMD. Together, subcellular fractionation, proteomics, and fluorescence microscopy form a robust combination to more rigorously define IMD proteins, which will aid future investigations to decipher the synthesis, maintenance and functions of this membrane domain.