Abstract
The intracellular membrane domain (IMD) in mycobacteria is a spatially distinct region of the plasma membrane with diverse functions. Previous comparative proteomic analysis of the IMD suggested that menaquinone biosynthetic enzymes are associated with this domain. In the present study, we determined the subcellular site of these enzymes using sucrose density gradient fractionation. We found that the last two enzymes, the methyltransferase MenG, and the reductase MenJ, are associated with the IMD in Mycobacterium smegmatis. MenA, the prenyltransferase that mediates the first membrane-associated step of the menaquinone biosynthesis, is associated with the conventional plasma membrane. For MenG, we additionally showed the polar enrichment of the fluorescent protein fusion colocalizing with an IMD marker protein in situ. To start dissecting the roles of IMD-associated enzymes, we further tested the physiological significance of MenG. The deletion of menG at the endogenous genomic loci was possible only when an extra copy of the gene was present, indicating that it is an essential gene in M. smegmatis. Using a tetracycline-inducible switch, we achieved gradual and partial depletion of MenG over three consecutive 24 h sub-cultures. This partial MenG depletion resulted in progressive slowing of growth, which corroborated the observation that menG is an essential gene. Upon MenG depletion, there was a significant accumulation of MenG substrate, demethylmenaquinone, even though the cellular level of menaquinone, the reaction product, was unaffected. Furthermore, the growth retardation was coincided with a lower oxygen consumption rate and ATP accumulation. These results imply a previously unappreciated role of MenG in regulating menaquinone homeostasis within the complex spatial organization of mycobacterial plasma membrane.
Highlights
Mycobacterium smegmatis has a complex membrane organization
Combining evidence for the important roles of these enzymes with the new proteomic analysis suggesting that MenG and MenJ might be intracellular membrane domain (IMD)-associated, we examined if the membrane steps of menaquinone biosynthesis are compartmentalized within the plasma membrane in M. smegmatis
MenG and MenJ have no predicted transmembrane domains, but we previously showed by comparative proteomics that peptide fragments corresponding to known MenG and MenJ sequences were recovered at higher level in the IMD than in the conventional plasma membrane (PM-CW) (Hayashi et al, 2016)
Summary
In addition to the inner plasma membrane, long branched fatty acids known as mycolic acids are covalently anchored to the peptidoglycan-arabinogalactan cell wall core, and serve as a major component of the outer membrane In addition to these topologically distinct membrane layers, the plasma membrane has a spatially distinct membrane domain known as the Intracellular Membrane Domain (IMD) (Hayashi et al, 2016, 2018). The IMD can be separated and purified from the conventional plasma membrane by sucrose density gradient fractionation of mycobacterial crude cell lysate (Morita et al, 2005) In this gradient fractionation, the IMD appears as vesicles of phospholipids without significant enrichment of cell wall components. A more recent study revealed that the IMD is enriched in the polar regions of the live actively growing cell, and associated with more than 300 proteins, among which are enzymes involved in cell envelope biosynthesis (Hayashi et al, 2016). There are many IMD-associated enzymes that are not involved in the cell envelope biosynthesis, suggesting more general functions of the IMD as a spatially distinct area of mycobacterial membrane, including the possible regulation of cytoplasmic metabolites, which is largely unexplored
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