DOI: 10.1101/452839Oct 25, 2018Paper

Demethylmenaquinone methyl transferase is a membrane domain-associated protein essential for menaquinone homeostasis in Mycobacterium smegmatis

BioRxiv : the Preprint Server for Biology
Julia PuffalYasu S Morita

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. 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 hour subcultures. This partial MenG deple...Continue Reading

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