Compartmentalization of galactan biosynthesis in mycobacteria

• Published in: The Journal of biological chemistry Vol. 300; no. 3; p. 105768
• Main Authors: Savková, Karin, Danchenko, Maksym, Fabianová, Viktória, Bellová, Jana, Bencúrová, Mária, Huszár, Stanislav, Korduláková, Jana, Siváková, Barbara, Baráth, Peter, Mikušová, Katarína
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2024; American Society for Biochemistry and Molecular Biology
• Subjects: cell wall; galactosyltransferases; GlfT2; Mycobacterium smegmatis; proteomics; radiolabeling assays; subcellular fractionation; IMD; MOPS; GlfT2; proteomics; subcellular fractionation; CCE; radiolabeling assays; CEF; LLG; Mycobacterium smegmatis; PMf; galactosyltransferases; PM-CW; PIM; HA; cell wall
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2024.105768

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) – N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[14C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.