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Published: 14 December 2025

Protein O-mannosyltransferases (PMTs) are conserved endoplasmic reticulum membrane-embedded enzymes responsible for the transfer of mannose from dolichol phosphate-mannose (Dol-P-Man) to serine/threonine-rich protein substrates or unfolded proteins. PMTs from three subfamilies form obligate dimers with different substrate specificities and require the concerted action of their transmembrane domains (TMDs) and a luminal MIR domain for catalysis. Here, we present structures, native mass spectrometry, and structure-based mutagenesis of the fungal Pmt4 homodimer. The core fold of the TMDs and MIR domain is conserved with the Pmt1-Pmt2 heterodimer, indicating a shared catalytic mechanism. Distinct from Pmt4, the MIR domain interacts in cis with the TMDs of the same subunit and has a β-hairpin insertion required for O-mannosylation of substrates. We further identify a cytosolic binding site for substrate Dol-P-Man within the Pmt4 TMDs, which is conserved amongst PMTs and important for in vivo activity. Thus, we provide a framework to understand the substrate specificity and regulation of the Pmt4 homodimer.
 
Publication: McDowell MA, Wild K, Fiorentino F, Bausewein D, Metschies A, Chiapparino A, Hackmann Y, Bilsing FL, Brenske D, Mortensen S, Wu D, Robinson CV, Strahl S, Sinning I. Structural characterisation of the fungal Pmt4 homodimer. Nat Commun. 2025 Dec 14;16(1):11134. doi: 10.1038/s41467-025-67412-1.