biostudies-literatureMcDowell MADeutsche Forschungsgemeinschaft (German Research Foundation)Wellcome Trust11134https://www.ebi.ac.uk/biostudies/studies/S-EPMC12705809biostudies-literatureUnknown16(1)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 iNature communicationsStructural characterisation of the fungal Pmt4 homodimer.PMC12705809221795/Z/20/ZLeibniz Programme (SI 586/6-1), FOR2509 (SI 586/8-2)Bausewein DHackmann YBrenske DStrahl SBilsing FLMcDowell MAFiorentino FWu DRobinson CVMortensen SChiapparino AMetschies AWild KSinning IfalseStructural characterisation of the fungal Pmt4 homodimer.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 i2025-01-01T00:00:00Z2025 Dec2026-07-15T06:39:06.26Z2026-07-02T03:08:56.257ZS-EPMC127058094139231510.1038/s41467-025-67412-1