Abstract
Protein O-mannosyltransferases (PMTs) add mannose to serine/threonine (S/T)-rich proteins in the endoplasmic reticulum, facilitating proper folding and trafficking through the secretory pathway. These enzymes share a conserved architecture that includes a large transmembrane domain housing the catalytic pocket and a lumenal β-trefoil-folded MIR domain. Although S/T-rich regions in acceptor proteins are generally disordered, it remains unclear how PMTs selectively target these regions over other intrinsically disordered sequences. Here, using cryo-EM and X-ray crystallography, we demonstrate that the Saccharomyces cerevisiae Pmt4 dimer recognizes an S/T-rich peptide at two distinct sites. A groove above the catalytic pocket in the transmembrane domain binds the mannose-accepting S/T site, while the lumenal MIR domain engages an S/T-X-S/T motif. Notably, the substrate peptide is simultaneously bound by the catalytic pocket of one Pmt4 protomer and the MIR domain of the other, revealing an unexpected cooperative dual substrate recognition mechanism. This mechanism likely underpins the invariant dimeric architecture observed in all PMT family members.