Fig. 5: Dol-P-Man binding to a cytosolic pocket is functionally important for Pmt4.

A Western blot analysis of the reporter protein FUS-Axl2TM^ZZ isolated from yeast strain pmt4-G/AXL transformed with empty pREP3-adh vector (EV), wild-type ctPmt4 (WT) or Dol-P-Man binding mutants under the control of the constitutive ADH1 promoter. 40 μg total membrane protein from each strain was separated on an 8% SDS-PA gel and probed with peroxidase-coupled anti-rabbit-IgG antibody. A representative blot is shown from three independent experiments. B Growth of the yeast pmt4-G strain transformed with empty pMT929 vector (EV), wild-type ctPmt4 (WT) or Dol-P-Man binding mutants. Selective medium was supplemented with 2% galactose to overexpress variants from a GAL1/GAL10 promoter and OD_600nm was measured every 60 min. Average values from three independent experiments are shown on a logarithmic scale with error bars showing the standard deviation. C Side-by-side comparison of the cytosolic pockets in ctPmt4 and the scPmt4 AlphaFold DB^23,24 model after superimposition of their TMDs (RMSD 1.00 Å over 487 Cα-atoms). The electrostatic surface potentials (± 10 kT) are shown and positively charged residues are labelled. In ctPmt4 the pocket is occupied by Dol-P-Man, whilst in scPmt4 it is occupied by its N-terminus (Nter), shown in cartoon representation for clarity. D Western blot of Ccw5 isolated from a yeast Δpmt4 strain expressing FLAG-tagged Ccw5 (pmt4-G/CCW5) transformed with wild-type scPmt4 (pJK4-B1; WT), empty pRS423 vector (EV) and pJK4-B1 with mutations in positively charged residues in the cytosolic pocket. Anti-FLAG antibody was used to detect O-mannosylated and N-glycosylated isoforms of Ccw5 separated on a 10% SDS-PA gel. A representative blot is shown from three independent experiments. Source data for A, B and D are provided in the Source Data file.