Fig. 7: Red1 homo-dimerization.
From: Structural analysis of Red1 as a conserved scaffold of the RNA-targeting MTREC/PAXT complex
a Y2H mapping of the Red1 regions involved in its self-association. Growth was assayed on medium lacking histidine and with 30 and 50 mM 3AT. EV- empty vector. FL-Red1 (left panel) or Red1390–712 (right panel) were used as a bait. b Ribbon representation of the Red1 dimeric coiled coil as modeled by AlphaFold235. Residues in heptad positions indicated in c are shown as sticks. c Sequence alignment of Red1 proteins comparing different Schizosaccharomyces species. Only the sequence of the coiled-coil region is shown. Identical residues are in brown boxes, and conserved residues are highlighted in brown. Heptad positions are indicated. d Details of the Red1 dimeric coiled-coil interactions centered on L481 (right panel) and I485 (left panel). e Molecular mass determination of the His-MBP-Red452–524 by MALLS. The measured molecular mass of 88 kDa corresponds to a dimer. Calculated molecular mass of a monomer is 53 kDa. The sample was injected at 9 mg/ml. f Y2H assay showing that the L481R, I485R double mutant essentially disrupts the full-length Red1 dimerization when one or both protomers are mutated. Growth was assayed on medium lacking histidine and with 50 mM 3AT. EV- empty vector. g Co-immunoprecipitation experiments of Red1-Myc13 and Red1-HA3, Red1-∆CC-HA3 or Red1-L481R-I485R-HA3, analyzed by western blot. Soluble extracts (SE) were prepared from exponentially growing cells in minimal liquid medium. Source data are provided as a Source Data file.
