Fig. 4: Disrupting Red1-Pla1 interaction in vivo leads to ‘Pla1-truncated’ MTREC complex.
A Y2H experiments showing the effects of deletion of Red1 residues 288-345 or Red1W298A/F305A double mutant on Pla1-Red1 interaction in the context of full-length proteins. B Coomassie blue stained SDS polyacrylamide gel of tandem affinity purified MTREC complex using Red1 as bait, from respective mutant strains (a representative gel from two independent experiments is shown); and C heat map representation of mass-spectrometry (MS) analysis of these purifications. Heat map shows the changes in the amount of co-purifying MTREC subunits (except the Cbc1 subunit which was not conclusively quantified), in indicated mutant strains compared to WT. Co-purifying protein amounts were normalised to the corresponding purified bait protein (WT or mutant Red1) amount. D Co-Immunoprecipitation (co-IP) of HA-tagged Pla1 (Pla1-HA) using Red1 as bait in WT and Red1 mutant strains. Inputs and final eluates of the tandem affinity purifications of the indicated strains were blotted to nitrocellulose membrane and cut around the 100 kDa marker. The bottom part of the membrane was probed with α-HA to detect co-purifying Pla1-HA (top panel) and the top part was probed with α-Flag to detect the bait protein Red1-FTP (bottom panel). Note that the Red1 bait protein in the Flag eluate runs ~20 kDa lower than in the input, due to the cleavage of the C-terminal Protein-A tag by the TEV enzyme during the elution step from the IgG column (see details in Methods section). A representative membrane from two independent experiments is shown. Source data are provided in Source Data file.
