Supplementary Figure 5: In vitro and in vivo interaction of Yvh1 with the pre-60S nuclear export machinery.

(a) The different pre-60S ribosomal particles were affinity-purified using the indicated Flag-TEV-ProtA-tagged bait proteins (FTP), and SDS-PAGE (upper panel) and western blotting using the indicated antibodies (lower panel) was used to analyze the final Flag eluates. Asterisk (*) marks the position of the bait protein. Note that the western blot membrane probed with α-Mrt4 and α-Mex67 antibodies contained the marker lane (M), whereas the other western blot membranes had no marker between lane 4 and 5, and therefore were cut between lanes 4 and 5 for better figure arrangement. This figure corresponds to a related Figure 4a, but in this case the eluates were probed with additional antibodies (e.g. α-Nsa2, α-Nug2 and α-Nog1) (b) Quantification of the extent of recombinant Mex67-Mtr2 binding to the Yvh1 particle in the absence or presence of either Mrt4 or Rsa4. The Coomassie stained gel shown in Fig. 4b was scanned in LAS 4000 (GE). Using Image Quant TL software, the intensity of the Mex67 band (normalized with respect to the intensity of the corresponding Lsg1 band) was determined for each lane of the depicted gel (lower panel). The region of the Coomassie-stained gel used for image analysis (see Fig. 4b) is shown in a blow up (lower panel). Lsg1, Arx1 and Mex67 bands stained by Coomassie are labeled. The stars in lane 1, 4 and 7 mark the presence of Hsp70 proteins (as determined by mass spectrometry), which co-migrate with endogenous Mex67. (c) Efficient co-enrichment of Mex67 with Lsg1 pre-60S particles requires Yvh1. Lsg1-FTP was affinity-purified derived from either wild-type YVH1 or yvh1Δ strain and Flag eluates were analyzed by SDS-PAGE (upper panel) and western blotting using the indicated antibodies (lower panel). Two independent affinity-purifications of Lsg1-FTP from wild-type (WT) and yvh1∆ cells are shown.