Figure 1

Interaction between the R2TP complex and Nop58. (A) Protein components of the R2TP and box C/D snoRNP complexes are shown on top. On bottom are the Western blot analyses for the presence of R2TP proteins in FLAG-tagged Nop1, Snu13, Nop56, and Nop58 pulldown complexes from log phase cells. Cell lysates from same wet weight of log phase cells were used for each pulldown. (B) Western blot and silver stain analysis of Nop58-FLAG pulldown complex purified from nuclease-treated or untreated cell lysates. Nop56 and Snu13 were identified by mass spectrometry. (C) Western blot analysis of R2TP complex interaction with Nop58-FLAG complex purified from wildtype, pih1Δ, or tah1Δ background strains. (D) Schematic representations of Nop58 constructs generated for the in vitro binding assays are shown on top. The KKE/D charged region of Nop58 (residues 448–511) is shown in gray. In vitro binding assay for GST-Nop58 and Pih1 is shown on bottom. Ponceau S staining and Western blot analysis of GST pulldown assays using purified GST-Nop58 constructs incubated with Pih1 or Pih1(1–230). Stars indicate the location of the band of the GST-Nop58 construct being tested. (E) On the left are schematic representations of the Nop58-FLAG and GFP-FLAG constructs used for the pulldown assays from yeast cells. The KKE/D charged region (residues 448–511) of Nop58 is shown in gray. Western blot analysis of FLAG pulldown assays is shown on the right. Stars indicate the location of the band of Nop58-FLAG or GFP-FLAG constructs corresponding to the schematic on the left panel. The arrowhead shows the lane for Nop58(285–447)-FLAG construct which binds to R2TP but not snoRNP core proteins.