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. 2010 Apr 19;5(4):e10194. doi: 10.1371/journal.pone.0010194

Figure 5. Analyses of the protein composition of SSU precursors containing rpS5-ΔC.

Figure 5

(A–C) Cells were grown overnight in selective media, diluted in YP-galactose (YPG) and expression of GAL-RPS5 was shut down for 2 hours (3 hours in C) in YP-glucose (YPD) medium. (A) FLAG-rpS5-ΔC containing SSU precursors were affinity purified from yeast strain ToY323, transformed with vector ToP1101. Protein content of affinity purified fractions was analyzed by mass spectrometry as indicated in Materials and Methods. The numbers of identified peptides of the indicated proteins together with the total ion scores are indicated. These identified proteins are all major non-ribosomal components of yeast late 40S precursors. In addition to that, one peptide of a non-ribosomal component specific to 60S precursor particles was identified. (B) Semi-quantitative comparison of Rio2-TAP associated SSU precursors. Yeast strain ToY1739, in which RPS5 is ectopically expressed under control of a galactose inducible GAL1 promoter and in which Rio2p is expressed as TAP-tag fusion protein, was transformed with vector ToP1162 or vector ToP1156, coding for HA-tagged full length rpS5 or rpS5-ΔC under the control of a constitutive promoter. Rio2-TAP associated SSU precursors were affinity purified from both transformants and their protein composition was compared in a semi-quantitative way by mass spectrometry as described in Materials and Methods. The mean values and standard deviations of three independent biological replicates are shown. The number of analysed tryptic peptides for each protein is given in brackets after the protein name. (C) RNA co-immunoprecipitation of TAP-tagged NOB1. Yeast strain ToY1765, in which RPS5 is ectopically expressed under control of a galactose inducible GAL1 promoter and in which Nob1p is expressed as TAP-TAG fusion protein, was transformed with an empty vector (YEplac181), vector ToP1162 or vector ToP1156, coding for HA-tagged full length rpS5 or rpS5-ΔC under the control of a constitutive promoter. Nob1p-TAP was affinity purified and SSU pre-rRNA, contained in input (In) and immuno-purified (IP) fractions was analyzed by Northern blotting as indicated in Materials and Methods.