Figure 1.

The ribosome biogenesis factors Noc1p, Noc2p and Rrp5p interact in vivo. (A) Noc1p-TAP efficiently co-purifies Noc2p and Rrp5p from yeast extracts. Noc1-TAP was isolated from extracts of strain Y3572 applying the tandem affinity purification method under increasing concentration of NaCl. The composition of the Noc1p containing particles was analysed on a 4–12% gradient SDS–PAGE gel, stained with Coomassie Blue. Prominent co-purified proteins are indicated. (B) Overview of the rrp5 alleles analysed in this study and schematic presentation of the corresponding protein variants. Black bars illustrate the S1 RNA binding motifs, grey bars the tetratricopeptide repeats, respectively [adapted from (20); see ‘Introduction’ section]. Point mutations in the temperature sensitive rrp5-11 allele are indicated by asterisk. (C) Genetic interaction studies show synthetic lethal effects between rrp5 alleles with deletions of N-terminal S1-repeats and noc1-ts or noc2-ts mutants. The respective double shuffle strains (Y3668, Y4207 and Y3715) were transformed with plasmids carrying the indicated wild-type and mutant alleles of ProtA-RRP5, ProtA-NOC1, NOC2 or ProtA-NOC3 (Supplementary Figure S4). Transformants were spotted in a 10-fold dilution series on SDC + FOA plates and incubated at the indicated temperature and for the indicated time to analyse genetic interactions.