Protein composition of the Spb4-TAP and Spb4(R360A)-TAP complexes. Strain YDK37-1B/ pTAPC111-SPB4, which expresses a TAP-tagged Spb4 fusion protein as a sole source of Spb4, was grown in YPD at 30°C to an OD₆₀₀ of around 0.8. Strain W3031-A/pAS24-NTAP/2xFLAG-DNSPB4, which expresses the dominant-negative Spb4(R360A)-TAP protein under the control of a GAL promoter, was grown at 30°C in SD-Leu, transferred to SGly/Lac-Leu, and then shifted to SGal-Leu for 24 h. Spb4-TAP and Spb4(R360A)-TAP were affinity purified by the TAP procedure, and the final EGTA eluate was separated on a 4 to 12% SDS-polyacrylamide gradient gel and subjected to Coomassie blue staining. The wild-type W303-1A strain (Spb4) served as a negative control. The indicated proteins and those described in Tables S1 to S4 in the supplemental material were identified by mass spectrometry. Std, molecular mass protein standards. Despite the fact that the figure is divided into two pieces, all samples were run in the same gel. Note that TAP-tagged Spb4(R360A) carries two consecutive N-terminal Flag epitopes that are not present in the TAP-tagged wild-type Spb4 construct.

Wild-type Spb4 associates with pre-60S ribosomal particles, and the dominant-negative Spb4(R360A) protein stalls in 90S preribosomal particles. (A) The indicated GFP-tagged bait proteins were affinity purified by using the GFP-Trap_A procedure (see Materials and Methods) from cells expressing HA-Spb4 under control of its cognate promoter. (B and C) In addition, we also used cells expressing under control of the GAL promoter the wild-type HA-Spb4 (B) or the dominant-negative HA-Spb4(R360A) (C) protein. Cells were grown for 12 h in galactose-containing medium. Equivalent amounts of the corresponding purified complexes were separated on 4 to 12% gradient SDS-polyacrylamide gels and subjected to Western blot analysis using specific antibodies against the HA epitope and the Has1, Mrt4, and L1 proteins. The cross-reacting band (marked with an asterisk) in the panel for Has1 most likely corresponds to nonstripped HA-Spb4 signal.

Expression of the dominant-negative Spb4(R360A)-TAP protein inhibits pre-rRNA processing. YDK37-1A cells harboring YCplac111-SPB4 (SPB4) were grown at 30°C in SD-Leu medium or transferred to SGly/Lac-Leu and then shifted to SGal-Leu (Gal) for 24 h. W303-1A cells harboring either the pAS24-NTAP/2xFLAG-SPB4 (GAL::SPB4-TAP) or the pAS24-NTAP/2xFLAG-DNSPB4 [GAL::SPB4(R360A)-TAP] plasmid were also grown at 30°C in SD-Leu (Glc) or transferred to SGly/Lac-Leu and then shifted to SGal-Leu (Gal) for 24 h. Cells were harvested at the indicated times. Total RNA was isolated and analyzed by Northern blotting. Probes (in parentheses) are described in Fig. S1A and Table S7 in the supplemental material. Signal intensities were measured by phosphorimager scanning. Values, indicated below each lane, were normalized to those obtained for each strain in SD-Leu (Glc), which were arbitrarily set at 1.0.

The Spb4-TAP fusion protein is fully functional. (A) The W303-1A strain harboring the empty YCplac111 vector (wild type) and the YDK37-1B strain containing the YCplac111-SPB4 (SPB4) or pTAPC111-SPB4 (SPB4-TAP) plasmid were grown in liquid SD-Leu medium. Ten-fold serial dilutions were spotted on SD-Leu plates and incubated at 30°C for 2 days. (B) The SPB4 and SPB4-TAP strains were grown in SD-Leu medium at 30°C and harvested at an OD₆₀₀ of 0.8. Cell extracts were prepared, and 10 A₂₆₀ units of each extract was centrifuged in 7% to 50% sucrose gradients. The A₂₅₄ was read continuously. Sedimentation is from left to right. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes, and polysomes are indicated. (C) Total RNA was isolated from cell extracts of the strains described above and analyzed by Northern blotting. Probes (in parentheses) are described in Fig. S1A and Table S7 in the supplemental material. Signal intensities were measured by phosphorimager scanning. Values, indicated below each lane, were normalized to those obtained for the wild-type control, which were arbitrarily set at 1.0.

Wild-type Spb4 is associated mainly with early pre-60S ribosomal particles, but dominant-negative Spb4(R360A) also coenriches 90S preribosomal particles. (A) Immunoprecipitation experiments were carried out using IgG-Sepharose and whole-cell extracts from the YDK37-1B strain harboring the pTAPC111-SPB4 plasmid, which expressed Spb4-TAP (Spb4-TAP) from its cognate promoter (SPB4 pr). RNA was extracted from the pellets obtained after purification (lanes IP) or from an amount of total extract corresponding to 1/30 of that used for the purification (lanes T). Pre-rRNAs and mature rRNAs were analyzed by Northern blotting. Strain YDK37-1B harboring the YCplac111-SPB4 plasmid, which expressed nontagged Spb4 (Spb4) from its cognate promoter, was used as a negative control. (B) Immunoprecipitation experiments were carried out as described above on whole-cell extracts from the wild-type W303-1A strain harboring either the pAS24-NTAP/2xFLAG-SPB4 (Spb4-TAP) or the pAS24-NTAP/2xFLAG-DNSPB4 [Spb4(R360A)-TAP] plasmid, which expressed the wild-type or the DN form of Spb4-TAP, respectively, from the GAL promoter (GAL pr). Cells were grown in SGal-Leu for 24 h. RNA was extracted from whole-cell extracts (lanes T) and affinity-purified samples (IP) and subjected to Northern analysis as described above. Probes (in parentheses) are described in Fig. S1A and Table S7 in the supplemental material. For panels A and B, signal intensities were measured by phosphorimager scanning to derive the percentage of input RNA precipitated together with tagged Spb4 proteins (values are indicated below each IP lane).

Expression of the Spb4(R360A)-TAP protein leads to a dominant-negative growth phenotype and a deficit in 40S ribosomal subunits. (A) The W303-1A strain was transformed with plasmid YCplac111 (wild type), pAS24-NTAP/2xFLAG-SPB4 (GAL::SPB4-TAP), or pAS24-NTAP/2xFLAG-DNSPB4 [GAL::SPB4(R360A)-TAP]. Transformants were grown in liquid SD-Leu and spotted in 10-fold serial dilutions on SD-Leu (glucose) or SGal-Leu (galactose). SD-Leu plates were incubated for 2 days at 30°C and SGal-Leu plates for 3 days at 30°C. (B) The GAL::SPB4-TAP and GAL::SPB4(R360A)-TAP strains were grown at 30°C in liquid SD-Leu medium (Glc) or transferred to SGly/Lac-Leu and then shifted to SGal-Leu (Gal) for 12 or 24 h. Total protein extracts were prepared from each sample. Equivalent amounts of extracts were subjected to SDS-PAGE and Western blot analysis with PAP and anti-L1 antibodies. (C) Polysome profiles from the GAL::SPB4-TAP and GAL::SPB4(R360A)-TAP strains. Cells were grown at 30°C in SD-Leu medium (glucose) or shifted to SGal-Leu (galactose) for 12 h. Cell cultures were maintained at an OD₆₀₀ of around 0.5 to 0.8. Cells were harvested, and extracts were prepared. Ten A₂₆₀ units of each extract was centrifuged in 7% to 50% sucrose gradients. The A₂₅₄ was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S ribosomal subunits, 80S free couples/monosomes, and polysomes are indicated.