The C-terminal WD-40 domain of Ytm1 (i.e., Ytm1-C) interacts directly with Erb1. (A) Schematic representation of Ytm1 and truncation constructs. Shown are the notchless-like element (NLE, white rectangle), and WD40 repeats 1–7 (hatched boxes). Black triangles (▾) indicate two mutations: G398D and S442N. (B) Left, 40% of the input [³⁵S] labeled proteins in each assay and 100% of the pulled down GST proteins (Coomassie blue staining) are shown. Right, synthetic ³⁵S-methionine–labeled full-length Ytm1, Ytm1-N, or Ytm1-C proteins (*) were incubated with purified GST-Erb1 protein. Complexes were eluted from glutathione beads, subjected to SDS-PAGE, and detected by autoradiography.

Expression of Ytm1-C causes dominant negative effects on growth and ribosome biogenesis. (A) Wild-type cells or yeast expressing endogenous Ytm1 plus full-length or truncated Ytm1 proteins were grown to early log phase, serially diluted (10-, 100-, 1000 and 10,000-fold) onto Glu (glucose) or Gal (galactose) medium, and incubated at 30°C. (B) Wild-type cells or yeast overexpressing Ytm1-C or co-overexpressing Ytm1-C and full-length Ytm1 were grown to early log phase and serially diluted as above. (C) Whole cell extracts were subjected to centrifugation on sucrose gradients to separate 40S and 60S ribosomal subunits, 80S ribosomes, and polyribosomes. (D) Pre-rRNA processing pathway in the yeast S. cerevisiae. (E) RNA was extracted from wild-type yeast (WT) or from strains in which Ytm1 was depleted (GAL-YTM1) or Ytm1-C was overexpressed (YTM1-C). Pre-rRNAs were assayed by primer extension (left) or by Northern blotting (right), using specific oligonucleotide primers or probes, respectively.

Expression of Erb1-C2 causes dominant negative effects on growth and ribosome biogenesis. (A) Wild-type cells or yeast expressing endogenous Erb1 plus full-length or truncated Erb1 proteins were grown to early log phase and serially diluted as in Figure 4A. (B) Extracts from wild-type cells (WT) or from cells overexpressing the indicated forms of Erb1 were subjected to centrifugation on sucrose gradients to separate 40S and 60S ribosomal subunits, 80S ribosomes, and polyribosomes. (C) RNA was extracted from wild-type yeast (WT) or from strains in which Erb1 was depleted (GAL-ERB1) or Erb1-C2 was overexpressed (ERB1-C2). Pre-rRNAs were assayed by primer extension (left) or Northern blotting (right), using specific oligonucleotide primers or probes, respectively.

Interaction domains of Nop7. (A) Schematic representation of Nop7 and its truncation constructs. Shown are the N-terminal pescadillo domain of Nop7 (white rectangle), BRCT domain (dotted box), and two coil-coiled domains (gray boxes). (B) Pre-rRNPs were purified from cells expressing endogenous Nop7 plus HA-tagged full-length Nop7 or each Nop7 truncation or from wild-type cells only expressing endogenous Nop7 (WT) as in Figure 3. Proteins present in the affinity-purified preribosomes were resolved by SDS-PAGE and stained with silver (top), or subjected to Western blot analysis (bottom) using antibodies against the HA epitope, or ribosomal protein rpL5. (C) TAP-tagged Nop7-N, Nop7-C, Nop7-M, or full-length Nop7 was expressed from the GAL promoter by the addition of galactose to a final concentration of 1% for 4 h. Cell extracts from these strains were prepared and subjected to a high-speed spin (42K) for 3 h. Proteins associated with each tagged truncation were affinity-purified from the supernatants, resolved by SDS-PAGE, and stained with silver (top), or subjected to Western blot analysis using antibodies against the Myc tag on Ytm1 or Erb1, or the protein A tag on Nop7-TAP (bottom). The black arrow in each panel indicates the corresponding TAP-tagged Nop7 truncation. The asterisk (*) indicates Ste23, a protein not necessary for ribosome biogenesis (L. Tang, unpublished data). (D) Wild-type cells or yeast expressing full-length or truncated Nop7 proteins were grown to early log phase and serially diluted as in Figure 4A. (E) Cell extracts were subjected to centrifugation on sucrose gradients to separate 40S and 60S ribosomal subunits, 80S ribosomes, and polyribosomes. (F) RNA was extracted from wild-type yeast (WT) or from strains in which Nop7 was depleted (GAL-NOP7) or Nop7-M was overexpressed (NOP7-M). Pre-rRNAs were assayed by primer extension (top) or Northern blotting (bottom), using specific oligonucleotide primers or probes, respectively.

Interdependence of assembly of Erb1, Nop7, and Ytm1 into preribosomes. Yeast strains containing GAL-NOP7, GAL-ERB1, or GAL-YTM1 were grown at 30°C in galactose medium to 3·10⁷ cells/ml. A second culture of each strain was grown in galactose medium and shifted to glucose medium for 16 h to 3 × 10⁷ cells/ml, to deplete the respective proteins. Preribosomes were isolated from whole cell extracts by affinity purification, using Rpf2-TAP. (A) Proteins in whole cell extracts were assayed by Western blotting. (B) Proteins in affinity-purified preribosomes were resolved by SDS-PAGE and stained with silver (top) or subjected to Western blot analysis (bottom). Positions of stained Erb1, Nop7, and Ytm1 are indicated.

Ytm1-C assembles into preribosomes and the Nop7-subcomplex in vivo. Yeast strains containing each of the GAL-inducible full-length or truncated YTM1 genes were grown at 30°C in synthetic medium containing 1% raffinose to ∼1.5·10⁷ cells/ml. Galactose was added to each strain to a final concentration of 1% to induce the expression of Ytm1 proteins. Cells were harvested after 4-h induction (one doubling time). (A) Lysates were prepared from cells expressing endogenous Ytm1 plus HA-tagged full-length Ytm1, Ytm1-N, Ytm1-C, or Ytm1-1-C, or from cells expressing only endogenous Ytm1 (WT). Pre-rRNPs were purified using TAP-tagged Rpf2. Proteins present in the affinity-purified preribosomes were resolved by SDS-PAGE and stained with silver (top) or subjected to Western blot analysis (bottom) using anti-HA or anti-rpL5 antiserum. (B) Left, preribosomes purified from wild-type cells using TAP-tagged Ytm1 and purified from cells using TAP-tagged Ytm1-C. Right, preribosomes purified from cells expressing TAP-tagged Ytm1-N. Proteins in affinity-purified samples were resolved by SDS-PAGE and stained with silver. Protein bands in the Ytm1-C-TAP purified samples were excised and subjected to mass spectrometry to identify Erb1, Nop7, and Ytm1-C-TAP. The band indicated by an asterisk is Ste23, a protein not necessary for ribosome biogenesis (L. Tang, unpublished data).

The N-terminal conserved region of Erb1 interacts with both Nop7 and Ytm1 and assembles into preribosomes. (A) Schematic representation of full-length and truncated Erb1 constructs. Shown are the N-terminal conserved region of Erb1 (white rectangle) and the WD40 repeats 1–7 (hatched boxes). (B) Lysates were prepared from cells expressing endogenous Erb1 plus HA-tagged full-length Erb1 or each Erb1 truncation or from wild-type cells only expressing endogenous Erb1 (WT), as in Figure 3. Pre-rRNPs were purified using TAP-tagged Rpf2. Proteins present in the affinity-purified preribosomes were resolved by SDS-PAGE and stained with silver (top) or subjected to Western blot analysis (bottom) using antibodies against the HA tag, or ribosomal protein L5. (C) TAP-tagged Erb1-N, Erb1-C1, Erb1-C2, or Erb1-M was expressed using the GAL promoter by the addition of galactose to a final concentration of 1% for 4 h. Proteins associated with each tagged, truncated protein were affinity-purified, resolved by SDS-PAGE, and stained with silver (top) or subjected to Western blot analysis using antibodies against Nop7, the Myc tag on Ytm1, or the HA tag on the Erb1 proteins (bottom). The black arrow (→) in each panel indicates the corresponding TAP-tagged Erb1 truncation.

The C-terminal WD40 domain of Erb1 is not essential for growth or ribosome assembly. (A) Empty vector or a plasmid containing each ERB1 truncation driven by the ERB1 promoter was introduced into the GAL-ERB1 strain. Cells were grown to early log phase in galactose medium, serially diluted (10-, 100-, 1000-, and 10,000-fold) onto Glu (glucose) medium, to turn off the wild-type GAL-ERB1 gene, or Gal (galactose) medium, to express GAL-ERB1, and incubated at 30°C. (B) Erb1-N was expressed from its own promoter in a GAL-ERB1 strain. This GAL-ERB1 ERB1-N strain was grown at 30°C in galactose medium to 3 ×10⁷ cells/ml. A second culture was grown in galactose medium and shifted to glucose medium for 16 h to 3 ×10⁷ cells/ml, to deplete wild-type Erb1 protein. Whole cell extracts were subjected to centrifugation on sucrose gradients to separate 40S and 60S ribosomal subunits, 80S ribosomes, and polyribosomes. (C) RNA was extracted from the GAL-ERB1 ERB1-N strain grown in galactose, or shifted to glucose for 16 h. The 27SA₂, 27SA₃, 27SB_L, and 27SB_S pre-rRNAs present in each strain were identified by primer extension.

(A) Interaction domains within the Nop7-subcomplex. Light pink or light green boxes in each protein indicate the smallest region shown to interact with their full-length ligands. (B) Architecture of the Nop7-subcomplex.