The ytm1-1 mutant contains mutations in two of the WD40 repeat motifs of Ytm1.
Ytm1 is an essential 51-kDa yeast nucleolar protein that is highly conserved from fungi to humans. Seven WD repeats are clustered at the C terminus of the protein (Fig. ). WD repeat-containing proteins are predicted to share a common circularized beta-propeller structure (Fig. ) based on the crystal structure of the Gβ subunit of heterotrimeric G proteins (16
). A paramount feature of WD repeat proteins is their ability to participate in multiple interactions either simultaneously or sequentially. The WD repeat motif is not known to have any enzymatic activity. Therefore, WD repeat proteins acting in disparate pathways may share a common function of establishing and regulating interactions within multiprotein complexes.
FIG. 2. Ytm1 is a WD40 repeat-containing protein. (A) Predicted amino acid sequence of S. cerevisiae Ytm1. WD40 repeats are overlined. Amino acid residues altered in the ytm1-1 mutant are indicated by asterisks. (B) Ras Mol 2.6 was used to generate the top and (more ...)
To investigate the importance of WD40 motifs in Ytm1, we used the ytm1
temperature-sensitive mutant that contains two mutations, G398D and S442N, in WD40 repeats 6 and 7 of Ytm1 (Fig. ). This mutant strain was generated by random mutagenesis of cloned YTM1
and replacement of chromosomal YTM1
with the mutant allele. The counterpart of Ytm1 residue G398 in the Gβ protein contacts phosducin (54
). Thus, residues at this position may be important for mediating interactions with associated proteins. Within the conserved WD40 repeat consensus sequence is the structural tetrad, which contributes to local and global stability through inter- and intrablade hydrogen bonding (40
). The G398D substitution occurs in the position adjacent to the highly conserved aspartate residue in the structural tetrad. Similarly, the S442N substitution occurs in a residue that is next to the serine/threonine residue of the structural tetrad. Thus, the ability of Ytm1 to bind to ligands may be compromised in the ytm1
mutant, which may result in defects in ribosome assembly.
The ytm1-1 mutant was unable to grow at 37°C and grew slower than the wild-type control strain at all temperatures below 37°C (data not shown). The G398D and S442N mutations might inactivate Ytm1 at 37°C, perhaps by distorting the structure of Ytm1 or by disrupting interactions with ligands of Ytm1. The ytm1-1 mutant protein is relatively stable: amounts of Ytm1-1 protein did not change drastically when compared to those of wild-type Ytm1 in strains grown at 25°C or shifted from 25°C to 37°C for 5 h (data not shown).
The ytm1-1 mutant is deficient in 60S ribosomal subunits.
To determine the effect of inactivation of Ytm1 on ribosome biogenesis, we assayed levels of ribosomal subunits, monoribosomes, and polyribosomes in ytm1
cells grown at 25°C or shifted from 25°C to 37°C for 3 h. Upon shifting the ytm1
mutant cells to 37°C, amounts of 60S ribosomal subunits and 80S monosomes were greatly reduced and half-mer polyribosomes were apparent. At 25°C, ytm1
mutant cells contained fewer free 60S subunits and 80S monoribosomes and accumulated half-mer polyribosomes, compared to wild-type YTM1
cells (Fig. ). This suggests that the function of Ytm1-1 is compromised even at the permissive temperature and is consistent with the slow-growth phenotype at 25°C. These findings are consistent with previous results obtained when Ytm1 is metabolically depleted (21
FIG. 3. The ytm1-1 mutant is deficient in 60S ribosomal subunits. Free 40S and 60S ribosomal subunits, monoribosomes, and polyribosomes were assayed in yeast strains JWY3400 (YTM1) (left) or JWY7128 (ytm1-1) (center) grown at 25°C or JWY7128 grown at (more ...) Pre-rRNA processing is slowed when Ytm1 is inactivated or depleted.
The kinetics of pre-rRNA processing were analyzed by pulse-labeling YTM1
wild-type and ytm1
mutant cells, grown at 25°C, and shifted to 37°C for 5 h, as well as the GAL-YTM1
cells shifted from galactose- to glucose-containing medium, in which Ytm1 is depleted. Processing of 35S pre-rRNA to mature 25S and 18S rRNAs occurred rapidly in the YTM1
strain. The 27S pre-rRNA processing intermediate was completely converted to 25S rRNA by the 60-min chase point. In the ytm1
mutant, however, 27S pre-rRNA was still present at the 60-min chase point; 27SA pre-rRNAs were converted to 27SB pre-rRNA less efficiently than in wild-type cells (Fig. ). Consequently, 3.6-fold less mature 25S rRNA was produced relative to 18S rRNA in the ytm1
mutant than in the wild-type cells (Fig. , compare lanes 5 and 10). Both 35S pre-rRNA and 23S pre-rRNA accumulated in the ytm1
mutant. The effects on 35S and 23S are thought to be indirect and often are observed when 60S ribosomal subunit assembly is perturbed (4
). Effects on pre-rRNA processing observed in Ytm1-depleted cells are identical to those found in the ytm1
mutant (data not shown).
FIG. 4. Processing of pre-rRNAs is altered in the ytm1-1 mutant. (A) Oligonucleotide probes or primers used to detect rRNAs and pre-rRNAs. (B) Yeast strains JWY3400 (YTM1) and JWY7128 (ytm1-1) were grown in YEPD medium at 25°C and shifted to 37°C (more ...)
We analyzed the steady-state levels of rRNA intermediates and mature rRNAs by primer extension and Northern blotting (Fig. ). Ytm1 was depleted from GAL-YTM1 cells by shifting galactose-grown cells to glucose for 0 h, 10 h, 12 h, 15 h, or 18 h. The ytm1-1 cells were grown at 25°C and shifted to 37°C for 0 h, 3 h, or 6 h. Primer extension using an oligonucleotide that detects 27S pre-rRNAs (Fig. ) indicated that under nonpermissive conditions amounts of 27SA2 pre-rRNA decrease in ytm1-1 cells but increase in GAL-YTM1 cells (Fig. ). 27SA3 pre-rRNA strongly accumulated upon shifting GAL-YTM1 and ytm1-1 cells to the nonpermissive conditions (Fig. ). Amounts of 27SB pre-rRNAs were also affected: 27SBS pre-rRNA was drastically diminished relative to 27SBL pre-rRNA, making these species nearly equal in Ytm1-depleted or Ytm1-inactivated cells (Fig. ).
Phosphorimage analysis of Northern blots also indicated that 7S pre-rRNA and 5S rRNA decrease slightly upon shifting GAL-YTM1 and ytm1-1 cells to nonpermissive conditions (Fig. ). Despite the observed effects on processing of early and intermediate pre-rRNAs present in 66S pre-rRNPs, amounts of 25S and 5.8S rRNA were largely unaffected. Because preexisting 25S and 5.8S rRNAs are present in vast quantities, changes in steady-state amounts of 25S and 5.8S rRNAs may be masked and therefore difficult to observe by standard assays.
Taken together, these results indicate that in ytm1 mutants pre-rRNA processing delays begin early, at the step when 27SA2 pre-rRNA is converted to 27SA3 pre-rRNA. Subsequent steps in pre-rRNA processing are similarly slowed down, but no step in pre-rRNA processing is completely blocked. Mutation of Ytm1 through depletion or inactivation results in nearly identical phenotypes, suggesting that Ytm1-1 protein is largely inactive.
Ytm1 is necessary for release of 66S preribosomes from the nucleolus.
To further investigate the timing and role of Ytm1 in ribosome biogenesis, we assayed the ability of 60S preribosomes to exit the nucleolus and nucleus in the ytm1
mutant, using the ribosome export assay (28
), in which eGFP-tagged rpL25 functions as a reporter. In ytm1
cells grown at the permissive temperature, 66S preribosomes were released to the cytoplasm (Fig. ). In wild-type cells grown at 25°C or shifted from 25°C to 37°C, rpL25eGFP signal was cytoplasmic (data not shown). When the ytm1
mutant strain was grown at 25°C and shifted to 37°C for 5 h, rpL25eGFP was strongly retained in the nucleolus in most cells (Fig. , arrows), although in some cells signal was distributed throughout the nucleoplasm. Thus, Ytm1 is important for nucleolar release of 66S preribosomes and perhaps for subsequent nuclear export.
FIG. 5. Inactivation of Ytm1 in the ytm1-1 mutant causes 66S preribosomes to accumulate in the nucleolus. The ytm1-1 mutant strain JWY6790 expressing eGFP-tagged rpL25 was grown in C-Trp medium at 25°C, washed and suspended in YEPD, and grown at 25°C (more ...) Ytm1 is a component of 66S preribosomes.
Previously Ytm1 was identified in 66S pre-rRNPs purified using TAP-tagged ribosome assembly factors (3
). Consistent with this result, HA3-tagged Ytm1 peaks in sucrose gradient fractions 15 to 17 containing 66S preribosomes (21
) (Fig. ). A small amount of Ytm1 can be detected sedimenting at the top of the gradient in lighter fractions and larger amounts at the bottom of the gradient in heavier fractions. The significance of the sedimentation in heavier fractions is unclear.
FIG. 6. Ytm1-HA3 cosediments on sucrose gradients with 66S preribosomes. Whole-cell extracts were prepared from yeast strain JWY6770 (YTM1-HA3) and fractionated on 7 to 47% sucrose velocity gradients. Fractions containing 40S and 60S ribosomal subunits and 80S (more ...)
To examine in more detail ribosome assembly intermediates containing Ytm1, we identified the pre-rRNAs and proteins associated with affinity-purified Ytm1-TAP. TAP-tagged Ytm1 is fully functional: the tagged strain grows at wild-type rates and has a wild-type polysome profile, and Ytm1-TAP sediments on sucrose gradients with a peak at 66S (data not shown).
To determine in which preribosomes Ytm1 is present, we assayed which pre-rRNAs copurify with TAP-tagged Ytm1. The amounts of 27SA2, 27SA3, 27SB, 25.5S, and 7S pre-rRNAs recovered relative to each other were similar to those found in whole cells. Smaller relative amounts of 5.8S rRNA and no 35S or 20S pre-rRNA or 18S rRNA copurified with Ytm1-TAP (Fig. ). Enrichment of Ytm1 with these RNA molecules is consistent with our finding that Ytm1 is important for assembly of 60S ribosomal subunits and for processing of 27S pre-rRNA (Fig. and ).
FIG. 7. Ytm1 associates with pre-rRNAs in 66S preribosomes. (A) Whole-cell extracts were prepared from the YTM1-TAP strain JWY7124 and from untagged strain JWY3400 grown at 30°C in YEPD medium to 6 × 107 cells/ml. RNA was extracted from whole (more ...)
Fifty-three different proteins that copurify with TAP-tagged Ytm1 were identified by SDS-PAGE and mass spectrometry (Fig. ). Among these are ribosomal proteins from both the large and small ribosomal subunits. Since these ribosomal proteins frequently contaminate TAPs (3
), their significance cannot be assessed. Seventeen nonribosomal proteins specifically required for biogenesis of 60S ribosomal subunits are present in the Ytm1-containing particles (Fig. and Table ). Copurification of these pre-rRNAs and proteins with TAP-tagged Ytm1 indicates that Ytm1 first enters the ribosome assembly pathway by associating with 66S preribosomes and remains stably associated with 66S pre-rRNPs until late stages of maturation in the nucleoplasm.
FIG. 8. Nonribosomal proteins necessary for biogenesis of 60S ribosomal subunits, as well as ribosomal proteins, copurify with TAP-tagged Ytm1. Whole-cell extract was prepared from the YTM1-TAP strain JWY7124 grown at 30°C in YEPD medium to 6 × (more ...)
Nonribosomal proteins that copurify with Ytm1-TAP
Ribosome assembly factors Ytm1, Nop7, and Erb1 are present together in a subcomplex.
Several different experiments demonstrate that Ytm1, Erb1, and Nop7 form aheterotrimeric subcomplex that is present within 66S preribosomes and also exists independently of pre-rRNPs. (i) Affinity purification from whole-cell extracts using TAP-tagged Ytm1, Nop7, or Erb1 yielded 50 to 60 proteins present in 66S pre-rRNPs (Fig. ) (21
; data not shown). In each case, greater amounts of Ytm1, Nop7, and Erb1 (Fig. , bands 33, 37, and 40, respectively) were recovered than those of any of the other proteins. This suggests that a mixture of 66S pre-rRNPs and a heterotrimer of Ytm1, Nop7, and Erb1 copurify with each of these TAP-tagged proteins (Fig. and see Fig. ). (ii) Affinity purification using TAP-tagged Nop7 or Ytm1 from rrp1
mutants in which 66S pre-rRNPs are unstable (21
) yielded mostly Ytm1, Nop7, and Erb1, and greatly diminished amounts of molecules comprising 66S pre-rRNPs (21
) (Fig. ). Thus, under these conditions, many fewer 66S pre-rRNPs were recovered, but the heterotrimeric subcomplexes remained intact. (iii) The Ytm1/Nop7/Erb1 heterotrimer could be separated from 66S pre-rRNPs by sucrose gradient fractionation or differential centrifugation of whole-cell extracts. Affinity purification from such enriched fractions using TAP-tagged Ytm1, Nop7, or Erb1 yielded primarily Ytm1, Nop7, and Erb1 (8
) (Fig. , lane 1, and C, lane 2). (iv) Treatment of whole-cell extracts with a cocktail of phosphatase inhibitors caused pre-rRNPs to be disrupted (P. Harnpicharnchai, unpublished), while the Nop7/Ytm1/Erb1 subcomplex remained intact. Nop7-TAP or Ytm1-TAP under these conditions resulted in the recovery of only the Ytm1/Nop7/Erb1 heterotrimer (Fig. , lane 2) (5
; data not shown). (v) Nop7-TAP from sucrose gradient fractions containing 66S preribosomes yielded most protein components of the 66S pre-rRNPs (Fig. , lane 1). However, when gradient fractions containing 66S pre-rRNPs were treated with the phosphatase inhibitor cocktail prior to affinity purification with Nop7-TAP, mostly the heterotrimer was recovered (Fig. , lane 2). The last result indicates that Ytm1, Erb1, and Nop7 form a stable complex within preribosomes sedimenting at 66S and can be released from the 66S pre-rRNPs by treatment with the phosphatase inhibitor cocktail. The molecular basis of the effect(s) of the phosphatase inhibitors is unknown.
FIG. 11. 66S preribosomes are largely intact but lack Ytm1 in the ytm1-1 mutant. Wild-type YTM1 cells and mutant ytm1-1 cells expressing Nop7-TAP or Brx1-TAP were grown in YEPD medium at 25°C and shifted to 37°C for 5 h. (A) Silver staining or (more ...)
FIG. 9. Ytm1, Erb1, and Nop7 form a heterotrimeric subcomplex both within 66S preribosomes and independently of these particles. (A) Ytm1, Erb1, and Nop7 are enriched (relative to other proteins found in 66S pre-rRNPs) among proteins copurifying with Ytm1-TAP (more ...)
Our current data suggest that the components of the Ytm1/Nop7/Erb1 heterotrimer join preribosomes separately since significant or small amounts of 35S pre-rRNA coprecipitate with Nop7 and Erb1, respectively, although no 35S pre-rRNA copurifies with Ytm1 (Fig. ). Thus, Nop7 likely joins nascent preribosomes first, followed by Erb1. Ytm1 later associates with 66S preribosomes containing 27SA2 pre-rRNA (Fig. ).
Ytm1 and Nop7 directly interact with Erb1.
To assay pairwise interactions between components of the heterotrimer and to determine whether the interactions are direct, we carried out GST pull-down assays. Ytm1 bound specifically to GST-Erb1, and Erb1 bound to GST-Ytm1, while Nop7 displayed strong binding to GST-Erb1 but not to GST-Ytm1 (Fig. ) (data not shown). Consistent with these observations, Pes1 and Bop1, the mammalian homologues of Nop7 and Erb1, bind to each other in vitro and interact in two-hybrid assays in vivo (34
). The interactions between Erb1 and Ytm1 were corroborated by two-hybrid assays in vivo. Cells expressing AD-YTM1
displayed strong expression of the GAL-HIS3
reporter gene (growth on 50 mM 3-aminotriazide) (data not shown). Thus, Ytm1 and Nop7 each bind directly to Erb1 but not to one another (Fig. ). Erb1, like Ytm1, contains WD40 repeats. Nop7 also contains a known protein-protein interaction motif, the BRCT domain (1
). Further analysis is necessary to test whether these or other domains dictate the strong interactions among these three proteins.
FIG. 10. Ytm1 and Nop7 directly interact with Erb1. (A) Synthetic radiolabeled proteins (*) were incubated with GST fusion proteins (lanes 2, 5, and 8). As negative controls, synthetic peptides were incubated with GST beads only (lanes 1, 4, and 7) or (more ...) Association of heterotrimer with 66S preribosomes is significantly weakened in the ytm1-1 mutant.
Since the two ytm1
mutations are in residues that may be important for interactions with ligands, we determined the effects of these mutations on the integrity of the heterotrimer and 66S preribosomes. We used TAP-tagged Nop7 or Brx1 to purify 66S preribosomes from YTM1
strains, since both of these proteins are present in all seven different 66S pre-rRNPs (21
) (Fig. ). SDS-PAGE, silver staining, and Western blotting revealed that most of the proteins present in wild-type 66S pre-rRNPs are also present in equivalent amounts in the particles isolated from the ytm1
mutant, indicating that the 66S preribosomes are largely intact in the ytm1
mutant (Fig. ). However, Ytm1-1 mutant protein was greatly diminished or absent from the pool of purified 66S preribosomes (Fig. , lanes 2 and 4, and B, lanes 2 and 4). Nop7 and Erb1 were still present, but in reduced amounts (Fig. , lanes 2 and 4). Thus some of the mutant preribosomes contain Nop7 and/or Erb1 but not Ytm1, and others lack all three proteins. The relative amounts of each pre-rRNA copurifying with TAP-tagged Nop7 or Brx1 in 66S pre-rRNPs parallel those in whole-cell extracts (data not shown).
The Ytm1/Nop7/Erb1 heterotrimer is destabilized in the ytm1-1 mutant.
The effects of the ytm1-1 mutations on 66S pre-rRNPs and pre-rRNA processing could result from alterations of the heterotrimer containing Ytm1. Therefore, we purified the heterotrimer from the ytm1-1 mutant and examined its integrity, using three assays: sucrose gradient centrifugation, differential centrifugation, and treatment of whole-cell extracts with phosphatase inhibitors. In each case, only small amounts, if any, of Erb1 copurified with Nop7 and no Ytm1 could be detected (Fig. , lane 2; C, lane 4; and D, lane 4). GST-pulldown assays confirmed that Ytm1-1 does not bind to Erb1 in vitro at 37°C (Fig. ). These results suggest that at the nonpermissive temperature, Ytm1-1 fails to interact with Erb1 and the Nop7-Erb1 association is significantly weakened, leading to destabilization of the heterotrimer and perturbations of preribosome maturation.