Yvh1 is required for ribosome biogenesis
A recent genome-wide protein complementation assay (PCA) in yeast linked Yvh1 to Tif6, Lsg1, and Rei1 (Tarassov et al., 2008
). These factors are all required for biogenesis of the 60S subunit, and Lsg1 and Rei1 are strictly cytoplasmic proteins (for reviews see Fromont-Racine et al., 2003
; Zemp and Kutay, 2007
; Johnson, 2009
). PCA monitors proximity (≤80 Å) but does not report direct physical interaction between protein pairs (Tarassov et al., 2008
). Based on this PCA analysis, we speculated that Yvh1 functions in maturation of the large subunit. The two-hybrid interaction between Yvh1 and the nucleolar 60S biogenesis factor Nop7 further supported a role in 60S biogenesis (Sakumoto et al., 2001
is not essential, but its deletion (yvh1Δ
) causes a strong defect in growth rate (Guan et al., 1992
). To determine whether Yvh1 acts in ribosome biogenesis, we fractionated extracts from yvh1Δ
and wild-type cells by sedimentation through sucrose density gradients. We observed that the level of 60S subunits was reduced in the yvh1Δ
mutant (), which was indicated by reduced free 60S levels and the appearance of halfmers, 43S preinitiation complexes that contain 40S but not 60S subunits. We examined the effect of yvh1Δ
on the well-established rRNA-processing pathway using Northern blotting. Compared with wild type, yvh1Δ
cells showed a modest accumulation of 35S, 27SA/B, and 23S rRNAs, reduced levels of 25S, and no significant changes in 20S and 18S rRNAs (Fig. S1
). The effects on 27S and 25S levels are consistent with Yvh1 being involved in 60S biogenesis. yvh1Δ
cells also showed reduced 60S export, monitored by the localization of Rpl25-GFP (Fig. S2
), as was recently reported (Liu and Chang, 2009
Figure 1. The dual-specificity phosphatase Yvh1 is a ribosome biogenesis factor, and its C-terminal domain is crucial for 60S interaction. (A) Extracts were prepared from BY4741 and yvh1Δ (AJY2976) cells and fractionated by sedimentation through 7–47% (more ...)
Yvh1 has a dual specificity phosphatase domain in its N terminus and a Zn2+
-binding domain at the C terminus (). Interestingly, only the C-terminal domain is necessary to complement the slow-growth defect of a yvh1Δ
mutant (Liu and Chang, 2009
). To dissect which functional domain of Yvh1 is required in 60S biogenesis, several Yvh1 mutants were constructed (). Consistent with previous data (Liu and Chang, 2009
), Yvh1 containing a mutation in the catalytic site of the phosphatase domain (yvh1-C117S) or deleted of the entire phosphatase domain (yvh1Δ
N) complemented the deletion mutant, whereas Yvh1 lacking the zinc-binding domain (yvh1Δ
C) did not (unpublished data). Polysome profiles were analyzed in these Yvh1 mutants. yvh1-C117S and yvh1Δ
N showed essentially wild-type profiles, whereas yvh1Δ
C was indistinguishable from the complete deletion mutant (Liu and Chang, 2009
; unpublished data). These results clearly link the growth defect of yvh1
mutants to a 60S biogenesis defect.
To determine whether this growth defect correlated with the ability of Yvh1 to bind to the 60S subunit, we separated ribosome-bound and free protein pools and analyzed these for the presence of Yvh1. The majority of wild-type Yvh1 and Yvh1-C117S cosedimented with ribosomes, whereas ~70% of Yvh1ΔN protein was in the free pool, indicating a partial loss of ribosome binding (). Yvh1ΔC was found entirely in the free pool, indicating that it completely lost 60S binding (). Together, these observations indicate that the function of Yvh1 requires its 60S subunit binding but not its phosphatase activity.
RPL12 is a high copy suppressor of yvh1Δ
To better understand the function of Yvh1, we screened for high copy suppressors of the yvh1Δ
growth defect. RPL12B
was identified from this screen as a modest growth suppressor (). High copy RPL12A
, which encodes a protein identical to Rpl12B, was also a weak suppressor (unpublished data). Because yvh1Δ
cells exhibit defects not only in 60S biogenesis but also in other pathways (Park et al., 1996
; Beeser and Cooper, 2000
; Sakumoto et al., 2001
; Hanaoka et al., 2005
; Liu and Chang, 2009
), we asked whether RPL12B
specifically suppressed the defect in synthesis of large subunits. Extracts from yvh1Δ
mutant cells with empty vector or high copy RPL12B
were analyzed on sucrose density gradients. Increasing the copy number of RPL12B
gave a modest improvement in polysomes but, surprisingly, reduced the levels of free 60S ().
Figure 2. RPL12B is a high copy suppressor of yvh1Δ. (A) Serial dilutions of AJY2976 (yvh1Δ) with vector or pAJ2458 (2µ RPL12B) were spotted onto URA dropout medium and incubated at 30°C for 2 d. (B) Extracts from AJY2976 (yvh1Δ (more ...)
Yvh1 is required to release Mrt4
Yeast Rpl12 corresponds to bacterial L11 that, together with L10, forms the base of the L7/L12 stalk (Gonzalo and Reboud, 2003
; Diaconu et al., 2005
). The binding of L11 and L10 to domain II of 23S rRNA is cooperative (Rosendahl and Douthwaite, 1995
). This interaction between bacterial L11 and L10 led us to consider whether high copy suppression of yvh1Δ
was the result of a defect in stalk assembly in the yvh1
mutant. The eukaryotic counterpart to bacterial L10 is P0, which is encoded by RPP0
in yeast. Thinking that P0 assembly may be defective, we first tested whether P0 is also a high copy suppressor of yvh1Δ
. However, overexpression of P0 did not suppress the growth defect of a yvh1Δ
mutant (unpublished data).
Mrt4 is a highly conserved protein in eukaryotes that is closely related to P0 in sequence. However, the two proteins show distinct compartmentalization; Mrt4 is present in the nucleolus and nucleoplasm, whereas P0 is a constituent of the mature ribosome in the cytoplasm. The similarity between the two proteins and the distinct localization suggests that Mrt4 acts as a biogenesis factor for the ribosome in the nucleolus and is replaced by P0 at a later stage in assembly. Sequence alignment of Mrt4, P0 and bacterial L10 (Fig. S2), and the atomic model of bacterial L7/L12 stalk (Kavran and Steitz, 2007
) indicate that the conserved N-terminal domain of P0 and Mrt4 is responsible for RNA binding. Additionally, Mrt4 and P0 bind to the 60S subunit in a mutually exclusive fashion (Rodriguez-Mateos et al., 2009
), further supporting the notion that the two proteins bind to the same site on the ribosome.
We considered that Yvh1 may be required for the release of Mrt4 from the subunit to allow P0 loading. This would predict that in the absence of Yvh1, Mrt4 would fail to be released and remain on subunits in the cytoplasm. To explore this possibility, we monitored the localization of Mrt4-GFP in wild-type versus yvh1Δ
cells. In wild-type cells, Mrt4 was nuclear and enriched in the nucleolus. However, in the yvh1Δ
strain, Mrt4 was nearly completely mislocalized to the cytoplasm (). However, we saw no effect on the localization of Nop7 (unpublished data), a nuclear 60S biogenesis factor reported to interact with Yvh1 (Sakumoto et al., 2001
). This implies that yvh1Δ
mutants specifically mislocalize Mrt4.
Figure 3. Mrt4 persists on cytoplasmic ribosomes in the absence of Yvh1. (A) The localization of genomic Mrt4-GFP was visualized in AJY3040 (MRT4-GFP) and AJY3048 (MRT4-GFP yvh1Δ). DNA was stained with Hoechst 33342. (B) Localization of Mrt4-GFP in various (more ...)
To test whether the 60S binding or the phosphatase activity of Yvh1 is required for Mrt4 release, we monitored the Mrt4 localization in the different yvh1
mutants. Although the localization of Mrt4 was restored in yvh1Δ
N and phosphatase mutants that complement the slow-growth phenotype of yvh1Δ
(Liu and Chang, 2009
; data not shown), Mrt4 remained in the cytoplasm in yvh1Δ
C cells (). This result strongly connects the growth phenotype of yvh1Δ
cells and Mrt4 mislocalization. The slow-growth defect of yvh1Δ
may arise from a failure to recycle Mrt4 to the nucleus to support pre-60S assembly, a failure to release Mrt4 and load P0, or both (see and Discussion).
Figure 4. The persistence of Mrt4 on subunits prevents P0 loading. (A) 10-fold serial dilutions of cultures of BY4741 (wild-type [WT]), AJY2976 (yvh1Δ), AJY2551 (mrt4Δ), and AJY2553 (yvh1Δ mrt4Δ) were spotted on a YPD plate and incubated (more ...)
The mislocalized Mrt4 could remain bound to the large subunit, indicating a failure in its release, or could be free in the cytoplasm, indicating a failure in its reimport to the nucleus. To distinguish between these possibilities, we analyzed the cosedimentation of Mrt4 with ribosomes in sucrose density gradients. Mrt4 cosedimented exclusively at the position of free 60S subunits in wild-type cells (), which is consistent with its role as a trans-acting factor of 60S subunits (note that these gradients do not resolve the different pre-60S and 60S species). The sedimentation pattern of Mrt4 was unaltered in a yvh1Δ mutant (), indicating that the Mrt4 observed in the cytoplasm in the absence of Yvh1 remains bound to free 60S subunits.
For further evidence that Mrt4 remained on subunits in the cytoplasm, we asked whether we could detect a shift in Mrt4 association from nuclear to cytoplasmic complexes. Rlp24, Nmd3, and Lsg1 are essential trans-acting factors in 60S ribosome biogenesis. Both Rlp24 and Nmd3 shuttle, but their steady-state distributions are primarily nuclear and cytoplasmic, respectively, whereas Lsg1 is restricted to the cytoplasm. As shown in , Mrt4 was depleted from the Rlp24 complex but accumulated in Nmd3, and Lsg1 immunoprecipitated complexes in a yvh1Δ mutant. Thus, in the absence of Yvh1, Mrt4 is not efficiently released from the subunit and mislocalizes to the cytoplasm.
If the persistence of Mrt4 on the subunit is responsible for the slow growth of yvh1Δ cells, elimination of Mrt4 may alleviate the problem. As seen in , the mrt4Δ and yvh1Δ single mutants were almost indistinguishable from the mrt4Δ yvh1Δ double mutant in growth rate, indicating an epistatic relationship between mrt4Δ and yvh1Δ. Deletion of MRT4 did not improve the growth defect of the yvh1Δ mutant, probably because deletion of MRT4 itself results in a growth defect comparable to deletion of YVH1. We also tested the effect of increasing the levels of Mrt4 in yvh1Δ cells, reasoning that overexpressing Mrt4 could restore the nuclear pool of Mrt4 to support 60S biogenesis. However, if Yvh1 were absolutely required for release of Mrt4, increasing Mrt4 levels would be expected to drive more Mrt4 onto nascent subunits. Without a mechanism for its release, this could be deleterious in a yvh1 mutant. In fact, overexpression of MRT4 strongly inhibited cell growth in yvh1Δ cells () but not wild type (not depicted).
The persistence of Mrt4 on subunits in the cytoplasm could prevent the assembly of P0 onto the subunit, accounting for the growth defect of yvh1Δ cells. We tested whether Mrt4 prevents P0 loading by immunoprecipitating Lsg1 particles from wild-type and yvh1Δ cells and comparing their relative P0 content to Rpl8 as a marker for 60S subunits. shows that the amount of P0 in the Lsg1-bound 60S complex was reduced in yvh1Δ cells compared with wild type. Overexpression of Mrt4 further reduced the level of P0 in these particles. Sucrose gradient analysis of yvh1Δ cells overexpressing Mrt4 showed a marked reduction in polysomes and a more dramatic imbalance in free subunits (), which is consistent with a defect in stalk assembly and utilization of 60S subunits. Therefore, Yvh1 plays a critical role in releasing Mrt4 to allow assembly of the ribosome stalk.
Mrt4-G68D bypasses the requirement for Yvh1
Mutant Mrt4 containing a glycine to aspartate change at position 68 (Mrt4-G68D) was reported to be a dominant suppressor of the yvh1Δ growth defect (Nugroho, S., N. Sakumoto, and S. Harashima. 2003. International Conference on Yeast Genetics and Molecular Biology. Abstr. 10-42), but a molecular understanding of the suppression was not known. We confirmed this suppression of yvh1Δ by Mrt4-G68D (). To determine whether Mrt4-G68D suppresses the 60S ribosome biogenesis defects of yvh1Δ, we analyzed the polysome profile of yvh1Δ cells expressing Mrt4-G68D as the sole copy of MRT4. Expression of Mrt4-G68D in yvh1Δ cells fully restored the polysome profile of yvh1Δ cells, reversing the free subunit imbalance and eliminating the halfmers evident in the yvh1 deletion mutant (). Similarly, Mrt4-G68D reversed the rRNA-processing defects seen in yvh1Δ cells (Fig. S1). Suppression of yvh1Δ by a point mutation in Mrt4 strongly argues that Mrt4 is the target of Yvh1. If Mrt4-G68D suppresses the defects of yvh1Δ cells, we would also expect Mrt4-G68D to localize to the nucleus in a yvh1Δ mutant. Indeed, Mrt4-G68D was predominantly nuclear in the absence of Yvh1 (). Thus, this mutant Mrt4 appears to bypass Yvh1 function while maintaining Mrt4 function.
Figure 5. Mrt4-G68D has reduced the affinity for 60S subunits and bypasses the need for Yvh1. (A) Serial dilutions of BY4741 and AJY2976 (yvh1Δ) with vector or pAJ2461 (MRT4-G68D) were spotted onto selective media and incubated at 30°C. (B) Polysome (more ...)
Because of the similarity in sequence between Mrt4, P0, and bacterial L10 (Fig. S3
), we modeled the Mrt4-G68D mutation into the L10 structure of the Haloarcula
50S subunit (Protein Data Bank accession no. 2QA4
; Kavran and Steitz, 2007
). Gly68 is in a region of Mrt4 that is highly conserved among Mrt4, P0, and bacterial L10 (Fig. S3), and flanking residues make direct contact with 23S RNA in the H. marismortui
structure (Diaconu et al., 2005
). In this model, Gly68 is at the Mrt4–25S rRNA interface located in a tight turn of the peptide backbone (). Introduction of Asp at this position would likely give electrostatic repulsion or distort the local structure of Mrt4, thereby weakening its affinity for the RNA. A weakened interaction with the ribosome could allow Mrt4 to be displaced from the ribosome without the need for Yvh1, explaining the mechanism of suppression. This would be similar to mutations in Tif6 that weaken its binding to the ribosome and bypass the requirement for Efl1 and Sdo1 to release Tif6 (Senger et al., 2001
; Menne et al., 2007
To determine whether Mrt4-G68D has reduced affinity for the ribosome, as we predicted from the structure model, we compared the salt sensitivity of the association of Mrt4 and Mrt4-G68D with 60S subunits. We found that the binding of Mrt4-G68D to the ribosome was significantly more salt sensitive than Mrt4. At 100 mM NaCl, the majority of wild-type and mutant Mrt4 was ribosome bound. However, at 200 and 300 mM NaCl, the majority of Mrt4-G68D was released from the subunit, whereas wild-type Mrt4 was largely unaffected (). The weakened affinity of Mrt4-G68D for the 60S subunit likely obviates the need for Yvh1 to release Mrt4. Because Mrt4-G68D fully complements the growth defect of an mrt4Δ mutant (unpublished data), the affinity of Mrt4-G68D for the ribosome must be finely balanced between binding strongly enough to support ribosome biogenesis but not so strongly that it requires Yvh1 for its release. It may seem counterintuitive that mutant Mrt4 with weaker affinity for 60S subunits is dominant, implying that it competes efficiently with wild-type Mrt4 for binding to the ribosome. However, in yvh1 mutants, wild-type Mrt4 is depleted from the nucleus because it is not released from subunits in the cytoplasm. This allows mutant Mrt4-G68D to load onto subunits effectively without competition from wild-type Mrt4.
Rpl12 is required for the recruitment of Yvh1 to the ribosome
Based on the structure of the archaeal 50S subunit (), Rpl12 binds to the GTPase-associated domain of 25S rRNA, which is adjacent to Mrt4 in the pre-60S or P0 in the mature 60S subunit. Together, Rpl12 and P0 form the stalk base. Because Mrt4 is released by Yvh1 but Rpl12 remains in the Yvh1–60S complex (see ), we speculated that Rpl12 might be required for Yvh1 binding on the 60S subunits. This idea was tested in an rpl12 deletion strain. In yeast, Rpl12 is encoded by two genes, RPL12A and RPL12B. The double deletion (rpl12ΔΔ) is viable but slow growing. As shown in , the majority of Yvh1 in wild-type cells sediments with 60S subunits. However, Yvh1 was quantitatively lost from 60S subunits from the rpl12ΔΔ strain. This indicates that Rpl12 is required for Yvh1 binding to the large subunit, suggesting that Rpl12 comprises part or all of the binding site for Yvh1.
Figure 7. Yvh1 shuttles out of the nucleus bound to a 60S subunit that lacks both Mrt4 and P0. (A) Extracts were prepared from cultures of AJY3048 (MRT4-GFP yvh1Δ) with Yvh1-myc (pAJ2020) and AJY3040 (MRT4-GFP) with pAJ538 (NMD3-myc) or pAJ2002 (RLP24-myc). (more ...)
Figure 6. Rpl12 is required for Yvh1 binding to the 60S subunit. (A) The binding of Yvh1 to 60S subunits was assayed in W303 and 6EA1 (rpl12ΔΔ) expressing Yvh1-myc (pAJ2020) by sedimentation through sucrose cushions at 100 mM NaCl. Equal amounts (more ...)
If Yvh1 cannot be recruited to the 60S subunits in the rpl12ΔΔ mutant, Mrt4 should persist on the 60S ribosome as it does in a yvh1Δ mutant. To test this idea, we asked whether Mrt4 was increased in abundance in Nmd3-containing 60S particles. Nmd3 was immunoprecipitated from wild-type or rpl12ΔΔ cells, and the relative levels of Mrt4 were assayed by Western blotting. Indeed, Mrt4 was significantly enriched in the Nmd3 immunoprecipitation from the deletion mutant (), which is similar to what we observed in a yvh1Δ mutant ().
The order and location of loading Mrt4, Yvh1, and P0 onto 60S subunits
To dissect the order and localization of Mrt4 release, Yvh1 binding, and P0 loading, we used Yvh1 as bait to immunoprecipitate the pre-60S complex from wild-type cells. Surprisingly, we did not detect either Mrt4 or P0 in the Yvh1 immunoprecipitate (). As controls, we also immunoprecipitated Nmd3 and Rlp24, both of which shuttle but show a bias toward the cytoplasm and nucleus, respectively. Nmd3 showed enrichment for P0, whereas Rlp24 was strongly enriched for Mrt4, which is consistent with P0 being cytoplasmic and Mrt4 nuclear. All three bait proteins immunoprecipitated similar levels of 60S subunits, which were monitored by Western blotting for Rpl8. These results suggest a linear series of events in which Mrt4 is released when Yvh1 binds and the subsequent binding of P0 coincides with the release of Yvh1.
We next turned to the question of where these exchanges take place. The steady-state distribution of Yvh1 is cytoplasmic and nuclear, whereas P0 is cytoplasmic. The localization of Yvh1 suggests that it shuttles and is consistent with a report that Yvh1 interacts physically with the nuclear 60S biogenesis factor Nop7 (Sakumoto et al., 2001
). However, the cytoplasmic distribution of P0 does not preclude that it might also shuttle. If these proteins shuttle, we would expect their export to be linked to the 60S subunit, assuming that they are exported with the subunit. Because 60S export depends on Crm1 (Ho et al., 2000
; Gadal et al., 2001
), we treated cells with the Crm1 inhibitor leptomycin B (LMB) and tested whether Yvh1 or P0 was trapped in the nucleus. Under conditions where export of Nmd3 and Rpl25 was blocked, Yvh1 accumulated in the nucleus, although the degree to which Yvh1 accumulated appeared less than that of Nmd3 ( and not depicted). However, P0 remained in the cytoplasm (). Although this result is consistent with the idea that Yvh1 shuttles out of the nucleus while bound to the 60S subunit, it does not rule out the possibility that Yvh1 is exported separately from the subunit but in a Crm1-dependent fashion. As an additional test, we examined Yvh1 localization in an nmd3-1
mutant. The nmd3-1
allele expresses a mutant protein that binds to pre-60S particles in the nucleus but is truncated for its C-terminal, leucine-rich nuclear export signal (Ho et al., 2000
) and thus specifically impairs 60S export. This mutant showed strong nuclear accumulation of Yvh1 but not P0 (). These results suggest that Yvh1 shuttles and is exported from the nucleus bound to the nascent 60S particle, whereas P0 is restricted to the cytoplasm.
In an attempt to test whether Yvh1 could function in the nucleus, we fused the well-characterized nuclear localization sequence from the SV40 large T antigen to the amino terminus of Yvh1. This fusion protein fully complemented a yvh1Δ mutant () and localized predominantly to the nucleus (). Sucrose gradient analysis of cells containing the NLS-Yvh1 construct as their sole copy of Yvh1 were indistinguishable from cells expressing wild-type Yvh1 (unpublished data). Furthermore, this construct promoted the release of Mrt4, as the wild-type localization of Mrt4 in the nucleolus and nucleus was observed in the cells (). These data support the idea that Yvh1 can release Mrt4 in the nucleus. However, we cannot rule out the possibility that a residual pool of NLS-Yvh1 in the cytoplasm is sufficient for this activity.
Figure 8. Nuclear localized Yvh1 is functional. (A) Serial dilutions of yvh1Δ cells (AJY2976) expressing empty vector, Yvh1-GFP (pAJ2464), or NLS-Yvh1-GFP (pAJ2481) were plated on selective media and incubated at 30°C for 2 d. (B) yvh1Δ (more ...)
The function of Yvh1 in releasing Mrt4 is conserved in human cells
Human Yvh1 (DUSP12) complements the slow-growing phenotype of yvh1Δ
in budding yeast, which is consistent with previous work (; Muda et al., 1999
). Human Mrt4 (MRTO4) was also able to complement the growth defect of mrt4Δ
. However, there was no improvement in growth from coexpressing both human MRTO4 and DUSP12 in a yvh1Δ mrt4Δ
double mutant (unpublished data). We asked whether the complementation of yvh1Δ
with DUSP12 correlated with restoring the function that we have ascribed to Yvh1 of releasing Mrt4. We monitored the cellular localization of yeast Mrt4-GFP in yvh1Δ
cells expressing DUSP12. As shown in , the human orthologue of Yvh1 restored the normal nuclear and nucleolar localization of Mrt4 (see for comparison). This implies that DUSP12 can release yeast Mrt4 from 60S subunits.
Figure 9. The function of Yvh1 to release Mrt4 is conserved in human cells. (A) 10-fold serial dilutions of cultures of AJY2551 (mrt4Δ) cells with empty vector, pAJ2475 (MRT4-HA), or pAJ2477 (MRTO4) and AJY2976 (yvh1Δ) cells with empty vector, pAJ2020 (more ...)
To further test whether the maturation pathway of the ribosome stalk is conserved from yeast to human, we asked if DUSP12 functions in human cells to release MRTO4. We used RNAi to knock down DUSP12 in HeLa cells and monitored the localization of MRTO4 by indirect immunofluorescence. Consistent with the localization of Mrt4 in yeast, MRTO4 was localized in the nucleus and nucleolus in HeLa cells (, top). Upon RNAi depletion of DUSP12, MRTO4 was found in the cytoplasm and nucleolus in HeLa cells but depleted from the nucleoplasm (, middle). Similar results were observed in HEK293T cells (unpublished data). No mislocalization was detected in the cells transfected with control siRNA (, bottom). The relocalization of MRTO4 from the nucleoplasm to the cytoplasm is consistent with our observation in yeast that Yvh1 is required for the release of Mrt4. However, it was surprising to us that the nucleolar pool of MRTO4 was also not diminished. In wild-type cells, MRTO4 is concentrated in the nucleolus, suggesting that its residence time in the nucleolus is long compared with that in the nucleoplasm. Because the DUSP12 depletion was not complete, MRTO4 is presumably released from subunits, but at a reduced rate. Under these conditions, if the recycling of MRTO4 to the nucleolus is slower than its export from the nucleus, we would expect the observed depletion of the nucleoplasmic pool preferentially over the nucleolar pool.