Rei1 is physically linked to Rpl24
Most ribosomal proteins of the large subunit are loaded on 60S pre-ribosomal particles in the early nuclear steps of 60S biogenesis. However, a few proteins, such as Rpl10 and Rpl24, appear to associate later to the pre-60S particles (Kruiswijk et al., 1978
). Rpl24 is a late-associating ribosomal protein that shares significant homology with the essential pre-ribosomal factor Rlp24 (Saveanu et al., 2003
). Rlp24 is present on pre-60S particles from the nucleolus to the cytoplasm, where it is believed to dissociate when Rpl24 associates with the particle.
To identify potential partners required for the loading of Rpl24 to the particles, a two-hybrid screen using Rpl24b as bait was performed. The most frequent prey selected in the screen was REI1/YBR267W, which is found as seven distinct inserts. All of the inserts containing the REI1 ORF shared a minimal interacting domain from amino acids 270 to 342 ().
Figure 1. Rei1 is physically associated with nontranslating, Rpl24-containing complexes. (A) Rei1 is a two-hybrid prey of Rpl24b. YBR267W (REI1) was found as prey ORF in a genomic two-hybrid screen performed with RPL24B as bait. We represented the 393–amino (more ...)
Rei1 is a 393–amino acid–long protein that contains three conserved C2
Zn finger motifs extending from amino acids 7 to 31, 162 to 187, and 215 to 239. The domain extending from amino acids 57 to 202 displays similarity with the PFAM E-MAP-115 domain characteristic of ensconsin, a microtubule-associated protein expressed in higher eukaryotes (Masson and Kreis, 1993
). The minimal two-hybrid interaction domain between Rei1 and Rpl24b is located in the COOH-terminal part of the protein apart from all of these motifs. This region is quite conserved in Rei1 homologues among eukaryotes, mainly in the portion extending from amino acids 300 to 330 (unpublished data).
Rei1 is associated with Rpl24-containing 60S complexes but is absent from the polysomes
The two-hybrid link between Rei1 and Rpl24 suggested that Rei1 might participate in molecular events involving the large ribosomal subunit together with the late-associating ribosomal protein Rpl24. This hypothesis was supported by previous tandem affinity purifications (TAPs), which had revealed the presence of Rei1 in several pre-60S complexes (Gavin et al., 2002
; Nissan et al., 2002
). The clustering of these TAP data (for review see Fromont-Racine et al., 2003
) had already shed light on Rei1 as a putative late pre-60S factor. Therefore, we further investigated the possible role of Rei1 in the cytoplasmic events of 60S biogenesis.
In addition to its cytoplasmic localization (Iwase and Toh-e, 2004
), the interaction of Rei1 with Rpl24 prompted us to determine whether this factor was associated with ribosomal or pre-ribosomal complexes in the cytoplasm. We performed TAP experiments on strains producing a Rei1 protein fused to a 13Myc epitope and either Rlp24 or Rpl24b fused to the TAP tag. Rei1-13Myc was enriched in the Rpl24b-TAP–associated complexes compared with the Rlp24-TAP–associated complexes or to the strictly nuclear Ssf1-TAP–associated complexes used as negative controls (). In contrast, the presence of Nog1 was observed, as expected, in both Ssf1-TAP– and Rlp24-TAP–associated pre-ribosomal complexes but not in the Rpl24b-TAP–associated complex. These data suggest that Rei1 is specifically associated with Rpl24-containing cytoplasmic complexes.
To analyze a potential association of Rei1 with mature translating ribosomes, total cellular extracts from cells producing Rpl24b-TAP were fractionated on a sucrose gradient. As expected, Rpl24b-TAP was found in fractions corresponding to the 60S, 80S, and polysomes. We observed that Rei1 was absent from the 80S and polysomal fractions but present in the 60S fraction (), indicating that in contrast to Rpl24, Rei1 is not associated with mature ribosomes during translation. Exclusive association with 60S particles supported the hypothesis that Rei1 is a late pre-60S factor that transiently binds to pre-ribosomal complexes in the cytoplasm after the release of Rlp24 from the particles.
Rei1 is a late cytoplasmic 60S pre-ribosomal factor
The presence of Rei1 in the 60S fractions of a sucrose gradient and not in polysome fractions suggested that it might participate in the late events of the large subunit biogenesis or be involved in the very early steps of translation initiation.
To determine whether Rei1 could be involved in ribosome biogenesis or in translation, the rei1Δ
strain and the corresponding wild-type strain were used for sucrose gradient analysis. The rei1Δ
strain displayed a slow growth phenotype at 30°C and was cold sensitive as previously observed in another genetic background (Iwase and Toh-e, 2004
). In the rei1Δ
strain, the polysome profile was clearly affected. At 30°C, the amount of 60S subunit decreased significantly (unpublished data). At 23°C, not only a decrease of the 60S subunit amount was observed, but also a drop in the average level of polysomes and the appearance of half-mers, which can be explained by abortive 48S preinitiation complexes being formed but remaining complexes being blocked on mRNA because of a lack of mature large ribosomal subunits ().
Figure 2. Absence of Rei1 affects the 60S maturation. (A) The rei1Δ strain displays an altered polysome profile. The disrupted strain and wild-type strain were grown for 16 h at 23°C in yeast extract–peptone–d-glucose (YPD). Whole (more ...)
The clearly affected polysome profiles in the rei1Δ strain correlated with a defect in rRNA maturation (). Ethidium bromide staining of agarose gels, Northern blots, and primer extensions were performed to determine the relative levels of various rRNA intermediates and mature species. The rRNA defect was already significant at 30°C (unpublished data) but increased at 23°C. At this temperature, we observed a drop in the levels of total 27S species, 27SA2 and 7S nuclear pre-rRNAs, as well as mature 5.8S rRNA normalized relatively to U2 small nuclear RNA levels. The 27SB/27SA2 pre-rRNA ratio was increased by 3.1 ± 0.4, and the 27SB/7S ratio was increased by 4.7 ± 0.5. Altogether, these data are typical of a defect in the ITS2 processing step (). They are in correlation with the drastic decrease in the 60S peak on sucrose gradients, which results in a stoichiometric imbalance between the small/large ribosomal subunit ().
Curiously, although Rei1 appears to be a cytoplasmic protein, its absence displayed rRNA maturation defects at the nuclear level. To investigate whether Rei1 might be a shuttling factor, we monitored Rei1-GFP localization in a strain defective for the export of pre-60S particles (). We used a strain in which a version of the nmd3Δ100
dominant-negative mutant was overexpressed compared with the same strain overexpressing a wild-type NMD3
version (Ho et al., 2000b
). As a control, we monitored the localization of the ribosomal protein Rpl25-GFP. As expected, Rpl25-GFP, which is normally mainly cytoplasmic, was accumulated in the nucleus when nmd3Δ100
was overexpressed. In contrast, no accumulation was seen for Rei1-GFP, which appears to be an exclusively cytoplasmic pre-60S factor.
We additionally tested the shuttling ability of pre-ribosomal factor Rlp24-TAP and of ribosomal protein Rpl24b-TAP (). In the nmd3Δ100
dominant-negative mutant, Rlp24-TAP accumulated in the yeast nuclei compared with wild-type conditions. Meanwhile, Rpl24b-TAP remained exclusively cytoplasmic. This confirmed previous data (Saveanu et al., 2003
) in favor of Rlp24 being a shuttling nucleocytoplasmic pre-60S factor, whereas Rpl24 appears to be a ribosomal protein that associates with pre-60S particles in the very last cytoplasmic steps of ribosome biogenesis.
As a late pre-60S factor, Rei1 was a candidate for participating in the export of pre-60S particles from the nucleus to the cytoplasm. Therefore, we monitored the localization of an Rpl25-GFP reporter construct in a rei1Δ strain. Because no export defect was observed (unpublished data), we assume that Rei1 is not required for the export of precursors of the large ribosomal subunit.
Altogether, these results are consistent with Rei1 being involved in the large ribosomal subunit biogenesis. Though it is a cytoplasmic nonshuttling protein, its absence results in defects in nuclear steps of the 60S maturation, suggesting that Rei1 could be required for the recycling of shuttling pre-60S factors from the cytoplasm to the nucleus.
The absence of Rei1 affects nuclear import of the shuttling factors Arx1 and Tif6
We looked at the localization at 23°C of the Arx1-GFP, Tif6-GFP, or Rlp24-TAP fusion proteins in a rei1Δ strain compared with a wild-type strain (). In the wild-type strains, Arx1-GFP and Tif6-GFP were mainly observed in the nucleus, and a small fraction of the proteins was found in the cytoplasm. In the absence of Rei1, Arx1-GFP or Tif6-GFP were mainly cytoplasmic. This confirmed our hypothesis that nuclear import of some shuttling factors could be impaired in the absence of Rei1. In contrast, no recycling defect was observed for Rlp24-TAP, which even appeared somewhat concentrated in the nucleolus of the rei1Δ strain compared with the wild-type strain, where it appeared more equally distributed between the nucleolus, nucleoplasm, and cytoplasm.
Figure 3. Rei1 is functionally linked to the shuttling factors Arx1 and Tif6. (A) In the absence of Rei1, Arx1 and Tif6 are redistributed to the cytoplasm. The localization of Arx1-GFP, Tif6-GFP, or Rlp24-TAP was observed after the growth of rei1Δ or wild-type (more ...)
These data offer a simple explanation for the nuclear rRNA maturation defect observed in the rei1Δ
strain. Indeed, the absence of Rei1 affects the processing of ITS2 in a manner similar to the repression of Tif6 (Basu et al., 2001
). To better illustrate this, a strain in which TIF6
was placed under the control of a PGAL1
promoter was shifted to glucose from 0 to 6 h, and pre-rRNA species were analyzed and quantified along these kinetics (). The rRNA intermediates ratio of the rei1Δ
strain to the wild type were comparable with the effects of a TIF6
repression for 4–6 h.
We also tested the phenotype of an ARX1
deletion, but no obvious rRNA maturation impairment was detected (unpublished data). Therefore, the 60S maturation defects observed in the rei1Δ
strain are likely caused by a decrease of nuclear Tif6. We tested whether the overexpression of TIF6
could compensate this phenotype, but TIF6
overexpression appeared to be toxic in wild-type as well as in rei1Δ
strains (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200510080/DC1
As Arx1-GFP and Tif6-GFP accumulated in the cytoplasm in the absence of Rei1, we wondered whether they were still associated with pre-60S complexes. To address this question, we separated extracts from a rei1Δ or wild-type strain expressing chromosomal ARX1-GFP or TIF6-GFP on sucrose gradients (). In the wild-type strain, we could detect Arx1-GFP in the 60S fractions of the gradient. In the absence of Rei1, Arx1 appeared to sediment closer to the top of the gradient, which suggested that in such conditions, Arx1-GFP was present in the cytoplasm as a small complex or as an oligomeric protein. In contrast, in the absence of Rei1, the Tif6-GFP fusion protein was still associated with particles of the size of 60S. Although the absence of Rei1 leads to a cytoplasmic retention of both shuttling pre-60S factors Tif6 and Arx1, Tif6 is still stably associated with 60S particles, whereas Arx1 accumulates as a small complex in the cytoplasm.
Yjl122w/Alb1 is a novel 60S-associated factor that is tightly linked with Arx1
The presence of Arx1 in a small cytoplasmic complex in the absence of Rei1 suggested the possibility that Arx1 could associate and act together with other proteins. We performed a two-hybrid screen using Arx1 as bait to identify the physical partners of Arx1. The most frequent prey we selected was YJL122W
, which is hereafter referred to as ALB1
little brother 1). This prey was found as seven distinct inserts; all of the inserts shared a minimal interacting domain from amino acids 148 to 175 (). The deletion of ALB1
had little effect on growth and displayed no obvious rRNA maturation impairment. In the arx1Δ
double mutant, the growth phenotype as well as the polysome profile resembled that of the arx1Δ
single mutant (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.2005010080/DC1
Figure 4. Yjl122w/Alb1 is a physical partner of Arx1. (A) Alb1 is a two-hybrid prey of Arx1. YJL122W (ALB1) was found as prey ORF in a genomic two-hybrid screen performed with ARX1 as bait. The results are presented as in . (B) Rei1, Arx1, and Alb1 are (more ...)
To confirm the physical interaction between Arx1 and Alb1, we purified the complexes associated with Alb1 (). Arx1- and Alb1-TAP–associated complexes look very similar on the Coomassie staining compared with Rei1-TAP. We could detect Arx1 and Rei1 in the three complexes. These results corroborate previous affinity precipitations (Ho et al., 2002
; Krogan et al., 2004
) where Alb1 had been identified in both Rei1- and Arx1-associated complexes. They not only confirm that Arx1 and Alb1 are physically associated under physiological conditions but also suggest that Alb1 could be a pre-60S–associated factor. Note that Rlp24 was present in Arx1- and Alb1-TAP–associated complexes but was absent from Rei1-TAP complexes, which confirms that Rei1 loads onto the particles after the release of Rlp24. In addition, we observed that Alb1-TAP sedimented around the 60S peak but was absent from the polysomal fractions on a sucrose gradient (); therefore, it is likely to associate with precursors of the large ribosomal subunit.
To determine whether any direct interaction could be detected between these factors, we performed in vitro GST pull-down experiments with GST-tagged baits (GST-Alb1, GST-Rei1, or GST) and the His6-Arx1 fusion protein as prey (). His6-Arx1 copurified with GST-Alb1 but not with GST or GST-Rei1 used as negative controls. Thus, we conclude that Arx1 and Alb1 are able to interact directly even in the absence of other yeast components.
The absence of Rei1 affects nuclear import of Alb1 and its binding to pre-60S particles
Because the nuclear import of Arx1 was affected in the absence of Rei1, we wondered whether Alb1 participated with Arx1 in the small cytoplasmic complex detected in these conditions. Therefore, we assessed the effects of the absence of Rei1 on Alb1 localization and association with 60S particles.
First, we tested whether Alb1 was an additional shuttling factor by monitoring the localization of an Alb1-GFP reporter construct in a wild-type or rei1Δ strain at 23°C (). In the wild-type strain, Alb1-GFP localization was similar to that of Arx1-GFP, with a strong nuclear–nucleolar signal and a weak cytoplasmic signal. In the rei1Δ mutant, Alb1 was relocalized to the cytoplasm, as was the case for Arx1.
Figure 5. Alb1 is an additional shuttling pre-60S factor and target of Rei1. (A) In the absence of Rei1, Alb1 is blocked in the cytoplasm. The localization of Alb1-GFP was observed in rei1Δ or wild-type strains and grown for 8 h in minimal medium at 23°C. (more ...)
Second, in a sucrose gradient analysis (), Alb1-GFP could be detected in the 60S fractions in the wild-type strain together with Arx1, whereas in the rei1Δ strain, it was found mainly in the same fractions as Arx1 at the top of the gradient. Thus, the association of Alb1 to nuclear pre-60S particles was impaired, as it was for Arx1, in a rei1Δ strain.
We tried to further characterize the small complex observed in the absence of Rei1 by purifying the Arx1- and Alb1-TAP–associated complexes in these conditions (). The Coomassie staining showed a weak decrease of the levels of most proteins in the rei1Δ strain compared with the wild-type profiles, except for Arx1. In the absence of Rei1, immunoblots performed on the purified fractions revealed a loss of the pre-ribosomal factors Nog1 and Tif6. These results suggest that Arx1 and Alb1 can form cytoplasmic subcomplexes in the absence of Rei1 independently from pre-ribosomal particles.
To confirm these results, purified complexes associated with Alb1-TAP were separated on a sucrose gradient in the absence of Rei1 (). These complexes sedimented in the first five fractions of the gradient and contained Arx1 and Kap121, as discussed in the following paragraphs. In contrast to the other Ponceau-stained bands, these two proteins could be depleted by preincubating the TEV eluate with calmodulin affinity resin, which retains Alb1-CBP, indicating that they are strongly associated with this bait.
Reimport of shuttling factors into the nucleus in the absence of Rei1 restores growth
To gain more insights into the molecular events involved in the observed phenotypes in the absence of Rei1, we performed a high copy number suppressor screen with a strain deleted for REI1
grown at 20°C. In addition to REI1
itself, we selected the homologous gene REH1/YLR387C
. The overexpression of this gene was able to partially complement the cold-sensitive phenotype of rei1Δ
() as previously shown (Iwase and Toh-e, 2004
), yet we could not detect any defect in reh1Δ
strains on polysome profiles nor on rRNA maturation (not depicted). Thus, we assume that although it shares partial functional overlap with Rei1, Reh1 is not limiting for the biogenesis of the large ribosomal subunit under physiological conditions.
Figure 6. Kap121 is involved in the Rei1-dependent nuclear import of pre-60S factors. (A) KAP121 is a high copy number suppressor of rei1Δ. A rei1Δ strain was transformed with high copy number plasmids carrying distinct DNA inserts (REI1, REH1, (more ...)
The third gene identified in our screen was KAP121/YMR308C
(). It was selected as seven distinct DNA inserts containing the whole KAP121
ORF. The smallest insert constituted a DNA fragment extending from 589 bp upstream of the initiation codon to 762 bp downstream of the stop codon. Kap121, also known as Pse1, is a β-karyopherin, which was shown to be involved in the import of various ribosomal proteins (Rout et al., 1997
) as well as pre-ribosomal factors such as Nop1 and Sof1 from the cytoplasm to the nucleus (Leslie et al., 2004
). The mislocalization to the cytoplasm of Arx1-GFP, Alb1-GFP, or Tif6-GFP in a rei1Δ
strain could be compensated by overexpression of either REI1
(). Both constructs were able to relocalize the three fusion proteins to the nuclear compartment compared with a strain transformed with an empty vector.
Because overexpression of the β-karyopherin Kap121 was sufficient to overcome the import defect of all three shuttling factors in the rei1Δ
strain, one might wonder whether Kap121 was the physiological importin for these proteins. Such a hypothesis was supported by the presence of Kap121 in Arx1- and Alb1-TAP–associated complexes ( and ; Gavin et al., 2002
; Nissan et al., 2002
) and by two-hybrid data (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200510080/DC1
). In contrast, Kap121 was not detected in Tif6-TAP–associated complexes; therefore, we did not further assess the possibility that Kap121 is the specific importin for this factor. As a control, Rlp24 was as efficiently copurified with Tif6-TAP as with Alb1-TAP or Arx1-TAP compared with Rei1-TAP, which was associated with neither Kap121 nor Rlp24 ().
Further evidence for Arx1 and Alb1 being Kap121 cargoes was provided by localization of the Arx1-GFP and Alb1-GFP fusion proteins in strains in which the expression of KAP121 was repressed (). Both fusions were redistributed to the cytoplasm compared with wild-type strains, where we observed bright nuclear–nucleolar signals. This result suggested that the karyopherin Kap121 might be the physiological importin for Arx1 and Alb1. Altogether, these results show that the nuclear import of Arx1 and Alb1 is Kap121 dependent. In the absence of Rei1, the return of Arx1–Alb1 to the nucleus as well as that of Tif6 can be restored by overexpressing this karyopherin.
The accumulation of Alb1–Arx1 in the cytoplasm prevents dissociation and recycling of Tif6 in rei1Δ strains
In the absence of Rei1, Arx1 and Alb1 accumulate in the form of a small complex, whereas Tif6 remains bound to the pre-60S particle. We hypothesized that the nonphysiological levels of the Arx1–Alb1 cytoplasmic subcomplex could prevent the dissociation of Tif6 from the premature large ribosomal subunit and subsequent return of this factor to the nucleus.
The deletion of either ARX1 or ALB1 in the rei1Δ strain restored growth at 23°C almost up to the levels of arx1Δ or alb1Δ single mutant strains, respectively (). Therefore, the cytoplasmic accumulation of Arx1–Alb1 accounts for the observed cold-sensitive phenotype of the rei1Δ strain.
Figure 7. In the absence of Rei1, the cytoplasmic Arx1–Alb1 complex prevents the recycling of Tif6. (A) Disruptions of ARX1 or ALB1 compensate the cold-sensitive phenotype of a rei1Δ strain. Wild-type, rei1Δ, arx1Δ, or rei1Δ (more ...)
According to our hypothesis, Arx1–Alb1 might also explain the cytoplasmic retention of Tif6. Thus, we monitored the localization of Tif6-GFP in rei1Δ alb1Δ or rei1Δ arx1Δ double mutant strains (). In these mutants, Tif6 was mainly nuclear, as in the wild-type strain, compared with the mostly cytoplasmic signal in the rei1Δ strain. Thus, we conclude that in the absence of Rei1, Arx1 and Alb1 participate in a cytoplasmic subcomplex that prevents the adequate release of Tif6 from pre-60S particles and its correct return to the nucleus for a new round of biogenesis.