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Reduced ribosome biogenesis in response to environmental conditions is a key feature of cell adaptation to stress. For example, ribosomal genes are transcriptionally repressed when cells are exposed to tunicamycin, a protein glycosylation inhibitor that induces endoplasmic reticulum stress and blocks vesicular trafficking in the secretory pathway. Here, we describe a novel regulatory model, in which tunicamycin-mediated stress induces the accumulation of long-chain sphingoid bases and subsequent activation of Pkh1/2 signaling, which leads to decreased expression of ribosomal protein genes via the downstream effectors Pkc1 and Sch9. Target of rapamycin complex 1 (TORC1), an upstream activator of Sch9, is also required. This pathway links ribosome biogenesis to alterations in membrane lipid composition under tunicamycin-induced stress conditions. Our results suggest that sphingolipid/Pkh1/2-TORC1/Sch9 signaling is an important determinant for adaptation to tunicamycin-induced stress.

Secretory defects cause transcriptional repression of ribosome biogenesis in Saccharomyces cerevisiae. However, the molecular mechanism underlying secretory defect-induced transcriptional repression of ribosome biogenesis remains to be fully elucidated. In this study, we demonstrated that the Arp2/3 complex was required for reduction of ribosome protein gene expression in response to defective secretion by addition of tunicamycin. Two cmd1 mutants, cmd1-228 and cmd1-239 that cause mislocalization of calmodulin and defective mitotic spindle formation, respectively, failed to interact with Arc35, a component of the Arp2/3 complex. These mutants also caused defects in the reduction of ribosome protein gene expression induced by secretory blockade. A mutation in TUB4 (tub4-1), whose product has an essential function in microtubule organization, showed a similar response. In addition, we showed that the response to a secretory defect required SUN protein Mps3, which was localized at the nuclear envelope and involved in spindle pole body assembly. These results suggest that the Arp2/3 complex is required to transmit signals resulting from secretory blockade, and that the spindle pole body functions as a transit point from cytoplasm to Mps3 at the nuclear envelope. Copyright © 2016 John Wiley & Sons, Ltd.

The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature-sensitive fission yeast strains, rrs1-D14/22G and rrs1-L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1-84 (D22/30G) and rrs1-124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two-hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts.

Secretory defects cause transcriptional repression of both ribosomal proteins and ribosomal RNA genes in Saccharomyces cerevisiae. Rrs1, a trans-acting factor that participates in ribosome biogenesis, is involved in the signaling pathway induced by secretory defects. Here, we found that Rrs1 interacts with two homologs of the glycogen synthase kinase-3 (GSK-3), Rim11, and Mrk1. Rrs1 possesses a repetitive consensus amino acid sequence for phosphorylation by GSK-3, and mutation of this sequence abolished the interaction of Rrs1 with Rim11 and Mrk1. Although this mutation did not affect vegetative cell growth or secretory response, disruption of all four genes encoding GSK-3 homologs, especially Mck1, diminished the transcriptional repression of ribosomal protein genes in response to secretory defects. Among the four GSK-3 kinases, Mck1 appears to be the primary mediator of this response, while the other GSK-3 kinases contribute redundantly.

Twin-twin transfusion syndrome or related conditions affect fetal loading. We report monochorionic-diamniotic twins. Twin 1 had Ebstein anomaly with mild tricuspid regurgitation (TR) and slightly thickened tricuspid valve leaflets with plastering. Twin 2 had tricuspid valve dysplasia (with abnormal thickening but without plastering) with moderate TR and mild right atrial dilatation. After birth, the severity of TR was greatly reduced in the recipient but increased in the donor. Therefore, intravascular volume change which was due to twin-twin transfusion syndrome seemed to affect the severity of the valvar disease in fetuses. This case suggests that the intrinsic severity of fetal tricuspid valvular disease may be overestimated in the recipient and underestimated in the donor twin. These factors need to be taken into consideration in clinical decision-making.

Secretory defects cause transcriptional repression of ribosome biogenesis in Saccharomyces cerevisiae. However, the molecular mechanism underlying secretory defect-induced transcriptional repression of ribosome biogenesis remains to be fully elucidated. In this study, we demonstrated that the Arp2/3 complex was required for reduction of ribosome protein gene expression in response to defective secretion by addition of tunicamycin. Two cmd1 mutants, cmd1-228 and cmd1-239 that cause mislocalization of calmodulin and defective mitotic spindle formation, respectively, failed to interact with Arc35, a component of the Arp2/3 complex. These mutants also caused defects in the reduction of ribosome protein gene expression induced by secretory blockade. A mutation in TUB4 (tub4-1), whose product has an essential function in microtubule organization, showed a similar response. In addition, we showed that the response to a secretory defect required SUN protein Mps3, which was localized at the nuclear envelope and involved in spindle pole body assembly. These results suggest that the Arp2/3 complex is required to transmit signals resulting from secretory blockade, and that the spindle pole body functions as a transit point from cytoplasm to Mps3 at the nuclear envelope. Copyright © 2016 John WileySons, Ltd.

Two interacting ribosome biogenesis factors, Ebp2 and Rrs1, associate with Mps3, an essential inner nuclear membrane protein. Both are found in foci along the nuclear periphery, like Mps3, as well as in the nucleolus. Temperature-sensitive ebp2 and rrs1 mutations that compromise ribosome biogenesis displace the mutant proteins from the nuclear rim and lead to a distorted nuclear shape. Mps3 is known to contribute to the S-phase anchoring of telomeres through its interaction with the silent information regulator Sir4 and yKu. Intriguingly, we find that both Ebp2 and Rrs1 interact with the C-terminal domain of Sir4, and that conditional inactivation of either ebp2 or rrs1 interferes with both the clustering and silencing of yeast telomeres, while telomere tethering to the nuclear periphery remains intact. Importantly, expression of an Ebp2–Mps3 fusion protein in the ebp2 mutant suppresses the defect in telomere clustering, but not its defects in growth or ribosome biogenesis. Our results suggest that the ribosome biogenesis factors Ebp2 and Rrs1 cooperate with Mps3 to mediate telomere clustering, but not telomere tethering, by binding Sir4.

Rrs1 has an essential role in 60S ribosomal subunit assembly in Saccharomyces cerevisiae. We isolated a temperature-sensitive kcs1 mutant that suppresses the cold sensitivity of rrs1-1. The kcs1 allele, resulting in truncation of inositol 6 phosphate kinase domain, and kcs1 disruption suppress a defect of rrs1-1 in 60S ribosomal subunit assembly. These results suggest that inositol polyphosphate metabolism affects ribosome biogenesis in yeast.

We have found that Ebp2p is essential for maturation of 25S rRNA and assembly of 60S pre-ribosomal subunits in Saccharomyces cerevisiae. We obtained three temperature-sensitive ebp2 mutants by PCR. Polysome analysis revealed that the synthesis of 60S ribosomal subunits was compromised in each of the ebp2 mutants at the restrictive temperature. The ebp2 alleles affected the transcriptional repression of both rRNA and ribosomal protein genes due to a secretion block. Fluorescence microscopy showed that a secretion block led to condensation of nucleolar Ebp2p, whereas that was not the case with the ebp2 mutant. These results suggest that Ebp2p is implicated in the secretory response, including changes in nucleolar architecture.

ARV1 is involved in regulating lipid homeostasis but also in the biosynthesis of glycosylphosphatidylinositol (GPI) in Saccharomyces cerevisiae. Here, we examined whether human ARV1 can complement the role of yeast ARV1 in GPI biosynthesis. Overexpression of human ARV1 could rescue the phenotypes associated with GPI anchor synthesis defect in the yeast arv1Δ mutant. The results suggest that Arv1 function in GPI biosynthesis may be conserved in all eukaryotes, from yeast to humans.

The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature-sensitive fission yeast strains, rrs1-D14/22G and rrs1-L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1-84 (D22/30G) and rrs1-124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two-hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts.

In eukaryotic organisms including mammals, nematodes, and yeasts, the ends of chromosomes, telomeres are clustered at the nuclear periphery. Telomere clustering is assumed to be functionally important because proper organization of chromosomes is necessary for proper genome function and stability. However, the mechanisms and physiological roles of telomere clustering remain poorly understood. In this study, we demonstrate a role for sphingolipids in telomere clustering in the budding yeast Saccharomyces cerevisiae. Because abnormal sphingolipid metabolism causes down-regulation of expression levels of genes involved in telomere organization, sphingolipids appear to control telomere clustering at the transcriptional level. Additionally, the data presented here provide evidence that telomere clustering is required to protect chromosome ends from DNA-damage checkpoint signaling. As sphingolipids are found in all eukaryotes, we speculate that sphingolipid-based regulation of telomere clustering and the protective role of telomere clusters in maintaining genome stability might be conserved in eukaryotes.

Ebp2 is an assembly factor of the 60S ribosomal subunit in yeast. We demonstrate that overexpression of SMY2 or SYH1 partially suppresses defects in growth and ribosome biogenesis of ebp2 mutants, and that smy2Δ and syh1Δ exhibit synthetic growth defects with the ebp2 allele. These results suggest that Smy2 and Syh1 may be involved in ribosome biogenesis in relation to Ebp2.

Rrs1p, a ribosomal protein L11-binding protein, has an essential role in biogenesis of 60S ribosomal subunits. We obtained conditionally synthetic lethal allele with the rrs1-5 mutation and determined that the mutation is in REX1, which encodes an exonuclease. The highly conserved leucine at 305 was substituted with tryptophan in rex1-1. The rex1-1 allele resulted in 3 0 -extended 5S rRNA. Polysome analysis revealed that rex1-1 and rrs1-5 caused a synergistic defect in the assembly of 60S ribosomal subunits. In vivo and in vitro binding assays indicate that Rrs1p interacts with the ribosomal protein L5–5S rRNA complex. The rrs1-5 mutation weakens the interaction between Rrs1p with both L5 and L11. These data suggest that the assembly of L5–5S rRNA on 60S ribosomal subunits coordinates with assembly of L11 via Rrs1p.

Saccharomyces cerevisiae Rrs1p is a nuclear protein that is essential for the maturation of 25 S rRNA and the 60 S ribosomal subunit assembly. In two-hybrid screening, using RRS1 as bait, we have cloned YKR081c/RPF2. Rpf2p is essential for growth and is mainly localized in the nucleolus. The amino acid sequence of Rpf2p is highly conserved in eukaryotes from yeast to human. Similar to Rrs1p, Rpf2p shows physical interaction with ribosomal protein L11 and appears to associate with preribosomal subunits fairly tightly. Northern, methionine pulse-chase, and sucrose density gradient ultracentrifugation analyses reveal that the depletion of Rpf2p results in a delayed processing of pre-rRNA, a decrease of mature 25 S rRNA, and a shortage of 60 S subunits. An analysis of processing intermediates by primer extension shows that the Rpf2p depletion leads to an accumulation of 27 SB pre-rRNA, suggesting that Rpf2p is required for the processing of 27 SB into 25 S rRNA.

A secretory defect leads to transcriptional repression of both ribosomal protein and rRNA genes in yeast. To elucidate the mechanism of the signaling, we previously isolated rrs mutants that were unable to respond to a secretory defect, and we cloned RRS1 encoding a nuclear protein that was required for ribosome biogenesis (Tsuno, A., Miyoshi, K., Tsujii, R., Miyakawa, T., and Mizuta, K. (2000) Mol. Cell. Biol. 20, 2066-2074). We identified duplicated genes encoding ribosomal protein L11, RPL11B as a wild-type allele complementing the rrs2 mutation, and RPL11A in two-hybrid screening using RRS1 as bait. Rpl11p was copurified with Rrs1p in immunoprecipitation analysis. Ultracentrifugation analysis revealed that Rrs1p associated fairly tightly with 60 S preribosomal subunits. These results suggest that signaling in response to a secretory defect requires the normal assembly of 60 S ribosomal subunits including Rrs1p and Rpl11p.

In eukaryotic organisms, including mammals, nematodes and yeasts, the ends of chromosomes, telomeres are clustered at the nuclear periphery. Telomere clustering is assumed to be functionally important because proper organization of chromosomes is necessary for proper genome function and stability. However, the mechanisms and physiological roles of telomere clustering remain poorly understood. In this study, we demonstrate a role for sphingolipids in telomere clustering in the budding yeast Saccharomyces cerevisiae. Because abnormal sphingolipid metabolism causes downregulation of expression levels of genes involved in telomere organization, sphingolipids appear to control telomere clustering at the transcriptional level. In addition, the data presented here provide evidence that telomere clustering is required to protect chromosome ends from DNA-damage checkpoint signaling. As sphingolipids are found in all eukaryotes, we speculate that sphingolipid-based regulation of telomere clustering and the protective role of telomere clusters in maintaining genome stability might be conserved in eukaryotes.

Ebp2 plays an essential role in biogenesis of 60S ribosomal subunits. We determined the genetic interactions between EBP2 and RPL36A/B, which encodes ribosomal protein L36a/b. RPL36A/B was a multicopy suppressor to ebp2 mutants, and the suppression was not common to defects in ribosome biogenesis resulting from other mutations of assembly factors. Disruption of RPL36A or RPL36B caused synthetic enhancement of the growth defect of the ebp2-14 allele at high temperatures. Disruption of RPL36B led to a more severe growth defect than that of RPL36A due to imbalances in the expression levels of the duplicated genes. Primer-extension analysis revealed that L36a/b is required for the processing of 27SA2, 27SA3, and 27SBL pre-rRNAs. Two-hybrid analysis indicated that Ebp2 interacts with ribosomal proteins L36a/b, L34a/b, and L8, which in mature ribosomes are located adjacent to each other in close proximity to the 3′ end of 5.8S rRNA. These results suggest that Ebp2 functions cooperatively with ribosomal proteins L36, L34, and L8 in biogenesis of the 60S ribosomal subunit.

Background A defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis. Rrs1p, an essential nuclear protein, is required for the secretory response. Results EBP2, encoding the yeast homologue of a human protein that interacts with Epstein–Barr virus Nuclear Antigen 1, was cloned in a two-hybrid screen using RRS1 as a bait. The rrs1-1 mutation, which produces Rrs1p without the C-terminal half and causes a defect in the secretory response, almost abolished the interaction with Ebp2p. Ebp2p is essential for growth and is mainly localized in the nucleolus. The effects of Ebp2p depletion on ribosome biogenesis is quite similar to that of Rrs1p depletion; in the Ebp2p-depleted cells, the rate of pre-rRNA processing is slower, and significantly less mature 25S rRNA is produced compared to those in wild-type cells. The polysome pattern indicates that Ebp2p-depletion causes a decrease of 80S monosomes and polysomes, an accumulation of 40S subunits, and the appearance of half-mer polysomes. Conclusions Ebp2p is required for the maturation of 25S rRNA and 60S subunit assembly. Ebp2p may be one of the target proteins of Rrs1p for executing the signal to regulate ribosome biogenesis.

To identify new proteins involved in Mn2+ homeostasis, we isolated Mn(2+)-resistant mutants of Saccharomyces cerevisiae starting from a calcineurin-deficient, Mn2+ hypersensitive strain (delta cmp1 delta cmp2). The mutations were found to lie in the PMR1 gene, known to encode a "P-type" Ca(2+)-ATPase that transports Ca2+ and Mn2+ from the cytosol to the Golgi apparatus. A second gene, AHP1, was cloned as a suppressor of the Mn2+ tolerance of a delta cmp1 delta cmp2 pmr1 mutant. Ahp1p was recently described as a thioredoxin peroxidase type II, an antioxidant protein with alkyl hydroperoxide defense properties in yeast. AHP1 disruption in strain W303 decreased tolerance to Mn2+ and H2O2. We found that a GFP-Ahp1p fusion construct was in the cytosol when cells were grown in glucose, and in the mitochondria when cells were grown in oleate. Based on Mn2+ transport data, we concluded that Ahp1p is involved in cellular Mn2+ homeostasis in trafficking of Mn2+ from cytosol to mitochondria and from cytosol for export across the plasma membrane.

Ebp2 has an essential role in the biogenesis of 60S ribosomal subunits. Synthetic-sick alleles with the ebp2-14 mutation were screened. The mutations were localized to the ARD1 and NAT1 genes, which encode the catalytic subunit and the auxiliary subunit of N(α)-acetyltransferase NatA respectively. Polysome analyses revealed that ard1Δ and nat1Δ caused a synergistic defect with ebp2-14 in the assembly of 60S ribosomal subunits. To identify the proteins that functionally interact with NatA, we designed mutants in which the second amino acid was substituted for proline in Ebp2 and functionally related proteins: Brx1, a partner of Ebp2 in ribosome biogenesis, and the ribosomal protein L36a/b, overexpression of which suppresses a growth defect in ebp2-14. Among these, only brx1-S2P exhibited a synthetic defect with ebp2-14. These results suggest that optimal NatA function is important to the cooperative function of Brx1 with Ebp2 in 60S ribosomal subunit biogenesis.

We previously reported the sequence of YL8A, one of the two genes encoding yeast ribosomal protein YL8. With the aim of conducting an evolutionary study we have cloned and sequenced a second gene, YL8B. The disruption of both genes is lethal. Unlike other duplicated ribosomal protein genes, each open reading frame is interrupted by two introns containing long conserved sequences. A comparison of nucleotide and amino-acid sequences reveals that the duplication of the YL8 gene must have occurred very recently. Alignment and phylogenetic analysis of the amino-acid sequences of YL8-related proteins from various species show the existence not only of YL8 ribosomal proteins but also of a family of YL8-like proteins. These are present in at least three species of yeast and seem to be functionally distinct from ribosomal proteins.

The yeast protein Ebp2 is required for early steps in production of 60S ribosomal subunits. To search for cofactors with which Ebp2 functions, or substrates on which it acts, we screened for mutants that were synthetically lethal (sl) with the ebp2-14 mutation. Four different mutant alleles of the 60S ribosomal subunit assembly factor Brx1 were found. To investigate defects of the double mutant, we constructed strains conditional for the ebp2-14 brx1- synthetic lethal phenotype. These ebp2-14 brx1 mutants were defective in processing of 27S pre-rRNA and production of 60S subunits, under conditions where each single mutant was not. Ebp2 and Brx1 exhibit a strong two-hybrid interaction, which is eliminated by some combinations of brx1 and ebp2 mutations. In one such mutant, Ebp2 and Brx1 can still associate with pre-ribosomes, but subunit maturation is perturbed. Depletion of either Ebp2 or Brx1 revealed that Brx1 requires Ebp2 for its stable association with pre-ribosomes, but Ebp2 does not depend on the presence of Brx1 to enter pre-ribosomes. These results suggest that assembly of 60S ribosomal subunits requires cooperation of Ebp2 with Brx1, together with other molecules present in pre-ribosomes, potentially including several found in assembly subcomplexes with Brx1 and Ebp2.

The nucleolus, the most prominent structure observed in the nucleus, is often called a “ribosome factory.” Cells spend an enormous fraction of their resources to achieve the mass-production of ribosomes required by rapid growth. On the other hand, ribosome biogenesis is also tightly controlled, and must be coordinated with other cellular processes. Ribosomal proteins and ribosome biogenesis factors are attractive candidates for this link. Recent results suggest that some of them have functions beyond ribosome biogenesis. Here we review recent progress on ribosome biogenesis factors, Ebp2 and Rrs1, in yeast Saccharomyces cerevisiae. In this organism, Ebp2 and Rrs1 are found in the nucleolus and at the nuclear periphery. At the nuclear envelope, these proteins interact with a membrane-spanning SUN domain protein, Mps3, and play roles in telomere clustering and silencing along with the silent information regulator Sir4. We propose that a protein complex consisting Ebp2, Rrs1 and Mps3 is involved in a wide range of activities at the nuclear envelope.

Using α- and β-hemoglobin sequences, we made a maximum likelihood inference as to the phylogenetic relationship among carnivores, including the two pandas, giant and lesser. Molecular phylogenetic studies up to 1985 had consistently indicated that the giant panda is more closely related to the bear than to the lesser panda. In 1986, however, a contradictory tree was constructed, using hemoglobins and so on, in which the two pandas link together (Tagle et al. 1986). In contrast to that tree, our conclusion supports the close relationship between bear and giant panda. The surface impression of a close relationship between the two pandas drawn from pairwise amino acid differences is explained by a rapid rate of hemoglobin evolution in the bear compared to that in the two pandas.

We isolated and sequenced a gene, YL8A, encoding ribosomal protein YL8 of Saccharomyces cerevisiae. It is one of the two duplicated genes encoding YL8 and is located on chromosome VII while the other is on chromosome XVI. The haploid strains carrying disrupted YL8A grew more slowly than the parent strain. The open reading frame is interrupted with two introns. The predicted amino acid sequence reveals that yeast YL8 is a homolog of mammalian ribosomal protein L7, E.coli L30 and others.

Ebp2p is essential for the assembly of 60S ribosomal subunits, and it interacts with other ribosome assembly factors in Saccharomyces cerevisiae. Two-hybrid screening exhibited that Ebp2p interacted with a small ubiquitin-related modifier (SUMO)-ligase Siz2p and SUMO-related proteins, Ris1p and Wss1p. Mutations of SUMO attachment sites of Ebp2p led to significantly weak interactions with Siz2p, Wss1p, and Ris1p, whereas they exhibited positive interactions with ribosome assembly factors. A SUMO-binding motif of Ris1p was required for interaction with Ebp2p. These results suggest that SUMO mediates the interaction between Ebp2p and SUMO related proteins and that Ebp2p switches its interaction partners via sumoylation.

Three ribonucleases (RNases) with different molecular masses were isolated from human kidney. The enzymes were purified to an electrophoretically homogeneous state, and their respective molecular masses were found to be 18,000 (tentatively named RNase HK-1), 20,000 (RNase HK-2A), and 22,000 (RNase HK-2B) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the amino acid compositions, aminoterminal sequences, and enzymological properties of the enzymes indicate that RNase HK-1 is related to “nonsecretory” RNase, and that RNases HK-2A and HK-2B are both related to “secretory” RNase. Furthermore, RNase HK-1 showed cross-reactivity with an antibody specific to nonsecretory RNase from human urine, whereas RNases HK-2A and HK-2B showed cross-reactivity with another antibody specific to human urine secretory RNase. However, the carbohydrate compositions of RNases HK-2A and HK-2B were markedly different from that of the secretory urine RNase. This finding seems to indicate that the kidney is not the origin of the urine enzyme.

We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.

The amino-acid sequence of cytochrome c 552 (PH c 552) from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus, was more than 50% identical to that of cytochrome c from an extreme thermophile, Hydrogenobacter thermophilus (HT c 552), and from a mesophile, Pseudomonas aeruginosa (PA c 551). The PH c 552 gene was overexpressed as a correctly processed holoprotein in the Escherichia coli periplasm. The overexpressed PH c 552 has been crystallized by vapour diffusion from polyethylene glycol 4000 pH 6.5. The crystals belong to space group C2221, with unit-cell parameters a = 48.98, b = 57.99, c = 56.20 A. The crystals diffract X-rays to around 2.1 A resolution.

Ebp2p, the yeast homolog of human Epstein-Barr virus nuclear antigen 1-binding protein 2, is essential for biogenesis of the 60 S ribosomal subunit. Two-hybrid screening exhibited that, in addition to factors necessary for assembly of the 60 S subunit, Ebp2p interacts with Rps16p, ribosomal protein S16, and the 40 S ribosomal subunit assembly factor, Utp11p, as well as Yil019w, the function of which was previously uncharacterized. Depletion of Yil019w resulted in reduction in levels of both of 18 S rRNA and 40 S ribosomal subunit without affecting levels of 25 S rRNA and 60 S ribosomal subunits. 35 S pre-rRNA and aberrant 23 S RNA accumulated, indicating that pre-rRNA processing at sites A(0)-A(2) is inhibited when Yil019w is depleted. Each combination from Yil019w, Utp11p, and Rps16p showed two-hybrid interaction.

Rrs1p is a ribosomal protein L11-binding protein in Saccharomyces cerevisiae. We have obtained temperature-sensitive rrs1 mutants by random PCR mutagenesis. [3H]Methionine pulse-chase analysis reveals that the rrs1 mutations cause a defect in maturation of 25S rRNA. Ribosomal protein L25-enhanced green fluorescent protein, a reporter of the 60S ribosomal subunit, concentrates in the nucleus with enrichment in the nucleolus when the rrs1 mutants are shifted to the restrictive temperature. These results suggest that Rrs1p stays on the pre-60S particle from the early stage to very late stage of the large-subunit maturation and is required for export of 60S subunits from the nucleolus to the cytoplasm.

We demonstrate that the genes encoding trans- acting factors essential for pre-rRNA processing/ribosomal subunit assembly are responsive to various kinds of stresses such as heat shock, nitrogen deprivation and a secretory defect, in coordination with ribosomal protein genes in Saccharomyces cerevisiae. The rap1-17 mutation, which produces the C-terminally truncated protein of a transcriptional factor Rap1p, affects transcriptional repression of the trans-acting factor genes due to a secretory defect as shown previously for both ribosomal protein and rRNA genes.

A secretory defect causes specific transcriptional repression of both ribosomal protein and ribosomal RNA genes, suggesting the coupling of plasma membrane and ribosome syntheses. We previously reported that the rap1-17 allele, which produced C-terminally truncated Rap1p, derepressed transcription of ribosomal protein genes when the secretory pathway was blocked. In this paper, we demonstrate that the rap1-17 mutation also leads to significant attenuation of transcriptional repression of rRNA genes due to a secretory defect. In contrast, the rap1-2 temperature-sensitive allele containing a unique missense mutation in the middle of the coding sequence has only a weak effect on repression. These results suggest that the C-terminal silencing domain of Rap1p is required for transcriptional repression of rDNA in response to a secretory defect. We also demonstrated that transcriptional regulation of ribosomal protein genes in response to nitrogen limitation was not affected by the rap1-17 allele, suggesting that the mechanism of nitrogen response is distinct from that of the secretory response.

A new variation in the group-specific component (GC) system, which was presumed to have arisen from duplication of the GC IF and GC 1A2 genes, was previously reported from our laboratory. Through a genetic survey of a Japanese population, a second example of the same variation was found in a family.

We have previously shown that a functional secretory pathway is essential for continued ribosome synthesis in Saccharomyces cerevisiae. When a temperature-sensitive mutant defective in the secretory pathway is transferred to the non-permissive temperature, transcription of both rRNA genes and ribosomal protein genes is nearly abolished. In order to define the cis -acting element(s) of ribosomal protein genes sensitive to a defect in the secretory pathway, we have constructed a series of fusion genes containing the CYH2 promoter region, with various deletions, fused to lacZ. Each fusion gene for which transcription is detected is subject to the repression. Rap1p is the transcriptional activator for most ribosomal protein genes, as well as having an important role in silencing in the vicinity of telomeres and at the silent mating-type loci. To assess its role in the repression of transcription by the defect in the secretory pathway, we have introduced rap1 mutations. The replacement of wild-type Rap1p by Rap1p truncated at the C-terminal region caused substantial attenuation of the repression. Furthermore, we have demonstrated that the Rap1p-truncation affects the repression of TCM1 , encoding ribosomal protein L3, which has no Rap1p-binding site in its upstream regulatory region. These results suggest that the repression of transcription of ribosomal protein genes by a secretory defect is mediated through Rap1p, but does not require a Rap1p-binding site within the UAS.

We isolated a temperature-sensitive mutant of Saccharomyces cerevisiae in which transcription both of ribosomal protein genes and of ribosomal RNA is defective at the non-permissive temperature. Temperature-sensitivity for growth is recessive and segregates 2:2. The wild type gene, termed RIC1 (for ribosome control) was cloned by complementation of the temperature-sensitive phenotype from a genomic DNA library based on the CEN plasmid. RIC1 encodes a protein of 1056 amino acid (aa) residues including a putative nuclear localization sequence. Data base searches revealed that RIC1 is a novel gene and predicted aa sequence share some sequence similarity with viral transcriptional regulatory proteins.

From the view point of biological evolution, we have been interested in analyzing sequence similarities of ribosomal proteins (r-proteins) from various organisms. According to Doolittle (1981) the ultimate goal of the study of protein evolution is the reconstruction of the past events that gave rise to the vast inventory of proteins in existence today. In order to reach the goal, we believe that studying the r-proteins is a very attractive way.

We isolated and sequenced YL16A and YL16B encoding ribosomal protein YL16 of Saccharomyces cerevisiae. The two nucleotide sequences within coding regions retain 91.1% identity, and their predicted sequences of 176 amino acids show 93.8% identity. Out of the ribosomal protein sequences from various organisms currently available, no counterpart to YL16 could be found.

We isolated and sequenced a gene, YS11A, encoding ribosomal protein YS11 of Saccharomyces cerevisiae. YS11A is one of two functional copies of the YS11 gene, located on chromosome XVI and transcribed in a lower amount than the other copy which is located on chromosome II. The disruption of YS11A has no effect on the growth of yeast. The 5'-flanking region contains a similar sequence to consensus UASrpg and the T-rich region. The open reading frame is interrupted with an intron located near the 5'-end. The predicted amino acid sequence reveals that yeast YS11 is a homologue to E. coli S4, one of the ram proteins, three chloroplast S4s and others out of the ribosomal protein sequences currently available.

Genetic polymorphism of GC (vitamin D-binding protein) in human urine was revealed by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels containing 2 M urea. Urine samples from 530 unrelated Japanese from the Fukui district, being only 1–2 ml of original urine, were examined, and correct GC typing was achieved by comparison with the results of direct grouping using plasma. Six common and twelve rare phenotypes were observed. The frequencies of the genes were 0.473 for GC ∗ 1F , 0.241 for GC ∗ 1S , 0.254 for GC ∗ 2 , and 0.032 for the total of six rare alleles.

Two ribonucleases (RNases), one active against RNA as well as poly(C) and the other more markedly against poly(C), were isolated from human erythrocytes by acetone fractionation in the presence of 0.25 M H2SO4, followed by a series of column chromatographies. The purified enzymes appeared homogeneous as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), and were tentatively designated RNase HE-1 and RNase HE-2. The content of RNase HE-1 in erythrocytes was much higher than that of RNase HE-2. The molecular mass of RNase HE-1 was determined to be 18,000 and 16,000 Da, and that of RNase He-2 39,000 and 31,000 Da, by SDS-PAGE and gel filtration, respectively. The catalytic properties and structural features of RNase HE-1 including the amino acid composition and N-terminal amino acid sequence indicated that its protein moiety is strictly related to a nonsecretory RNase purified from human urine (Yasuda et al., 1988, Biochim. Biophys. Acta 965, 185-195). In particular, the N-terminal amino acid sequence up to the 32nd residue was identical with that of urine nonsecretory RNase reported recently (Beintema et al., 1988, Biochemistry 27, 4530-4538). Furthermore, RNase HE-1 was immunologically indistinguishable from urine nonsecretory RNase, but clearly differed from urine secretory RNase. On the other hand, erythrocyte RNase HE-2 was enzymologically and immunologically similar to urine secretory RNase.

Genetic polymorphism of transferrin (TF) was revealed in human urine by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels. Using this technique more than 300 urine samples were examined, and correct TF typing from a small volume of urine (approx. 0.5 ml) was achieved, in comparison with the results of direct grouping for plasma. Three common phenotypes, TF C1, C2-1 and C2, were differentiated. In addition, the rare types TF C1D, C2D, and C1B were observed. The frequencies of the TF alleles in our samples were found to be: TF∗C1 = 0.7265, TF∗C2 = 0.2624, TF∗D = 0.0083 and TF∗B = 0.0028.

A ribonuclease (RNase) was isolated from the urine of a 35-year-old male and purified to electrophoretic homogeneity. The enzyme was tentatively designated RNase 2. A rabbit antibody produced by injection of the purified RNase 2 was able to distinguish RNase 2 from another type of RNase coexisting in body fluids. With this antibody it was possible to detect RNase 2 isozymes in human serum and urine without difficulty using isoelectric focusing or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting. Both RNase 2 in serum and urine seemed to exist in multiple forms with regard to their molecular masses and pI values. This technique may prove to be useful in genetic and forensic studies of RNase polymorphism.