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Functional analysis in yeast of the Brix protein superfamily involved in the biogenesis of ribosomes
FEMS Yeast Research 3 (2003) 35^43
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Functional analysis in yeast of the Brix protein superfamily involved in the biogenesis of ribosomes Edith Bogengruber a , Peter Briza a , Edith Doppler a , Herbert Wimmer a , Lore Koller a , Franco Fasiolo b , Bruno Senger b , Johannes H. Hegemann c , Michael Breitenbach a; b
a Institute of Genetics and General Biology, University of Salzburg, 5020 Salzburg, Austria UPR 9002 du CNRS, Institut de Biologie Mole¤culaire et Cellulaire du CNRS, 15, rue Rene¤ Descartes, 67084 Strasbourg Cedex, France c Heinrich-Heine-Universita«t Du«sseldorf, Institut fu«r Mikrobiologie, Universita«tsstr. 1, 40225 Du«sseldorf, Germany
Received 8 April 2002; received in revised form 2 July 2002; accepted 2 July 2002 First published online 20 November 2002
Abstract An extensive homology search based on the sequence of the yeast protein Brx1p (biogenesis of ribosomes in Xenopus, YOL077c) revealed that it is a member of a superfamily of proteins sharing remarkable sequence similarities. Previous work on Brx1p showed that this protein is involved in the process of ribosome biogenesis [Kaser et al., Biol. Chem. 382 (2001) 1637^1647]. Brx1p is the founding member of one of the five existing eukaryotic subfamilies which are all present in yeast. Four of them are represented by one essential gene each and one family is represented by two closely related genes which can functionally replace each other but are essential together for survival. We created conditional alleles of four of the five genes which allowed us to study the effect of depletion of the respective proteins on the ribosome profiles of the strains. In this study we show that not only Brx1p but also three additional superfamily members, namely YHR088w (Rpf1p), YKR081c (Rpf2p) and the homologous proteins Ssf1p (YHR066w)/Ssf2p (YDR312w) are all involved in the multistep process of the assembly of the large ribosomal subunit. This agrees well with the fact that these three proteins, like Brx1p, are located in the nucleolus. Moreover, all four proteins closely interact functionally, because all four mutants are suppressed by the same multicopy suppressor gene. = 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ribosome biogenesis; Saccharomyces cerevisiae ; Nucleolus
1. Introduction Ribosome biogenesis exclusively takes place in a specialized compartment of the nucleus, the nucleolus. It represents a distinct nuclear region where a number of chromosomal regions with multiple copies of genes encoding ribosomal RNA approach each other and ribosomal RNA is transcribed. After rDNA transcription, the pre-rRNA is extensively processed, modi¢ed and assembled with ribosomal proteins (for a review see [2]). Three of the four yeast ribosomal RNAs, namely the 25S and the 5.8S rRNA of the large ribosomal subunit and the 18S rRNA of the small subunit, are part of the
* Corresponding author. Tel. : +43 (662) 8044-5786; Fax : +43 (662) 8044-144. E-mail address : [email protected] (M. Breitenbach).
primary rRNA transcript generated by RNA polymerase I. In yeast, the third RNA component of the large 60S particle, the 5S rRNA, is present on the same rDNA repeat but is transcribed by RNA polymerase III in the opposite direction to the polymerase I transcript. The primary transcript containing the large rRNAs has to be extensively processed to yield mature ribosomal RNAs. These maturation steps like methylation, pseudouridylation, as well as endo- and exonucleolytic cleavages mainly occur in the nucleolus and require various enzymes/proteins including RNA helicases [3], RNA 3P-terminal phosphate cyclases [4], so-called assembly factors, and the family of snoRNPs (small nucleolar ribonucleoproteins) [5,6]. To date, only a few of these factors have been functionally characterized in detail. It is, however, generally believed that most of them play essential roles in di¡erent steps of pre-rRNA maturation and ribosome assembly. Recently, Brx1p (biogenesis of ribosomes in Xenopus) was identi¢ed as an essential nucleolar protein. Depletion
1567-1356 / 02 / $22.00 = 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 9 3 - 9
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of Brx1p in a conditional yeast mutant leads to defects in rRNA processing and a block in the assembly of large ribosomal subunits [1]. Homology searches based on Brx1p led to the identi¢cation of a protein superfamily consisting of more than 40 members [7]. The superfamily consists of six families, ¢ve of them eukaryotic and one archaeal. In yeast, four of the ¢ve families are represented by a single essential gene and one family consists of a duplicated gene, the double knockout of which is also lethal. The common feature of this family is a sequence domain of 150^180 amino acid residues called the Brix domain. Most members of this family are not yet functionally characterized. One of the Brix domain-containing proteins, Imp4p, is already known to be a component of the eukaryotic U3 snoRNP complex, which plays an important role in pre-rRNA processing. Some of the proteins of the complex have known enzymatic functions. Others may function as pre-rRNA chaperones [8]. Together with Imp3p, Imp4p is essential for pre-18S rRNA processing at sites A0, A1 and A2, and hence is involved in biogenesis of the small ribosomal subunit [9]. Both essential proteins, Imp3p and Imp4p, interact with Mpp10p and contain an RNA binding domain. A homology search starting from the IMP4 sequence [10] revealed protein families similar to the ones found by Eisenhaber et al. [7]. Very little information is available about the other members of the Brix domain protein superfamily. The essential gene pair, SSF1/SSF2 (suppressor of sterile four), has a potential role in mating [11] but the subcellular localization of Ssf1p was shown to be nucleolar [12], suggesting an additional function besides mating. The remaining two yeast gene family members, RPF1 and RPF2 (ribosome production factor), were largely uncharacterized with no annotation in the databases at the time of preparation of this article. In this communication we present experiments demonstrating that the in silico identi¢ed protein superfamily members Brx1p, Rpf1p, Rpf2p and the homologous proteins Ssf1p/Ssf2p are essential for the biogenesis of the large ribosomal subunit. Our results demonstrate that the proteins are located in vivo in the nucleolar compartment and interact functionally, as mutations in the respective genes are phenotypically suppressed by the yeast gene eIF4G2 (TIF4632) encoding translation initiation factor 4G, which acts as a multicopy suppressor.
2. Materials and methods 2.1. Yeast strains and media Heterozygous diploid disruption strains of BRX1, RPF1, RPF2, SSF1 and SSF2 were generated by the Saccharomyces Genome Deletion Project, and distributed via Euroscarf (strain i.d. : Y21768 (YOL077c, BRX1),
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Y21916 (YHR088w, RPF1), Y25997 (YKR081c, RPF2), Y21894 (YHR066w, SSF1), Y23671 (YDR312w, SSF2), http://www.uni-frankfurt.de/fb15/mikro/euroscarf/col_index. html). The corresponding isogenic wild-type strains were BY4741 (MATa, his3v1, leu2v0, met15v0, ura3v0), BY4742 (MATK, his3v1, leu2v0, lys2v0, ura3v0) and the diploid obtained by mating of the two isogenic haploid strains, BY4743. For the construction of the conditional mutant strains YEB088, YEB081 and YEBssf1 carrying alleles of RPF1, RPF2 and SSF1 with the natural promoter replaced by the tetO7 -CYC1 hybrid promoter [13] we generated a tetO promoter-containing polymerase chain reaction (PCR) fragment with homologous sequences to the promoter and the coding regions of the genes of interest, thus suitable for in vivo recombination. The following primers were used to amplify the tetO promoter from plasmid pAH3 : YHR088/kan (5P-cgatgagatgtaaaaagaaatgcgatgccataagctatactttacgattggcataggccactagtggatctg-3P) and YHR088/tet (5P-tcttgcctctttagcttgtttgtgatgtttatctcattaccgagagccatcgaattgatccggtaatttag-3P), YKR081/kan (5P-ttcttatagtgtaatgcattattggaaagtgatttggtatatcgatgagtgcataggccactagtggatctg-3P) and YKR081/tet (5P-accaaagctctcttggctcttgcattcttgggttttacggttctaatcatcgaattgatccggtaatttag-3P), SSF1/kan (5P-catctcatcgcgatattagttcgatatctgggtttgagtgagtagtatgtgcataggccactagtggatctg-3P) and SSF1/tet (5P-tcaggtgtaagctgtgcatgtgttcttttcttttgtcttctcttggccatcgaattgatccggtaatttag-3P). The respective PCR products were transformed into YUG37 (MATa, ura3-52, trp1-63, leu2-v1: :tTA-LEU2) (J.H. Hegemann, unpublished), a FY1679 strain derivative in which the tTA transactivator under the control of the CMV promoter has been integrated at the genomic LEU2 locus rendering the strain leucine-prototrophic. Selection for positive clones was performed on YPD supplemented with 200 mg l31 G418 (Calbiochem, San Diego, CA, USA). To generate strain YEBssf1vssf2 the loxP-£anked kanMX marker of the tetO cassette was removed by expression of the cre recombinase from plasmid pSH47 [14] and the resulting strain was crossed with the haploid SSF2 deletion strain obtained from Euroscarf (Y13671, BY4742 ; MATK, his3v1, leu2v0, lys2v0, ura3v0, YDR312w: :kanMX4). Tetrad dissection yielded the haploid strain YEBssf1vssf2 harboring a conditional allele of SSF1 and a SSF2 knockout. Media and culture conditions were maintained as described previously [15]. Cells were grown on rich medium (YPD) containing 2% (w/v) peptone, 1% (w/v) yeast extract and 2% (w/v) glucose. 2% (w/ v) agar was added as required for solid media. Drop-out media for selection of plasmids contained 2% (w/v) glucose, 0.5% (w/v) ammonium sulfate, 0.17% (w/v) yeast nitrogen base without amino acids and the required auxotrophic supplements. Cells were sporulated on solid sporulation medium containing 1% (w/v) potassium acetate, 0.1% (w/v) yeast extract, 0.025% (w/v) glucose and the required amino acids. Medium components were obtained
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from Serva (Heidelberg, Germany) and Gibco (Paisley, UK). 2.2. Plasmid constructions The centromeric cognate clones YCplac33-RPF1 and YCplac33-RPF2 and the multicopy cognate clones YEplac195-RPF1 and YEplac195-RPF2 were constructed by cloning PCR fragments generated with primers 88-1 (5Ptaggatccctctaccacctctagtct-3P) and 88-2 (5P-tactaagcttcttactatatctgccgaagc-3P) or 81-1 (5P-taggatcccgcaatgggtacgctaag-3P) and 81-2 (5P-tactaagcttgactggtaaagttaccattg-3P) via BamHI and HindIII into the respective vectors [16]. For localization studies we used the single-copy vector pUG35 (Gu«ldener and Hegemann, http://mips.gsf.de/proj/ yeast/info/tools/hegemann/gfp.html) and generated C-terminal yEGFP fusion constructs termed pUG35-RPF1, pUG35-RPF2 and pUG35-SSF2 which contain the coding sequences under the control of the regulatable MET25 promoter. RPF1 was ampli¢ed using oligonucleotides 88-3 (5P-taggatccggaagaggtaaagcaggc-3P) and 88-4 (5P-cagaattcgtccatctcaggtttatgc-3P) and cloned via BamHI and EcoRI in front of yEGFP. For plasmid pUG35-RPF2 we used the primers 81-3 (5P-taggatcccaggagaacactggcag3P) and 81-4 (5P-tactaagcttctgtcttttagcagagggc-3P) equipped with BamHI and HindIII sites. pUG35-SSF2 was created by cloning the coding region of SSF2 ampli¢ed with oligos SSF-3 (5P-taggatccgtcaccatcccgcacata-3P) and SSF-4 (5Ptactaagcttactgaataagtcactatcc-3P) via BamHI and HindIII into vector pUG35. Restriction enzymes were obtained from Promega (Madison, WI, USA). 2.3. GFP £uorescence microscopy Yeast cells were ¢xed for £uorescence microscopy with 70% ethanol. The cells were mounted in DAPI solution (1 Wg ml31 ) to allow for observation of the nucleus. DAPI (4P,6-diamidino-2-phenylindole) was obtained from Sigma (Vienna, Austria). The green £uorescent protein (GFP) signal of the tagged proteins was examined using the £uorescein ¢lter of a Zeiss Axioskop £uorescence microscope. 2.4. Polysome analysis Yeast strains YEB088, YEB081, YEBssf1vssf2 and YUG37 were ¢rst grown in YPD. The cultures were then split into two parts : one was kept in YPD, the other was cultured overnight in the presence of 100 mg l31 doxycycline (Sigma, St. Louis, MO, USA) to an OD600 of 0.8. Cells were harvested 15 min after addition of cycloheximide (100 Wg ml31 culture). The pellets were washed once with water, and suspended in 5 ml g31 wet weight of lysis bu¡er (25 mM Tris^HCl, pH 7.2, 50 mM KCl, 5 mM MgCl2 , 300 mM sorbitol, 5.6 mM 2-mercaptoethanol, 100 Wg ml31 cycloheximide, 1 mM phenylmethylsulfonyl £uoride). After disruption of the cells with glass
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beads, cell debris was removed by centrifugation. Aliquots of 10 OD260 units were loaded onto sucrose gradients (11 ml, 7^47%) prepared in lysis bu¡er (lacking sorbitol, cycloheximide, and 2-mercaptoethanol) and centrifuged at 170 000Ug for 3.5 h. Gradients were fractionated with a Bio-Rad fraction collector 2118 with continuous monitoring at 280 nm. Fractions of 0.3 ml were treated with 1 volume 20% trichloroacetic acid overnight to precipitate proteins for Western blot analysis. The respective fusion proteins to yEGFP were visualized with a polyclonal rabbit antiGFP antibody diluted 1:600. As a secondary antibody we used horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:2000 dilution) from Sigma (St. Louis, MO, USA). Detection of the luminescence was performed with the ECL1 Western blotting kit from Amersham (Little Chalfont, UK). 2.5. Revertant analysis and construction of the Rev77/7 DNA library Standard yeast genetic methods [17] were used to analyze a set of revertants isolated in the conditionally lethal (very slowly growing) strain YEB77 after ethyl methanesulfonate (EMS) mutagenesis. The revertants were crossed to strain JS619-3A (MATK, leu2-v1: :tTA-LEU2, his3, TRP1, ura3, tetO: :BRX1) to determine the dominant or recessive nature of the revertant loci. The diploids that resulted were homozygous for the conditional allele tetO-BRX1 and heterozygous for the suppressor mutations. Tetrad analysis was then performed to test for regular segregation of the suppressor mutation. The revertants were then crossed to strain JS617-3A (MATK, leu2v1: :tTA-LEU2, his3, TRP1, ura3) in order to test whether the corresponding mutations in the revertant strains were intra- or extragenic. The appearance of mutant phenotypes in the tetrads indicated an extragenic suppressor locus. A genomic library was then constructed from one dominant extragenic revertant by partially digesting chromosomal DNA with Sau3A, precipitating with 5% polyethylene glycol and inserting fragments greater than 2 kb into the BamHI site of the yeast vector, pVT103-U (URA3, 2W) [18] resulting in a genomic DNA library of 36 000 recombinant clones. Suppressor clones were isolated by transforming strain YEB77 with the library and selection of fast growing colonies on plates containing doxycycline.
3. Results 3.1. RPF1, RPF2 and the gene pair SSF1/SSF2 are essential genes in yeast Heterozygous deletion strains of the respective open reading frames were generated by members of the Saccha-
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romyces Genome Deletion Project in the diploid strain BY4743. Using tetrad analysis of the corresponding heterozygous disruption strains we veri¢ed that RPF1, as well as RPF2, whose molecular functions are largely unknown, are essential for viability as already mentioned [19,20]. In each tetrad, we obtained only two viable G418-sensitive haploid segregants. Inference determined that the nongrowing spores contained the kanMX disruption allele. The terminal phenotype of the inviable disruption strains was a microcolony of three to seven cells in both cases. We assume that the residual gene product of the heterozygous mother cell was distributed equally to the four spores and allowed spore germination and the ¢rst mitotic divisions to occur. In both cases the centromeric cognate clones YCplac33-RPF1 and YCplac33-RPF2 as well as the multicopy cognate clones, YEplac195-RPF1 and YEplac195RPF2, complemented the lethal phenotype of the disruption. In the case of the gene pair SSF1/SSF2 the viability of single disruption strains is not a¡ected. By means of tetrad analysis of a diploid disruption strain doubly heterozygous for the ssf1 and ssf2 disruptions we found that doubly deleted haploid strains perform only one to two mitotic divisions and are inviable con¢rming earlier ¢ndings [11]. 3.2. Subcellular localization of Rpf1p, Rpf2p and Ssf2p To determine the subcellular distribution of Rpf1p, Rpf2p and Ssf2p in living cells we constructed C-terminal fusion proteins to yEGFP, termed pUG35-RPF1, pUG35RPF2 and pUG35-SSF2, respectively. All three fusion proteins turned out to be functionally active as assessed by tetrad analysis of the heterozygous deletion strains transformed with the respective construct. Haploid deletion strains containing the GFP fusion constructs mentioned above displayed normal growth on YPD and were used for £uorescence microscopy. Rpf1p, Rpf2p and Ssf2p exclusively stained a subnuclear region, the nucleolus (see Fig. 1). To determine whether localization of Ssf2p depended on the presence of a functional Ssf1p we transformed a vssf1 deletion strain with pUG35-SSF2 and analyzed the subcellular distribution of Ssf2p by GFP £uorescence microscopy. We detected no di¡erence in the localization of Ssf2p in the vssf1 deletion strain. This suggests that Ssf1p is not necessary for correct localization of Ssf2p. 3.3. Depletion of Rpf1p, Rpf2p and Ssf1/Ssf2p a¡ects ribosome biogenesis Sequence similarity of the three genes with Brx1p, an essential protein recently shown to be responsible for biogenesis of the large ribosomal subunit, and their subcellular localization led us to test whether Rpf1p, Rpf2p and Ssf1p/Ssf2p were involved in the complex multistep pathway of ribosome biogenesis. Since all three proteins are
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essential for cell viability we constructed conditionally lethal yeast mutant strains that are alive but produce decreased levels of the protein of interest. We used a system that allowed us to modulate expression levels simply by adding doxycycline to the growth medium. We exchanged the native promoter of RPF1, RPF2 and SSF1 to the regulatable tetO promoter [13]. Doxycycline leads to a suppression of transcription from the tetO promoter within 2 h after addition to the medium [1]. In the case of the homologous gene pair SSF1/SSF2 we constructed strain YEBssf1vssf2 harboring the SSF1 open reading frame under control of the tet promoter, removed the loxP-£anked kanMX cassette (included in the tetO promoter construct) by expression of the cre recombinase and crossed the resulting strain with the haploid SSF2 deletion strain obtained from Euroscarf. After tetrad dissection we selected for a strain containing the conditional allele of SSF1 in one instance and the SSF2 knockout construct in another. Both genomic rearrangements were checked by analytical PCR. Doxycycline does not change the growth characteristics of the wild-type strain (Fig. 2A). The growth characteristics of the conditional strain YEB088 (RPF1) changed upon addition of doxycycline to the medium. As expected, the strain grew more slowly when RPF1 was transcriptionally suppressed (Fig. 2B). However, a similar transcriptional suppression of RPF2 and SSF1 did not change the doubling time of the strains (Fig. 2C,D). Serial dilution tests of all three conditional strains revealed a small colony phenotype indicating decreased vitality (not shown). The conditional mutant strains were used to perform polysome analysis using sucrose gradients. When grown in YPD medium the wild-type YUG37 (Fig. 3A) as well as the mutant strains containing the tetO promoter constructs looked identical with a ladder of polysomes, a large peak of 80S monoribosomes and about equimolar amounts of 60S and 40S subunits (not shown for the mutant strains). In contrast, YEB088 (RPF1), YEB081 (RPF2) and YEBssf1vssf2, when analyzed after growth in the presence of doxycycline, show aberrant ribosomal pro¢les. We observed a reduced amount of polysomes, an accumulation of the 40S ribosomal subunits and a marked decrease in free 60S ribosomal subunits (see Fig. 3C^E). The wild-type strain YUG37 showed a normal pro¢le (Fig. 3B). To test whether Rpf1p, Rpf2p and Ssf1/Ssf2p are structural ribosomal proteins we performed Western blot analysis of sucrose gradient fractions. Strains harboring the conditional alleles just described were transformed with the respective GFP fusion constructs and ribosomal pro¢les of the transformed cells grown in the presence of doxycycline were analyzed. In all three cases the ribosome pro¢les were like those of wild-type cells demonstrating that the GFP fusion constructs are fully functional in terms of ribosome biogenesis. The results of the Western blots revealed that all three fusion proteins were present
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Fig. 1. Rpf1p, Rpf2p and Ssf2p localize to the nucleolus. BY4741 cells expressing the respective yEGFP fusion proteins were visualized by £uorescence and di¡erential interference contrast light microscopy. DNA was stained with DAPI. The blue DAPI color was changed to red for optical reasons. Rpf1p-yEGFP, Rpf2p-yEGFP and Ssf2p-yEGFP are shown in green and localize to a specialized region within the nucleus, the nucleolus. Row I: Localization of Rpf1p. Row II: Localization of Rpf2p. Row III: Localization of Ssf2p.
only in the supernatants but not in any fractions containing mature ribosomes or ribosomal subunits (data not shown). We therefore conclude that Rpf1p, Rpf2p and Ssf1p/ Ssf2p do play an essential role in the biogenesis of the large ribosomal subunit but are not structural components of mature ribosomes. 3.4. In£uence of depletion of Rpf1p, Rpf2p, Ssf1p/Ssf2p and Brx1p on subcellular localization of the remaining Brix protein superfamily members To test whether the localization of any one of the proteins is dependent on the presence of physiological amounts of each of the other proteins, we investigated the subcellular distribution of the corresponding GFP fusion proteins in the conditional strains YEB088 (tetORPF1), YEB081 (tetO-RPF2) and YEBssf1vssf2 (tetOSSF1) as well as in YEB77 (tetO-BRX1). We monitored cells grown on plates containing doxycycline to inhibit the
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expression of the tetO promoter-controlled genes and did not ¢nd a redistribution of the fusion protein in any case. For instance, strain YEB088 harvested after growth with expression of only low amounts of Rpf1p still showed nucleolar localization of Rpf2p, Ssf2p and Brx1p. We conducted the same experiment for all four conditional strains and in all cases Rpf1p, Rpf2p, Ssf2p and Brx1p were found to be exclusively located to the nucleolus as are the proteins in wild-type cells. We conclude that the presence of all four Brix family member proteins is not needed for correct localization of any one of them. 3.5. Isolation of a suppressor clone and investigation of genetic interactions between BRX1, RPF1, RPF2 and SSF1/SSF2 To better understand the role of the Brix domain protein family members we used the slow-growth phenotype of YEB77 (a conditional mutant strain harboring BRX1 under the control of the tetO promoter [1]) for the isola-
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Fig. 2. Depletion of Rpf1p, Rpf2p and Ssf1/Ssf2p in£uences the growth behavior. The wild-type strain YUG37 (A) and the conditional mutant strains YEB088 (B), YEB081 (C) and YEBssf1vssf2 (D) were grown at 28‡C in YPD or in YPD supplemented with doxycycline. In the absence of doxycycline the wild-type and the conditional strains show a similar growth behavior (open triangles). Upon addition of doxycycline the wild-type strain continues to grow whereas the conditional strains change their growth characteristics (¢lled circles).
tion of suppressors. Using EMS mutagenesis, about 20 revertants that grew normally on doxycycline plates were isolated. Genetic analysis showed that all but two revertants harbored dominant mutations which all resided in a
gene other than BRX1. A genomic library constructed from one dominant revertant strain (called Rev7) was transformed into YEB77 and complementing plasmids were isolated by selection of fast-growing colonies on
Fig. 3. Depletion of Rpf1p, Rpf2p and Ssf1p/Ssf2p leads to aberrant ribosomal pro¢les. YUG37 (wild-type) and the conditional mutant strains YEB088, YEB081 and YEBssf1vssf2 were grown overnight at 28‡C in YPD or in YPD supplemented with doxycycline. Cell extracts were fractionated on sucrose density gradients. Peaks of free 40S and 60S ribosomal subunits, of 80S ribosomes and polysomes are indicated. Half-mer polyribosomes are labelled with asterisks. A: YUG37 grown in YPD. B: YUG37 grown in YPD+doxycycline. C: YEB088 (tetO-RPF1) grown in YPD+doxycycline. D : YEB081 (tetO-RPF2) grown in YPD+doxycycline. E: YEBssf1vssf2 grown in YPD+doxycycline.
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Fig. 4. TIF4632 acts as multicopy suppressor of BRX1, RPF1, RPF2 and SSF1/SSF2 mutant strains. The conditional mutant strains YEB77, YEB081, YEB088 and YEBssf were transformed with a multicopy plasmid containing TIF4632. Their growth behavior in comparison with untransformed cells was tested on YPD plates (A) and on YPD plates supplemented with doxycycline (B). TIF4632 restores wild-type-like growth in all four mutant strains.
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plates containing doxycycline. Sequencing of one positive clone revealed that it contained only one complete yeast gene (TIF4632, YGL049c) encoding the mRNA cap binding protein eIF4G2 involved in initiation of translation [21]. Since the sequence of eIF4G2 on the suppressor clone was identical to the published wild-type sequence, we conclude that it functions as a multicopy suppressor. Moreover, genetic analysis (not shown in detail) indicates that eIF4G2 is not the genetic suppressor of strain YEB77/ Rev7. Since the depletion of Rpf1p, Rpf2p and Ssf1/2p closely resembles the phenotype of the brx1 mutant strain [1], we tested whether the suppressor clone containing TIF4632 also suppressed the defect of the conditional alleles of RPF1, RPF2 and SSF1/SSF2. Growth tests on doxycycline plates clearly showed (Fig. 4) that the suppressor clone, but not the empty library plasmid pVT103-U, restored wild-type-like growth properties in the conditional mutant strains (see also Table 1). At the same time, we also tested whether the library plasmid containing TIF4632 was able to suppress an e£1v1 strain [22]. EFL1 encodes a cytoplasmic GTPase that is required for 60S subunit biogenesis. The phenotype of an e£1v1 strain presents very slow growth and a defect in the 60S ribosome subunit biogenesis which is quite similar to that of the strains investigated in the present study. Senger et al. [23] have shown that EFL1 genetically interacts with TIF6, encoding a nucle(ol)ar protein associated with 60S subunits. Wild-type Tif6p, which can act as a multicopy suppressor of the e£1v1 strain, and a mutant allele of TIF6, called tif6*, was isolated as a suppressor clone of an e£1 deletion strain. To test whether the Brix domain family members Brx1p, Rpf1p, Rpf2p and Ssf1p/ Ssf2p follow the same pathway as E£1p, we transformed the e£1 deletion strain with the Brix suppressor clone harboring TIF4632 and, vice versa, the conditional mutant strains YEB77, YEB081, YEB088 and YEBssf1vssf2 were transformed with a plasmid harboring tif6*, the suppressor allele of TIF6. These crosswise suppression tests showed negative results (absence of suppression) in both directions (Table 1). This ¢nding clearly shows that E£1p
Table 1 Depletion of Brix proteins and E£1p cannot be compensated by the same suppressors Strain
YEB77 YEB081 YEB088 YEBssf1vssf2 e£1v1
Growth (suppression) phenotype of cells transformed with Untransformed control
TIF4632
pVT103-U (empty vector)
tif6*
3 3 3 3 3
+ + + + 3
3 3 3 3 3
3 3 3 3 +
Growth tests on YPD plates were performed with an e£1v1 strain transformed with plasmids harboring tif6* or TIF4632, and compared with the Brix family mutants transformed with the same suppressor plasmids. TIF4632 restores wild-type-like growth in Brix mutant strains (indicated as +) as tested on plates containing doxycycline but not in an e£1v1 strain (indicated as 3). In contrast, tif6* positively in£uences the growth behavior of e£1v1 but not of Brix mutant strains.
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and the Brix domain protein family act independently of each other.
4. Discussion In this communication, we present the characterization of four of the ¢ve yeast members of the Brix domain protein superfamily [7]. This superfamily consists of ¢ve eukaryotic and one archaeal subfamily. Our recent experimental data obtained for the yeast protein Brx1p, a member of the Brix homology group, demonstrated involvement of these proteins in ribosomal biogenesis. Whereas in eukaryotes ¢ve subfunctions seem to have evolved with each being carried out by one subfamily member, one single protein seems to perform the task in Archaea. The role for these proteins in Archaea which contain neither nuclei nor nucleoli remains unknown, while the suggested function in ribosome biogenesis of the eukaryotic proteins is now also shown for Rpf1p, Rpf2p and Ssf1p/Ssf2p using the results presented in this paper. All yeast members of the Brix domain protein superfamily are essential for growth. Conditional mutants expressing low amounts of protein were generated to investigate the cellular function of the previously uncharacterized genes RPF1, RPF2 and the gene pair SSF1/SSF2. Depletion of the individual proteins Brx1p, Rpf1p, Rpf2p and Ssf1/2p led to slow growth and aberrant ribosomal pro¢les supporting the hypothesis that all of them are required for the production of mature ribosomes. Since a lack of any one of the investigated proteins leads to an underaccumulation of free 60S ribosomal subunits we propose that the Brix domain protein family speci¢cally affects biogenesis and/or stability of the large ribosomal subunit. Each of these proteins is essential for cell viability showing that they cannot functionally replace each other. We assume that the Brix domain proteins interact only transiently with pre-ribosomal particles, as all three proteins were found to be present only in the supernatant but not in any fractions containing mature ribosomes or ribosomal subunits. Recent results by Gavin et al. [24] have identi¢ed multiprotein complexes in yeast by tandem a⁄nity puri¢cation and mass spectrometry. A search in their database (accessible at http://yeast.cellzome.com) showed that all four members of the Brix domain protein family, Brx1p, Rpf1p, Rpf2p and Ssf1p/2p, are part of one multiprotein complex (complex 163) with an assigned function in RNA metabolism. This result strengthens our interpretation that all four proteins are involved in the same step of ribosome assembly assuming that proteins working together in one complex usually ful¢ll similar functions. Proteins also copuri¢ed in the same complex include Ebp2p and Rlp7p which are both required for processing of the 27S prerRNA. This ¢nding further strengthens our assumption that members of the Brix domain protein family are re-
FEMSYR 1533 13-2-03
quired for processing and/or early assembly steps in ribosome biogenesis. More speci¢cally, Brx1p, Rpf1p, Rpf2p and Ssf1p/Ssf2p are involved in early assembly steps of the large ribosomal subunit, while Imp4p participates in assembly of the small ribosomal subunit [9]. The defective phenotypes of all four conditional mutants presented here are very similar to each other. Moreover, overexpression of eIF4G2 equally suppresses these phenotypes in all four mutants, while it does not suppress the phenotype of e£1v1, a mutant which also confers a defect in 60S ribosomal subunit biogenesis. Taken together these experimental results provide further evidence that the four proteins are functionally related. Two alternative hypotheses come to mind which would explain the experimental results. First, the four genes could act in a dependent consequent sequence, and TIF4632 could act downstream of all four Brix domain-containing proteins. Alternatively, and more probably, the four Brix domain proteins could form a heterotetrameric complex (part of complex 163 [24]), which could be an assembly factor (without specifying the precise biochemical function) for 60S ribosomal subunit biogenesis. Again, the suppressor must act downstream of the four proteins. The proteins are, however, localized in the nucleolus even if the putative heterotetrameric complex cannot be completely formed due to deletion of one of the four proteins. Brx1p is also a member of a di¡erent (partly overlapping) complex that contains the suppressor protein Tif4632 (complex 132 [24]). The relationship of the macromolecular complexes discussed so far to the pre-ribosomal particles isolated on sucrose gradients in [25] and [26] is presently unclear. However, these pre-60S particles contain both Brx1p and Tif6p. The latter protein was found to be a suppressor of e£1v1 [22], a mutant blocking another step in 60S ribosomal subunit biogenesis di¡erent from the one mediated by the Brix family proteins. All four proteins shown here to be assembly factors for the large ribosomal subunit have also recently been shown to be RNA binding proteins speci¢cally binding to the 35S and 27S precursor RNAs of the large ribosomal subunit [27]. They are, however, not endonucleases. Taken together, the presented data and the literature discussed above demonstrate that the four Brix superfamily proteins, Brx1p, Rpf1p, Rpf2p, and Ssf1p/Ssf2p, are localized in the nucleolus and together (probably as a heterotetrameric complex) mediate a distinct step in the early assembly of the 60S ribosomal subunit of yeast. A defect in this step is suppressed by overexpression of Tif4632, a protein previously known as a translation initiation factor but which, according to the MIPS database (http:// mips.gsf.de/proj/yeast/) also contains a degenerate RNA recognition motif. This fact and the identi¢cation of large multiprotein assemblies [24] point towards a combinatorial principle (network hypothesis) necessary to explain physiological regulation in a eukaryotic cell with only about 5700 genes.
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Acknowledgements This work was ¢nancially supported by the Austrian Science Fund FWF, Project P12103-MOB to M.B.
[16]
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[1] Kaser, A., Bogengruber, E., Hallegger, M., Doppler, E., Lepperdinger, G., Jantsch, M., Breitenbach, M. and Kreil, G. (2001) Brix from Xenopus laevis and Brx1p from yeast de¢ne a new family of proteins involved in the biogenesis of large ribosomal subunits. Biol. Chem. 382, 1637^1647. [2] Venema, J. and Tollervey, D. (1999) Ribosome synthesis in Saccharomyces cerevisiae. Annu. Rev. Genet. 33, 261^311. [3] Burger, F., Daugeron, M.C. and Linder, P. (2000) Dbp10p, a putative RNA helicase from Saccharomyces cerevisiae, is required for ribosome biogenesis. Nucleic Acids Res. 28, 2315^2323. [4] Billy, E., Wegierski, T., Nasr, F. and Filipowicz, W. (2000) Rcl1p, the yeast protein similar to the RNA 3P-phosphate cyclase, associates with U3 snoRNP and is required for 18S rRNA biogenesis. EMBO J. 19, 2115^2126. [5] Olson, M.O., Dundr, M. and Szebeni, A. (2000) The nucleolus: an old factory with unexpected capabilities. Trends Cell Biol. 10, 189^ 196. [6] Kressler, D., Linder, P. and de La Cruz, J. (1999) Protein trans-acting factors involved in ribosome biogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 7897^7912. [7] Eisenhaber, F., Wechselberger, C. and Kreil, G. (2001) The Brix domain protein family ^ a key to the ribosomal biogenesis pathway ? Trends Biochem. Sci. 26, 345^347. [8] Gerbi, S.A. (1995) Small nucleolar RNA. Biochem. Cell Biol. 73, 845^858. [9] Lee, S.J. and Baserga, S.J. (1999) Imp3p and Imp4p, two speci¢c components of the U3 small nucleolar ribonucleoprotein that are essential for pre-18S rRNA processing. Mol. Cell. Biol. 19, 5441^ 5452. [10] Mayer, C., Suck, D. and Poch, O. (2001) The archaeal homolog of the Imp4 protein, a eukaryotic U3 snoRNP component. Trends Biochem. Sci. 26, 143^144. [11] Yu, Y. and Hirsch, J.P. (1995) An essential gene pair in Saccharomyces cerevisiae with a potential role in mating. DNA Cell Biol. 14, 411^418. [12] Kim, J. and Hirsch, J.P. (1998) A nucleolar protein that a¡ects mating e⁄ciency in Saccharomyces cerevisiae by altering the morphological response to pheromone. Genetics 149, 795^805. [13] Gari, E., Piedra¢ta, L., Aldea, M. and Herrero, E. (1997) A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast 13, 837^848. [14] Gu«ldener, U., Heck, S., Fielder, T., Beinhauer, J. and Hegemann, J.H. (1996) A new e⁄cient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24, 2519^2524. [15] Bogengruber, E., Eichberger, T., Briza, P., Dawes, I.W., Breitenbach, M. and Schricker, R. (1998) Sporulation-speci¢c expression of the yeast DIT1/DIT2 promoter is controlled by a newly identi¢ed repres-
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sor element and the short form of Rim101p. Eur. J. Biochem. 258, 430^436. Gietz, R.D. and Sugino, A. (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking sixbase pair restriction sites. Gene 74, 527^534. Rose, M.D., Winston, F. and Hieter, P. (1990) Methods in Yeast Genetics. A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Vernet, T., Dignard, D. and Thomas, D.Y. (1987) A family of yeast expression vectors containing the phage f1 intergenic region. Gene 52, 225^233. Winzeler, E.A., Shoemaker, D.D., Astromo¡, A., Liang, H., Anderson, K., Andre¤, B., Bangham, R., Benito, R., Boeke, J.D., Bussey, H., Chu, A.M., Connelly, C., Davis, K., Dietrich, F., Dow, S.W., El Bakkoury, M., Foury, F., Friend, S.H., Gentalen, E., Giaever, G., Hegemann, J.H., Jones, T., Laub, M., Liao, H. and Davis, R.W. et al. (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901^906. Zuniga, S., Boskovic, J., Garcia-Cantalejo, J.M., Jimenez, A., Ballesta, J.P. and Remacha, M. (1999) Deletion of 24 open reading frames from chromosome XI from Saccharomyces cerevisiae and phenotypic analysis of the deletants. Gene 233, 141^150. Goyer, C., Altmann, M., Lee, H.S., Blanc, A., Deshmukh, M., Woolford Jr., J.L., Trachsel, H. and Sonenberg, N. (1993) TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function. Mol. Cell. Biol. 13, 4860^4874. Senger, B., Despons, L., Walter, P., Jakubowski, H. and Fasiolo, F. (2001) Yeast cytoplasmic and mitochondrial methionyl-tRNA synthetases: two structural frameworks for identical functions. J. Mol. Biol. 311, 205^216. Basu, U., Si, K., Warner, J.R. and Maitra, U. (2001) The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis. Mol. Cell. Biol. 21, 1453^1462. Gavin, A.C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., Schultz, J., Rick, J.M., Michon, A.M., Cruciat, C.M., Remor, M., Hofert, C., Schelder, M., Brajenovic, M., Ru¡ner, H., Merino, A., Klein, K., Hudak, M., Dickson, D., Rudi, T., Gnau, V., Bauch, A., Bastuck, S., Huhse, B., Leutwein, C., Heurtier, M.A., Copley, R.R., Edelmann, A., Querfurth, E., Rybin, V., Drewes, G., Raida, M., Bouwmeester, T., Bork, P., Seraphin, B., Kuster, B., Neubauer, G. and Superti-Furga, G. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141^147. Harnpicharnchai, P., Jakovljevic, J., Horsey, E., Miles, T., Roman, J., Rout, M., Meagher, D., Imai, B., Guo, Y., Brame, C.J., Shabanowitz, J., Hunt, D.F. and Woolford Jr., J.L. (2001) Composition and functional characterization of yeast 66S ribosome assembly intermediates. Mol. Cell 8, 505^515. Fatica, A., Cronshaw, A.D., Dlakic, M. and Tollervey, D. (2002) Ssf1p prevents premature processing of an early pre-60S ribosomal particle. Mol. Cell 9, 341^351. Wehner, K.A. and Baserga, S.J. (2002) The sigma(70)-like motif. A eukaryotic RNA-binding domain unique to a superfamily of proteins required for ribosome biogenesis. Mol. Cell 9, 329^339.
Cyaan Magenta Geel Zwart
www.fems-microbiology.org
Functional analysis in yeast of the Brix protein superfamily involved in the biogenesis of ribosomes Edith Bogengruber a , Peter Briza a , Edith Doppler a , Herbert Wimmer a , Lore Koller a , Franco Fasiolo b , Bruno Senger b , Johannes H. Hegemann c , Michael Breitenbach a; b
a Institute of Genetics and General Biology, University of Salzburg, 5020 Salzburg, Austria UPR 9002 du CNRS, Institut de Biologie Mole¤culaire et Cellulaire du CNRS, 15, rue Rene¤ Descartes, 67084 Strasbourg Cedex, France c Heinrich-Heine-Universita«t Du«sseldorf, Institut fu«r Mikrobiologie, Universita«tsstr. 1, 40225 Du«sseldorf, Germany
Received 8 April 2002; received in revised form 2 July 2002; accepted 2 July 2002 First published online 20 November 2002
Abstract An extensive homology search based on the sequence of the yeast protein Brx1p (biogenesis of ribosomes in Xenopus, YOL077c) revealed that it is a member of a superfamily of proteins sharing remarkable sequence similarities. Previous work on Brx1p showed that this protein is involved in the process of ribosome biogenesis [Kaser et al., Biol. Chem. 382 (2001) 1637^1647]. Brx1p is the founding member of one of the five existing eukaryotic subfamilies which are all present in yeast. Four of them are represented by one essential gene each and one family is represented by two closely related genes which can functionally replace each other but are essential together for survival. We created conditional alleles of four of the five genes which allowed us to study the effect of depletion of the respective proteins on the ribosome profiles of the strains. In this study we show that not only Brx1p but also three additional superfamily members, namely YHR088w (Rpf1p), YKR081c (Rpf2p) and the homologous proteins Ssf1p (YHR066w)/Ssf2p (YDR312w) are all involved in the multistep process of the assembly of the large ribosomal subunit. This agrees well with the fact that these three proteins, like Brx1p, are located in the nucleolus. Moreover, all four proteins closely interact functionally, because all four mutants are suppressed by the same multicopy suppressor gene. = 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ribosome biogenesis; Saccharomyces cerevisiae ; Nucleolus
1. Introduction Ribosome biogenesis exclusively takes place in a specialized compartment of the nucleus, the nucleolus. It represents a distinct nuclear region where a number of chromosomal regions with multiple copies of genes encoding ribosomal RNA approach each other and ribosomal RNA is transcribed. After rDNA transcription, the pre-rRNA is extensively processed, modi¢ed and assembled with ribosomal proteins (for a review see [2]). Three of the four yeast ribosomal RNAs, namely the 25S and the 5.8S rRNA of the large ribosomal subunit and the 18S rRNA of the small subunit, are part of the
* Corresponding author. Tel. : +43 (662) 8044-5786; Fax : +43 (662) 8044-144. E-mail address : [email protected] (M. Breitenbach).
primary rRNA transcript generated by RNA polymerase I. In yeast, the third RNA component of the large 60S particle, the 5S rRNA, is present on the same rDNA repeat but is transcribed by RNA polymerase III in the opposite direction to the polymerase I transcript. The primary transcript containing the large rRNAs has to be extensively processed to yield mature ribosomal RNAs. These maturation steps like methylation, pseudouridylation, as well as endo- and exonucleolytic cleavages mainly occur in the nucleolus and require various enzymes/proteins including RNA helicases [3], RNA 3P-terminal phosphate cyclases [4], so-called assembly factors, and the family of snoRNPs (small nucleolar ribonucleoproteins) [5,6]. To date, only a few of these factors have been functionally characterized in detail. It is, however, generally believed that most of them play essential roles in di¡erent steps of pre-rRNA maturation and ribosome assembly. Recently, Brx1p (biogenesis of ribosomes in Xenopus) was identi¢ed as an essential nucleolar protein. Depletion
1567-1356 / 02 / $22.00 = 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 9 3 - 9
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of Brx1p in a conditional yeast mutant leads to defects in rRNA processing and a block in the assembly of large ribosomal subunits [1]. Homology searches based on Brx1p led to the identi¢cation of a protein superfamily consisting of more than 40 members [7]. The superfamily consists of six families, ¢ve of them eukaryotic and one archaeal. In yeast, four of the ¢ve families are represented by a single essential gene and one family consists of a duplicated gene, the double knockout of which is also lethal. The common feature of this family is a sequence domain of 150^180 amino acid residues called the Brix domain. Most members of this family are not yet functionally characterized. One of the Brix domain-containing proteins, Imp4p, is already known to be a component of the eukaryotic U3 snoRNP complex, which plays an important role in pre-rRNA processing. Some of the proteins of the complex have known enzymatic functions. Others may function as pre-rRNA chaperones [8]. Together with Imp3p, Imp4p is essential for pre-18S rRNA processing at sites A0, A1 and A2, and hence is involved in biogenesis of the small ribosomal subunit [9]. Both essential proteins, Imp3p and Imp4p, interact with Mpp10p and contain an RNA binding domain. A homology search starting from the IMP4 sequence [10] revealed protein families similar to the ones found by Eisenhaber et al. [7]. Very little information is available about the other members of the Brix domain protein superfamily. The essential gene pair, SSF1/SSF2 (suppressor of sterile four), has a potential role in mating [11] but the subcellular localization of Ssf1p was shown to be nucleolar [12], suggesting an additional function besides mating. The remaining two yeast gene family members, RPF1 and RPF2 (ribosome production factor), were largely uncharacterized with no annotation in the databases at the time of preparation of this article. In this communication we present experiments demonstrating that the in silico identi¢ed protein superfamily members Brx1p, Rpf1p, Rpf2p and the homologous proteins Ssf1p/Ssf2p are essential for the biogenesis of the large ribosomal subunit. Our results demonstrate that the proteins are located in vivo in the nucleolar compartment and interact functionally, as mutations in the respective genes are phenotypically suppressed by the yeast gene eIF4G2 (TIF4632) encoding translation initiation factor 4G, which acts as a multicopy suppressor.
2. Materials and methods 2.1. Yeast strains and media Heterozygous diploid disruption strains of BRX1, RPF1, RPF2, SSF1 and SSF2 were generated by the Saccharomyces Genome Deletion Project, and distributed via Euroscarf (strain i.d. : Y21768 (YOL077c, BRX1),
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Y21916 (YHR088w, RPF1), Y25997 (YKR081c, RPF2), Y21894 (YHR066w, SSF1), Y23671 (YDR312w, SSF2), http://www.uni-frankfurt.de/fb15/mikro/euroscarf/col_index. html). The corresponding isogenic wild-type strains were BY4741 (MATa, his3v1, leu2v0, met15v0, ura3v0), BY4742 (MATK, his3v1, leu2v0, lys2v0, ura3v0) and the diploid obtained by mating of the two isogenic haploid strains, BY4743. For the construction of the conditional mutant strains YEB088, YEB081 and YEBssf1 carrying alleles of RPF1, RPF2 and SSF1 with the natural promoter replaced by the tetO7 -CYC1 hybrid promoter [13] we generated a tetO promoter-containing polymerase chain reaction (PCR) fragment with homologous sequences to the promoter and the coding regions of the genes of interest, thus suitable for in vivo recombination. The following primers were used to amplify the tetO promoter from plasmid pAH3 : YHR088/kan (5P-cgatgagatgtaaaaagaaatgcgatgccataagctatactttacgattggcataggccactagtggatctg-3P) and YHR088/tet (5P-tcttgcctctttagcttgtttgtgatgtttatctcattaccgagagccatcgaattgatccggtaatttag-3P), YKR081/kan (5P-ttcttatagtgtaatgcattattggaaagtgatttggtatatcgatgagtgcataggccactagtggatctg-3P) and YKR081/tet (5P-accaaagctctcttggctcttgcattcttgggttttacggttctaatcatcgaattgatccggtaatttag-3P), SSF1/kan (5P-catctcatcgcgatattagttcgatatctgggtttgagtgagtagtatgtgcataggccactagtggatctg-3P) and SSF1/tet (5P-tcaggtgtaagctgtgcatgtgttcttttcttttgtcttctcttggccatcgaattgatccggtaatttag-3P). The respective PCR products were transformed into YUG37 (MATa, ura3-52, trp1-63, leu2-v1: :tTA-LEU2) (J.H. Hegemann, unpublished), a FY1679 strain derivative in which the tTA transactivator under the control of the CMV promoter has been integrated at the genomic LEU2 locus rendering the strain leucine-prototrophic. Selection for positive clones was performed on YPD supplemented with 200 mg l31 G418 (Calbiochem, San Diego, CA, USA). To generate strain YEBssf1vssf2 the loxP-£anked kanMX marker of the tetO cassette was removed by expression of the cre recombinase from plasmid pSH47 [14] and the resulting strain was crossed with the haploid SSF2 deletion strain obtained from Euroscarf (Y13671, BY4742 ; MATK, his3v1, leu2v0, lys2v0, ura3v0, YDR312w: :kanMX4). Tetrad dissection yielded the haploid strain YEBssf1vssf2 harboring a conditional allele of SSF1 and a SSF2 knockout. Media and culture conditions were maintained as described previously [15]. Cells were grown on rich medium (YPD) containing 2% (w/v) peptone, 1% (w/v) yeast extract and 2% (w/v) glucose. 2% (w/ v) agar was added as required for solid media. Drop-out media for selection of plasmids contained 2% (w/v) glucose, 0.5% (w/v) ammonium sulfate, 0.17% (w/v) yeast nitrogen base without amino acids and the required auxotrophic supplements. Cells were sporulated on solid sporulation medium containing 1% (w/v) potassium acetate, 0.1% (w/v) yeast extract, 0.025% (w/v) glucose and the required amino acids. Medium components were obtained
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from Serva (Heidelberg, Germany) and Gibco (Paisley, UK). 2.2. Plasmid constructions The centromeric cognate clones YCplac33-RPF1 and YCplac33-RPF2 and the multicopy cognate clones YEplac195-RPF1 and YEplac195-RPF2 were constructed by cloning PCR fragments generated with primers 88-1 (5Ptaggatccctctaccacctctagtct-3P) and 88-2 (5P-tactaagcttcttactatatctgccgaagc-3P) or 81-1 (5P-taggatcccgcaatgggtacgctaag-3P) and 81-2 (5P-tactaagcttgactggtaaagttaccattg-3P) via BamHI and HindIII into the respective vectors [16]. For localization studies we used the single-copy vector pUG35 (Gu«ldener and Hegemann, http://mips.gsf.de/proj/ yeast/info/tools/hegemann/gfp.html) and generated C-terminal yEGFP fusion constructs termed pUG35-RPF1, pUG35-RPF2 and pUG35-SSF2 which contain the coding sequences under the control of the regulatable MET25 promoter. RPF1 was ampli¢ed using oligonucleotides 88-3 (5P-taggatccggaagaggtaaagcaggc-3P) and 88-4 (5P-cagaattcgtccatctcaggtttatgc-3P) and cloned via BamHI and EcoRI in front of yEGFP. For plasmid pUG35-RPF2 we used the primers 81-3 (5P-taggatcccaggagaacactggcag3P) and 81-4 (5P-tactaagcttctgtcttttagcagagggc-3P) equipped with BamHI and HindIII sites. pUG35-SSF2 was created by cloning the coding region of SSF2 ampli¢ed with oligos SSF-3 (5P-taggatccgtcaccatcccgcacata-3P) and SSF-4 (5Ptactaagcttactgaataagtcactatcc-3P) via BamHI and HindIII into vector pUG35. Restriction enzymes were obtained from Promega (Madison, WI, USA). 2.3. GFP £uorescence microscopy Yeast cells were ¢xed for £uorescence microscopy with 70% ethanol. The cells were mounted in DAPI solution (1 Wg ml31 ) to allow for observation of the nucleus. DAPI (4P,6-diamidino-2-phenylindole) was obtained from Sigma (Vienna, Austria). The green £uorescent protein (GFP) signal of the tagged proteins was examined using the £uorescein ¢lter of a Zeiss Axioskop £uorescence microscope. 2.4. Polysome analysis Yeast strains YEB088, YEB081, YEBssf1vssf2 and YUG37 were ¢rst grown in YPD. The cultures were then split into two parts : one was kept in YPD, the other was cultured overnight in the presence of 100 mg l31 doxycycline (Sigma, St. Louis, MO, USA) to an OD600 of 0.8. Cells were harvested 15 min after addition of cycloheximide (100 Wg ml31 culture). The pellets were washed once with water, and suspended in 5 ml g31 wet weight of lysis bu¡er (25 mM Tris^HCl, pH 7.2, 50 mM KCl, 5 mM MgCl2 , 300 mM sorbitol, 5.6 mM 2-mercaptoethanol, 100 Wg ml31 cycloheximide, 1 mM phenylmethylsulfonyl £uoride). After disruption of the cells with glass
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beads, cell debris was removed by centrifugation. Aliquots of 10 OD260 units were loaded onto sucrose gradients (11 ml, 7^47%) prepared in lysis bu¡er (lacking sorbitol, cycloheximide, and 2-mercaptoethanol) and centrifuged at 170 000Ug for 3.5 h. Gradients were fractionated with a Bio-Rad fraction collector 2118 with continuous monitoring at 280 nm. Fractions of 0.3 ml were treated with 1 volume 20% trichloroacetic acid overnight to precipitate proteins for Western blot analysis. The respective fusion proteins to yEGFP were visualized with a polyclonal rabbit antiGFP antibody diluted 1:600. As a secondary antibody we used horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:2000 dilution) from Sigma (St. Louis, MO, USA). Detection of the luminescence was performed with the ECL1 Western blotting kit from Amersham (Little Chalfont, UK). 2.5. Revertant analysis and construction of the Rev77/7 DNA library Standard yeast genetic methods [17] were used to analyze a set of revertants isolated in the conditionally lethal (very slowly growing) strain YEB77 after ethyl methanesulfonate (EMS) mutagenesis. The revertants were crossed to strain JS619-3A (MATK, leu2-v1: :tTA-LEU2, his3, TRP1, ura3, tetO: :BRX1) to determine the dominant or recessive nature of the revertant loci. The diploids that resulted were homozygous for the conditional allele tetO-BRX1 and heterozygous for the suppressor mutations. Tetrad analysis was then performed to test for regular segregation of the suppressor mutation. The revertants were then crossed to strain JS617-3A (MATK, leu2v1: :tTA-LEU2, his3, TRP1, ura3) in order to test whether the corresponding mutations in the revertant strains were intra- or extragenic. The appearance of mutant phenotypes in the tetrads indicated an extragenic suppressor locus. A genomic library was then constructed from one dominant extragenic revertant by partially digesting chromosomal DNA with Sau3A, precipitating with 5% polyethylene glycol and inserting fragments greater than 2 kb into the BamHI site of the yeast vector, pVT103-U (URA3, 2W) [18] resulting in a genomic DNA library of 36 000 recombinant clones. Suppressor clones were isolated by transforming strain YEB77 with the library and selection of fast growing colonies on plates containing doxycycline.
3. Results 3.1. RPF1, RPF2 and the gene pair SSF1/SSF2 are essential genes in yeast Heterozygous deletion strains of the respective open reading frames were generated by members of the Saccha-
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romyces Genome Deletion Project in the diploid strain BY4743. Using tetrad analysis of the corresponding heterozygous disruption strains we veri¢ed that RPF1, as well as RPF2, whose molecular functions are largely unknown, are essential for viability as already mentioned [19,20]. In each tetrad, we obtained only two viable G418-sensitive haploid segregants. Inference determined that the nongrowing spores contained the kanMX disruption allele. The terminal phenotype of the inviable disruption strains was a microcolony of three to seven cells in both cases. We assume that the residual gene product of the heterozygous mother cell was distributed equally to the four spores and allowed spore germination and the ¢rst mitotic divisions to occur. In both cases the centromeric cognate clones YCplac33-RPF1 and YCplac33-RPF2 as well as the multicopy cognate clones, YEplac195-RPF1 and YEplac195RPF2, complemented the lethal phenotype of the disruption. In the case of the gene pair SSF1/SSF2 the viability of single disruption strains is not a¡ected. By means of tetrad analysis of a diploid disruption strain doubly heterozygous for the ssf1 and ssf2 disruptions we found that doubly deleted haploid strains perform only one to two mitotic divisions and are inviable con¢rming earlier ¢ndings [11]. 3.2. Subcellular localization of Rpf1p, Rpf2p and Ssf2p To determine the subcellular distribution of Rpf1p, Rpf2p and Ssf2p in living cells we constructed C-terminal fusion proteins to yEGFP, termed pUG35-RPF1, pUG35RPF2 and pUG35-SSF2, respectively. All three fusion proteins turned out to be functionally active as assessed by tetrad analysis of the heterozygous deletion strains transformed with the respective construct. Haploid deletion strains containing the GFP fusion constructs mentioned above displayed normal growth on YPD and were used for £uorescence microscopy. Rpf1p, Rpf2p and Ssf2p exclusively stained a subnuclear region, the nucleolus (see Fig. 1). To determine whether localization of Ssf2p depended on the presence of a functional Ssf1p we transformed a vssf1 deletion strain with pUG35-SSF2 and analyzed the subcellular distribution of Ssf2p by GFP £uorescence microscopy. We detected no di¡erence in the localization of Ssf2p in the vssf1 deletion strain. This suggests that Ssf1p is not necessary for correct localization of Ssf2p. 3.3. Depletion of Rpf1p, Rpf2p and Ssf1/Ssf2p a¡ects ribosome biogenesis Sequence similarity of the three genes with Brx1p, an essential protein recently shown to be responsible for biogenesis of the large ribosomal subunit, and their subcellular localization led us to test whether Rpf1p, Rpf2p and Ssf1p/Ssf2p were involved in the complex multistep pathway of ribosome biogenesis. Since all three proteins are
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essential for cell viability we constructed conditionally lethal yeast mutant strains that are alive but produce decreased levels of the protein of interest. We used a system that allowed us to modulate expression levels simply by adding doxycycline to the growth medium. We exchanged the native promoter of RPF1, RPF2 and SSF1 to the regulatable tetO promoter [13]. Doxycycline leads to a suppression of transcription from the tetO promoter within 2 h after addition to the medium [1]. In the case of the homologous gene pair SSF1/SSF2 we constructed strain YEBssf1vssf2 harboring the SSF1 open reading frame under control of the tet promoter, removed the loxP-£anked kanMX cassette (included in the tetO promoter construct) by expression of the cre recombinase and crossed the resulting strain with the haploid SSF2 deletion strain obtained from Euroscarf. After tetrad dissection we selected for a strain containing the conditional allele of SSF1 in one instance and the SSF2 knockout construct in another. Both genomic rearrangements were checked by analytical PCR. Doxycycline does not change the growth characteristics of the wild-type strain (Fig. 2A). The growth characteristics of the conditional strain YEB088 (RPF1) changed upon addition of doxycycline to the medium. As expected, the strain grew more slowly when RPF1 was transcriptionally suppressed (Fig. 2B). However, a similar transcriptional suppression of RPF2 and SSF1 did not change the doubling time of the strains (Fig. 2C,D). Serial dilution tests of all three conditional strains revealed a small colony phenotype indicating decreased vitality (not shown). The conditional mutant strains were used to perform polysome analysis using sucrose gradients. When grown in YPD medium the wild-type YUG37 (Fig. 3A) as well as the mutant strains containing the tetO promoter constructs looked identical with a ladder of polysomes, a large peak of 80S monoribosomes and about equimolar amounts of 60S and 40S subunits (not shown for the mutant strains). In contrast, YEB088 (RPF1), YEB081 (RPF2) and YEBssf1vssf2, when analyzed after growth in the presence of doxycycline, show aberrant ribosomal pro¢les. We observed a reduced amount of polysomes, an accumulation of the 40S ribosomal subunits and a marked decrease in free 60S ribosomal subunits (see Fig. 3C^E). The wild-type strain YUG37 showed a normal pro¢le (Fig. 3B). To test whether Rpf1p, Rpf2p and Ssf1/Ssf2p are structural ribosomal proteins we performed Western blot analysis of sucrose gradient fractions. Strains harboring the conditional alleles just described were transformed with the respective GFP fusion constructs and ribosomal pro¢les of the transformed cells grown in the presence of doxycycline were analyzed. In all three cases the ribosome pro¢les were like those of wild-type cells demonstrating that the GFP fusion constructs are fully functional in terms of ribosome biogenesis. The results of the Western blots revealed that all three fusion proteins were present
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Fig. 1. Rpf1p, Rpf2p and Ssf2p localize to the nucleolus. BY4741 cells expressing the respective yEGFP fusion proteins were visualized by £uorescence and di¡erential interference contrast light microscopy. DNA was stained with DAPI. The blue DAPI color was changed to red for optical reasons. Rpf1p-yEGFP, Rpf2p-yEGFP and Ssf2p-yEGFP are shown in green and localize to a specialized region within the nucleus, the nucleolus. Row I: Localization of Rpf1p. Row II: Localization of Rpf2p. Row III: Localization of Ssf2p.
only in the supernatants but not in any fractions containing mature ribosomes or ribosomal subunits (data not shown). We therefore conclude that Rpf1p, Rpf2p and Ssf1p/ Ssf2p do play an essential role in the biogenesis of the large ribosomal subunit but are not structural components of mature ribosomes. 3.4. In£uence of depletion of Rpf1p, Rpf2p, Ssf1p/Ssf2p and Brx1p on subcellular localization of the remaining Brix protein superfamily members To test whether the localization of any one of the proteins is dependent on the presence of physiological amounts of each of the other proteins, we investigated the subcellular distribution of the corresponding GFP fusion proteins in the conditional strains YEB088 (tetORPF1), YEB081 (tetO-RPF2) and YEBssf1vssf2 (tetOSSF1) as well as in YEB77 (tetO-BRX1). We monitored cells grown on plates containing doxycycline to inhibit the
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expression of the tetO promoter-controlled genes and did not ¢nd a redistribution of the fusion protein in any case. For instance, strain YEB088 harvested after growth with expression of only low amounts of Rpf1p still showed nucleolar localization of Rpf2p, Ssf2p and Brx1p. We conducted the same experiment for all four conditional strains and in all cases Rpf1p, Rpf2p, Ssf2p and Brx1p were found to be exclusively located to the nucleolus as are the proteins in wild-type cells. We conclude that the presence of all four Brix family member proteins is not needed for correct localization of any one of them. 3.5. Isolation of a suppressor clone and investigation of genetic interactions between BRX1, RPF1, RPF2 and SSF1/SSF2 To better understand the role of the Brix domain protein family members we used the slow-growth phenotype of YEB77 (a conditional mutant strain harboring BRX1 under the control of the tetO promoter [1]) for the isola-
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Fig. 2. Depletion of Rpf1p, Rpf2p and Ssf1/Ssf2p in£uences the growth behavior. The wild-type strain YUG37 (A) and the conditional mutant strains YEB088 (B), YEB081 (C) and YEBssf1vssf2 (D) were grown at 28‡C in YPD or in YPD supplemented with doxycycline. In the absence of doxycycline the wild-type and the conditional strains show a similar growth behavior (open triangles). Upon addition of doxycycline the wild-type strain continues to grow whereas the conditional strains change their growth characteristics (¢lled circles).
tion of suppressors. Using EMS mutagenesis, about 20 revertants that grew normally on doxycycline plates were isolated. Genetic analysis showed that all but two revertants harbored dominant mutations which all resided in a
gene other than BRX1. A genomic library constructed from one dominant revertant strain (called Rev7) was transformed into YEB77 and complementing plasmids were isolated by selection of fast-growing colonies on
Fig. 3. Depletion of Rpf1p, Rpf2p and Ssf1p/Ssf2p leads to aberrant ribosomal pro¢les. YUG37 (wild-type) and the conditional mutant strains YEB088, YEB081 and YEBssf1vssf2 were grown overnight at 28‡C in YPD or in YPD supplemented with doxycycline. Cell extracts were fractionated on sucrose density gradients. Peaks of free 40S and 60S ribosomal subunits, of 80S ribosomes and polysomes are indicated. Half-mer polyribosomes are labelled with asterisks. A: YUG37 grown in YPD. B: YUG37 grown in YPD+doxycycline. C: YEB088 (tetO-RPF1) grown in YPD+doxycycline. D : YEB081 (tetO-RPF2) grown in YPD+doxycycline. E: YEBssf1vssf2 grown in YPD+doxycycline.
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Fig. 4. TIF4632 acts as multicopy suppressor of BRX1, RPF1, RPF2 and SSF1/SSF2 mutant strains. The conditional mutant strains YEB77, YEB081, YEB088 and YEBssf were transformed with a multicopy plasmid containing TIF4632. Their growth behavior in comparison with untransformed cells was tested on YPD plates (A) and on YPD plates supplemented with doxycycline (B). TIF4632 restores wild-type-like growth in all four mutant strains.
41
plates containing doxycycline. Sequencing of one positive clone revealed that it contained only one complete yeast gene (TIF4632, YGL049c) encoding the mRNA cap binding protein eIF4G2 involved in initiation of translation [21]. Since the sequence of eIF4G2 on the suppressor clone was identical to the published wild-type sequence, we conclude that it functions as a multicopy suppressor. Moreover, genetic analysis (not shown in detail) indicates that eIF4G2 is not the genetic suppressor of strain YEB77/ Rev7. Since the depletion of Rpf1p, Rpf2p and Ssf1/2p closely resembles the phenotype of the brx1 mutant strain [1], we tested whether the suppressor clone containing TIF4632 also suppressed the defect of the conditional alleles of RPF1, RPF2 and SSF1/SSF2. Growth tests on doxycycline plates clearly showed (Fig. 4) that the suppressor clone, but not the empty library plasmid pVT103-U, restored wild-type-like growth properties in the conditional mutant strains (see also Table 1). At the same time, we also tested whether the library plasmid containing TIF4632 was able to suppress an e£1v1 strain [22]. EFL1 encodes a cytoplasmic GTPase that is required for 60S subunit biogenesis. The phenotype of an e£1v1 strain presents very slow growth and a defect in the 60S ribosome subunit biogenesis which is quite similar to that of the strains investigated in the present study. Senger et al. [23] have shown that EFL1 genetically interacts with TIF6, encoding a nucle(ol)ar protein associated with 60S subunits. Wild-type Tif6p, which can act as a multicopy suppressor of the e£1v1 strain, and a mutant allele of TIF6, called tif6*, was isolated as a suppressor clone of an e£1 deletion strain. To test whether the Brix domain family members Brx1p, Rpf1p, Rpf2p and Ssf1p/ Ssf2p follow the same pathway as E£1p, we transformed the e£1 deletion strain with the Brix suppressor clone harboring TIF4632 and, vice versa, the conditional mutant strains YEB77, YEB081, YEB088 and YEBssf1vssf2 were transformed with a plasmid harboring tif6*, the suppressor allele of TIF6. These crosswise suppression tests showed negative results (absence of suppression) in both directions (Table 1). This ¢nding clearly shows that E£1p
Table 1 Depletion of Brix proteins and E£1p cannot be compensated by the same suppressors Strain
YEB77 YEB081 YEB088 YEBssf1vssf2 e£1v1
Growth (suppression) phenotype of cells transformed with Untransformed control
TIF4632
pVT103-U (empty vector)
tif6*
3 3 3 3 3
+ + + + 3
3 3 3 3 3
3 3 3 3 +
Growth tests on YPD plates were performed with an e£1v1 strain transformed with plasmids harboring tif6* or TIF4632, and compared with the Brix family mutants transformed with the same suppressor plasmids. TIF4632 restores wild-type-like growth in Brix mutant strains (indicated as +) as tested on plates containing doxycycline but not in an e£1v1 strain (indicated as 3). In contrast, tif6* positively in£uences the growth behavior of e£1v1 but not of Brix mutant strains.
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and the Brix domain protein family act independently of each other.
4. Discussion In this communication, we present the characterization of four of the ¢ve yeast members of the Brix domain protein superfamily [7]. This superfamily consists of ¢ve eukaryotic and one archaeal subfamily. Our recent experimental data obtained for the yeast protein Brx1p, a member of the Brix homology group, demonstrated involvement of these proteins in ribosomal biogenesis. Whereas in eukaryotes ¢ve subfunctions seem to have evolved with each being carried out by one subfamily member, one single protein seems to perform the task in Archaea. The role for these proteins in Archaea which contain neither nuclei nor nucleoli remains unknown, while the suggested function in ribosome biogenesis of the eukaryotic proteins is now also shown for Rpf1p, Rpf2p and Ssf1p/Ssf2p using the results presented in this paper. All yeast members of the Brix domain protein superfamily are essential for growth. Conditional mutants expressing low amounts of protein were generated to investigate the cellular function of the previously uncharacterized genes RPF1, RPF2 and the gene pair SSF1/SSF2. Depletion of the individual proteins Brx1p, Rpf1p, Rpf2p and Ssf1/2p led to slow growth and aberrant ribosomal pro¢les supporting the hypothesis that all of them are required for the production of mature ribosomes. Since a lack of any one of the investigated proteins leads to an underaccumulation of free 60S ribosomal subunits we propose that the Brix domain protein family speci¢cally affects biogenesis and/or stability of the large ribosomal subunit. Each of these proteins is essential for cell viability showing that they cannot functionally replace each other. We assume that the Brix domain proteins interact only transiently with pre-ribosomal particles, as all three proteins were found to be present only in the supernatant but not in any fractions containing mature ribosomes or ribosomal subunits. Recent results by Gavin et al. [24] have identi¢ed multiprotein complexes in yeast by tandem a⁄nity puri¢cation and mass spectrometry. A search in their database (accessible at http://yeast.cellzome.com) showed that all four members of the Brix domain protein family, Brx1p, Rpf1p, Rpf2p and Ssf1p/2p, are part of one multiprotein complex (complex 163) with an assigned function in RNA metabolism. This result strengthens our interpretation that all four proteins are involved in the same step of ribosome assembly assuming that proteins working together in one complex usually ful¢ll similar functions. Proteins also copuri¢ed in the same complex include Ebp2p and Rlp7p which are both required for processing of the 27S prerRNA. This ¢nding further strengthens our assumption that members of the Brix domain protein family are re-
FEMSYR 1533 13-2-03
quired for processing and/or early assembly steps in ribosome biogenesis. More speci¢cally, Brx1p, Rpf1p, Rpf2p and Ssf1p/Ssf2p are involved in early assembly steps of the large ribosomal subunit, while Imp4p participates in assembly of the small ribosomal subunit [9]. The defective phenotypes of all four conditional mutants presented here are very similar to each other. Moreover, overexpression of eIF4G2 equally suppresses these phenotypes in all four mutants, while it does not suppress the phenotype of e£1v1, a mutant which also confers a defect in 60S ribosomal subunit biogenesis. Taken together these experimental results provide further evidence that the four proteins are functionally related. Two alternative hypotheses come to mind which would explain the experimental results. First, the four genes could act in a dependent consequent sequence, and TIF4632 could act downstream of all four Brix domain-containing proteins. Alternatively, and more probably, the four Brix domain proteins could form a heterotetrameric complex (part of complex 163 [24]), which could be an assembly factor (without specifying the precise biochemical function) for 60S ribosomal subunit biogenesis. Again, the suppressor must act downstream of the four proteins. The proteins are, however, localized in the nucleolus even if the putative heterotetrameric complex cannot be completely formed due to deletion of one of the four proteins. Brx1p is also a member of a di¡erent (partly overlapping) complex that contains the suppressor protein Tif4632 (complex 132 [24]). The relationship of the macromolecular complexes discussed so far to the pre-ribosomal particles isolated on sucrose gradients in [25] and [26] is presently unclear. However, these pre-60S particles contain both Brx1p and Tif6p. The latter protein was found to be a suppressor of e£1v1 [22], a mutant blocking another step in 60S ribosomal subunit biogenesis di¡erent from the one mediated by the Brix family proteins. All four proteins shown here to be assembly factors for the large ribosomal subunit have also recently been shown to be RNA binding proteins speci¢cally binding to the 35S and 27S precursor RNAs of the large ribosomal subunit [27]. They are, however, not endonucleases. Taken together, the presented data and the literature discussed above demonstrate that the four Brix superfamily proteins, Brx1p, Rpf1p, Rpf2p, and Ssf1p/Ssf2p, are localized in the nucleolus and together (probably as a heterotetrameric complex) mediate a distinct step in the early assembly of the 60S ribosomal subunit of yeast. A defect in this step is suppressed by overexpression of Tif4632, a protein previously known as a translation initiation factor but which, according to the MIPS database (http:// mips.gsf.de/proj/yeast/) also contains a degenerate RNA recognition motif. This fact and the identi¢cation of large multiprotein assemblies [24] point towards a combinatorial principle (network hypothesis) necessary to explain physiological regulation in a eukaryotic cell with only about 5700 genes.
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Acknowledgements This work was ¢nancially supported by the Austrian Science Fund FWF, Project P12103-MOB to M.B.
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