Interactions of the eIF-4F Subunits in the Yeast Saccharomyces cerevisiae”

Recognition of the cap structure at the 5’ end of mRNA is one of the first events in initiation of eukaryotic translation. This step is mediated by the transla- tion initiation factor 4F (eIF-4F). In mammalian cells this factor is composed of the cap-binding protein eIF- 4E, eIF-4A, and a 220-kDa polypeptide. In yeast Saccharomyces cerevisiae, eIF-4E is found associated with a 150-kDa protein (p150) and a 20-kDa protein (p20). The resulting protein complex is proposed to represent yeast eIF-4F. To study the functions of p150 and p20 and their interaction with eIF-4E, we disrupted the genes encoding pl50 and p20 and analyzed the effects on protein complex formation and cell viability. Yeast cells with single and double disruptions of the genes encoding p150 and p20 are viable, but p150 single and p150/p20 double disruptions show a slow growth phenotype. Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E. Eukaryotic cellular mRNAs carry at their 5’ nitrocellulose was for 45 min at 70 V, 220 mA in a Mini Trans Blot Cell (Bio-Rad). Blots were saturated in TBS containing 5% BSA for 30 min at 4 "C, followed by incubation in 1:lOOO diluted rat polyclonal anti-eIF-4F antibody (Altmann et al., 1987; this antibody also recognizes the 70-kDa heat shock protein concomitantly purified with eIF-4F) in TBS containing 0.5% BSA for 2 h to overnight at 4 "C. After washing in TBS for 30 min, the filters were decorated with rabbit anti-rat antibody conjugated with peroxydase (DAKO, Denmark) and with TBS containing chloronaphthol (0.018%) and H202 (0.006%). When the color reaction was faint, the filters were rewashed with TBS, 0.5% BSA for 1 h, decorated with alkaline phosphatase-conjugated swine anti-rabbit antibody (DAKO, Denmark), and stained with 5-bromo-4-chloro-indolylphosphate (X-phosphate)/nitroblue tetrazolium chloride according to the manufac- turer (Boehringer Mannheim).

Recognition of the cap structure at the 5' end of mRNA is one of the first events in initiation of eukaryotic translation. This step is mediated by the translation initiation factor 4F (eIF-4F). In mammalian cells this factor is composed of the cap-binding protein eIF-4E, eIF-4A, and a 220-kDa polypeptide. In yeast Saccharomyces cerevisiae, eIF-4E is found associated with a 150-kDa protein (p150) and a 20-kDa protein (p20). The resulting protein complex is proposed to represent yeast eIF-4F. To study the functions of p150 and p20 and their interaction with eIF-4E, we disrupted the genes encoding p l 5 0 and p20 and analyzed the effects on protein complex formation and cell viability. Yeast cells with single and double disruptions of the genes encoding p150 and p20 are viable, but p150 single and p150/p20 double disruptions show a slow growth phenotype. Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E.
Eukaryotic cellular mRNAs carry at their 5' end the cap structure m7GpppX ( X means any ribonucleotide) (for a review, see Shatkin, 1976). The cap structure protects mRNAs against exonucleases (Furuichi et al., 1977) and facilitates initiation of translation (for a review, see Shatkin, 1985). During initiation the cap structure is recognized by eukaryotic initiation factor 4F (eIF-4F). In mammalian cells this factor is composed of three subunits: the cap-binding protein eIF-4E (24 kDa), eIF-4A (46 kDa), and a 220-kDa polypeptide (p220) (for reviews, see Sonenberg, 1988;. Both, eIF-4A and eIF-4E are also found as free polypeptides not associated with eIF-4F. While eIF-4E and p220 are stably associated, eIF-4A can be separated from the complex by phosphocellulose chromatography, suggesting that its interaction with the complex is weaker (Ray et al., 1985). It is believed that binding of eIF-4E to the cap structure is the first step in translation initiation (for a review, see Thach, 1992). Binding of eIF-4F or formation of this complex at the cap structure are thought to stimulate the binding of free eIF-4A and of eIF-4B (Thach, 1992). Together, these factors have RNA helicase activity and are involved in the melting of RNA secondary structure in the leader region of mRNA (Rozen et al., 1990). Cap recognition and melting of RNA secondary structure are essential for the subsequent binding of the 40 S ribosomal subunit to most mRNAs .
Factor eIF-4E and eIF-4F are present in limiting amounts in mammalian cells (Duncan and Hershey, 1987;Hiremath et al., 1985), and eIF-4F was shown to have translational discriminatory activity in in vitro translation systems (Ray et al., 1983). For these reasons binding of eIF-4F to or assembly of eIF-4F at the cap structure is thought to be rate limiting for translation initiation and therefore to represent a key target for translational regulation (Jagus et al., 1981;Thach, 1992). Indeed, cap binding activity in uninfected and virusinfected mammalian cells was shown to be regulated by phosphorylation of eIF-4E and p220 (Lazaris-Karatzas et al., 1990;Huang andSchneider, 1991, for reviews, see Huembelin andThomas, 1991;Thach, 1992) or by degradation of p220 (Etchison et al., 1982, reviewed in Sonenberg, 1987. The availability of powerful genetic techniques has made the yeast Saccharomyes cerevisiae an attractive system for studies of eukaryotic translation (for reviews, see Mueller and Trachsel, 1990;Linder and Prat, 1990). The cap-dependent translation initiation pathway appears to be highly conserved between yeast and mammals. This is demonstrated most impressively by the finding that mouse eIF-4E can substitute for its yeast homologue in vivo (Altmann et al., 1989a). Further elucidation of the functions of individual eIF-4F subunits in translation initiation may be easier to achieve in yeast than in higher eukaryotes. Yeast eIF-4E has been cloned and sequenced (Altmann et al., 1987). Comigration of eIF-4E and a 150-kDa protein (p150) in sucrose density gradients and during DEAE-cellulose chromatography (Goyer et al., 1989) and coelution of eIF-4E with p150 and a 20-kDa protein (p20) from 7-methyl-guanosine-diphosphate (m7GDP)-Sepharose columns  suggest that these polypeptides form a protein complex. Furthermore, p150 and p20 could be shown to cross-link specifically to capped mRNA (Goyer et al., 1989).' Based on these findings, we consider it to be likely that the proteins eIF-4E, p150, and p20 represent subunits of the yeast homologue of mammalian eIF-4F. In contrast to mammalian eIF-4F, yeast eIF-4F does not contain eIF-4A (Goyer et al., 1989).
In the experiments described below, we investigated the interactions of the putative yeast eIF-4F subunits p150 and p20 with eIF-4E by disrupting the genes encoding p150 and p20 and analyzing the effects on cell viability and protein complex formation.

MATERIALS AND METHODS
Strains and Gene Disruptions-The strains used in this work are listed in Table I. If not stated otherwise, restriction enzymes and DNA manipulating enzymes were purchased from Boehringer Mann-'S. Lanker, P. P. Muller, M. Altmann, C. Goyer, N. Sonenberg, and H. Trachsel, unpublished results. Interactions of Subunits in the Yeast Cap-binding Complex heim, and methods to manipulate DNA were according to Sambrook et al. (1989). For growth rate studies, standard complete medium (YPD, Sherman et al., 1986) was used. The gene CAF20 encoding protein p20 has previously been subcloned as a 2.2-kb2 genomic DNA fragment into the Bluescript-M13+vector (Altmann et al., 1989a). This plasmid was cut with BclI in the translation initiation ATG codon, and a 1.2-kb DNA fragment with BglII-restricted ends carrying the URA3 gene was inserted, thus disrupting the CAF20 reading frame. A 3.3-kb EcoRI fragment of this plasmid carrying the disrupted CAF20 gene was used to transform uracil auxotroph (Ura-) haploid (T105D) and diploid (T210A) strains. Transformants were selected for Ura+ prototrophy.
The gene TZF4631-encoding protein p150 has been cloned and ~equenced.~ A 3.2-kb EcoRI cDNA fragment containing the complete TZF4631 open reading frame (ORF) was inserted into the EcoRI-cut vector pUC8 (Sigma). This plasmid was digested with BglII, which cuts out a 2.2-kb fragment of the 2.9-kb TZF4631 ORF, and a 1.2-kb BgUI fragment carrying the URA3 gene was inserted, thus destroying the reading frame of TIF4631. A 3.5-kb EcoRI fragment of this construct containing the deleted/disrupted TIF4631 was used to transform Ura-haploid (T105D) and diploid (T210A) strains, and transformants were selected for growth on minimal medium lacking uracil.
To construct CAF20 TZF4631 double disruptions, we first selected for a spontaneous mutant carrying tif4631::ura3 (Le. a strain which had its genomic TZF4631 gene disrupted by an inactive URA3 gene). This was achieved by growing strain T105D, transformed with a plasmid carrying the tif463l::URA3 disruption allele, on minimal agar medium containing uracil and 5-fluoroorotic acid. 5-Fluoroorotic acid is converted by the URA3 gene product orotidine-5'-phosphate decarboxylase into a toxic product (Boeke et al., 1984). Colonies which were able to grow on 5-fluoroorotic acid-containing medium, but were unable to grow on media without uracil indicating an inactive URA3 allele, were selected. A tif4631::ura3 strain was then crossed with the caf2O::URA3 strain T149B and resulting diploids were sporulated.
Ribosomal Salt Wash Preparation-Yeast cultures were grown to a n ODm of 1.5-2. Cells were collected by centrifugation for 10 min at 4000 X g and washed twice with ice-cold HzO. Cells were resuspended in 1 ml of buffer K (100 mM KCl, 20 mM HEPES-KOH (pH 7.0), 0.2 mM EDTA, 0.05 mM ATP, 10 mM P-mercaptoethanol, 5 mM MgC12, and 0.5 mM phenylmethylsulfonyl fluoride) per g wet cell weight, glass beads (0.4-mm diameter) were added (1 g/ml suspension), and the cells were broken by 5-10 strokes of 20 s each in a Brown MSK cell homogenizer. The resulting extract was centrifuged at 28,000 X g for 20 min at 4 "C, and the supernatant recentrifuged for 4 h at 4 "C in a TST41.14 rotor (Centrikon) a t 200,000 X g. The ribosomal pellet was resuspended in buffer K (4 times pellet volume) adjusted with 4 M KC1 to a final concentration of 0.5 M and dissolved in a Dounce homogenizer with 20 strokes. This suspension was then centrifuged at 4 "C in a TST60.4 rotor (Centrikon) for 1 h at 260,000 X g, the resulting supernatant (ribosomal salt wash, RSW) adjusted to 20% glycerol, quick-frozen in liquid Nz, and stored at -70 "C.
C. Goyer, unpublished observations. eluted with buffer A at a flow rate of 0.1-0.2 ml/min, and 1-ml fractions were collected, adjusted to 10% glycerol, quick-frozen, and stored at -70 "C.
SDS-Polyacrylamide Gel Electrophoresis and Western Blotting-SDS-PAGE with 12 and 15% gels (Anderson et al., 1973) and Western blotting (Towbin et al., 1979) were performed as described. Transfer of proteins to nitrocellulose was for 45 min at 70 V, 220 mA in a Mini Trans Blot Cell (Bio-Rad). Blots were saturated in TBS containing 5% BSA for 30 min a t 4 "C, followed by incubation in 1:lOOO diluted rat polyclonal anti-eIF-4F antibody (Altmann et al., 1987; this antibody also recognizes the 70-kDa heat shock protein concomitantly purified with eIF-4F) in TBS containing 0.5% BSA for 2 h to overnight a t 4 "C. After washing in TBS for 30 min, the filters were decorated with rabbit anti-rat antibody conjugated with peroxydase (DAKO, Denmark) and stained with TBS containing chloronaphthol (0.018%) and H202 (0.006%). When the color reaction was faint, the filters were rewashed with TBS, 0.5% BSA for 1 h, decorated with alkaline phosphatase-conjugated swine anti-rabbit antibody (DAKO, Denmark), and stained with 5-bromo-4-chloro-indolylphosphate (Xphosphate)/nitroblue tetrazolium chloride according to the manufacturer (Boehringer Mannheim).

RESULTS
Disruption of the Genes Encoding p150 and p20"Adsorption of yeast RSW fraction to m7GDP-agarose and elution of proteins from this affinity resin with m7GDP yields the proteins eIF-4E, p150 and p20. They are believed to represent yeast eIF-4F based on their coelution with m7GDP and the fact that they cannot be eluted with GDP . Fig. 1, lane 1, shows a Western blot of RSW fraction from a wild-type strain decorated with a polyclonal anti-yeast eIF-4F antibody (Altmann et al., 1987). This antibody recognizes the eIF-4F subunits (denote the pattern of  T138B was derived from a genetic cross between strain T105D (which was transformed with an integrating plasmid containing the TZF4631 gene disrupted with a URAJ marker gene) and strain A230 (MATa, urd-52). T149B was obtained in a similar cross, but strain T105D was transformed with a plasmid containing the CAF20 gene disrupted with a URA3 marker gene.  degradation products of p150) as well as the 70-kDa heat shock protein SSAl (Slater and Craig, 1989). The 70-kDa heat shock protein is bound to the eIF-4F complex but can be mainly eluted with GDP (Goyer et al., 1989): To study the functions of the yeast eIF-4F subunits p150 and p20, we disrupted the genes encoding p150 and p20 by the one-step procedure of Rothstein (1983, see "Materials and Methods"). Both genes were shown by Southern blotting and hybridization to be present in single copies/haploid genome (results not shown, Altmann et aL, 1989b). Following the terminology introduced by Linder and Slonimsky (1989), we term the gene encoding p20 CAF20 (for cap-associated factor) and the gene encoding p150 TIF4631 (for translation initiation factor). To disrupt the CAF20 gene, the diploid strain T105D and the haploid strain T210A (Table I) were transformed with a 3.3-kb EcoRI restriction fragment containing the entire CAF20 ORF interrupted at its BcZI site (which is located in the ATG translation initiation codon) with the URAJ gene as a genetic marker. Similar numbers of Ura+ transformants were obtained after transformation of haploid and diploid strains, indicating that the CAF20 gene is not essential for growth under normal growth conditions. Only haploid transformants were further analyzed. Genomic DNA of Ura3+ transformants was analyzed by Southern blotting and hybridization to a 32P-labeled CAF20 probe. The results demonstrated the disruption of the CAF20 gene (data not shown). Immunoblots performed with RSW fraction from caf20::URAS disrupted strains showed that p20 was absent, confirming that the disrupted strain did not express this protein (Fig. 1, lune 2 ) . The reduced amount of eIF-4E in lune 2 (also seen in lunes 3 and 4 ) compared to lune 1 is not due t o less protein loaded, since equal amounts of RSW fractions were loaded in each lane (see "Discussion").
Disruption of the TIF4631 gene was done by transforming haploid strain T105D and diploid strain T210A with a 3.5-kb EcoRI cDNA fragment which had part of the ORF of TIF4631 replaced by a 2.1-kb BgZII fragment carrying the URA3 gene (see "Materials and Methods"). Genomic DNA of Ura+ transformants was analyzed by Southern blotting and the disruption of the TIF4631 gene confirmed (data not shown). Both haploid and diploid Ura+ transformants were viable, but haploid transformants showed a slow growth phenotype (Table   ' M. Altmann, unpublished observations.

FIG. 2. AcA44 gel chromatography of RSW fraction of a
wild-type yeast strain. RSW fraction (see "Materials and Methods'') was prepared from wild-type strain T149D and 6 mg of total protein was fractionated by AcA44 gel chromatography as described under "Materials and Methods." Fractions of 1 ml were collected and aliquots of 20 pl were separated by SDS-PAGE and analyzed by Western blotting as described in the legend to Fig. 1. Positions in the gel corresponding to eIF-4E, p150, and p20 are denoted on the left; positions of molecular mass standards (in kDa) are marked on the right. 11). Thus, TIF4631 is not an essential gene. Immunoblots revealed that p150 was absent in tif4631::URA3 disrupted strains (Fig. 1, lune 3 ) .
Next, we tested whether disruption of both genes resulted in a more severe effect on the growth rate of yeast cells than disruption of single genes. To address this question, we first selected for spontaneous mutants with a tif4631::urcd phenotype (see "Materials and Methods"). One such mutant strain was then crossed with a caf20::URA3-disrupted strain, this diploid was sporulated, and the tetrads were analyzed. All tetrads revealed four viable spores with a 2:2 segregation of Ura+ and a slow growth phenotype. Colonies with Ura+/ slow growth phenotypes, indicating caf20::URAS tif4631::ura3 double disruptions, were further analyzed. Measurement of generation times showed a 1.6-fold slower growth rate than tif4631::URA3 strains (Table 11). When a RSW fraction from the double-disrupted strain was analyzed by Western blotting (Fig. 1, lune 4 ) , p150 and p20 were absent. Interestingly, the amount of eIF-4E was even more drastically reduced in this strain as compared to the single disrupted strains (lunes 2 and 3), an observation which will be discussed below (see "Discussion*). We conclude that CAF20 and TIF4631 are nonessential genes. However, disruption of TIF4631 or (more pronounced) CAF20 and TIF4631 results in a slow growth phenotype.
The Proteins p150 and p20 Bind to eIF-4E-The results of previous experiments  suggested that p150 and p20 bound through eIF-4E to the cap affinity column. To verify that p150 and p20 bind to eIF-4E, and to address the question of whether complex maintenance requires the presence of a cap structure or cap analog, we fractionated RSW preparations from wild-type strains and strains carrying caf20::URAS or tif4631::URA3 disruptions by molecular sieve chromatography (AcA44) and analyzed individual fractions by SDS-polyacrylamide gel electrophoresis

Interactions of Subunits in the Yeast Cap-binding Complex
and Western blotting. Fig. 2 shows a Western blot of the different fractions obtained after separation of RSW fraction from the wild-type strain T149D. Polypeptide p150 is only found in fractions eluting early from the column (fractions 3-5, high molecular weight fractions). In contrast, p20 is found in high molecular weight fractions (fractions 4-10) and low molecular weight fractions (fractions 18-21). The 70-kDa heat shock protein is distributed between fractions 4 and 11. Initiation factor eIF-4E appears in low molecular weight fractions and broadly distributed between fractions 3 and 14, indicating that it may form several different high molecular weight complexes.
To test whether p150 and p20 interact directly with eIF-4E, we performed immunoadsorption experiments, in which eIF-4E was reacted with a monoclonal anti-yeast eIF-4E antibody and antibody-antigen complexes adsorbed to protein G-Sepharose. In this way, polypeptides complexed to eIF-4E can be identified by their (indirect) adsorption to protein G-Sepharose. To assay the association of p150 and p20 with eIF-4E, AcA44 column fraction 3 containing all three proteins (Fig. 2, lane 3) was analyzed. Antigen-antibody complexes bound to protein G-Sepharose were eluted with SDS sample buffer, fractionated by SDS-PAGE, blotted onto nitrocellulose, and reacted with the polyclonal anti-eIF-4F antibody. Fig. 3 shows that p150 and p20 in this fraction ( l a n e 2) were bound to eIF-4E and could be adsorbed with the monoclonal antibody to protein G-Sepharose ( l a n e 3). Only minor amounts were not bound and remained in the supernatant ( l a n e 4). The 70-kDa protein did only bind partially to eIF-4E and appeared mainly in the supernatant ( l a n e 4, the distortion of the 70-kDa band in the supernatant is due to albumin in the monoclonal antibody solution). We conclude from these data that eIF-4E can bind both p150 and p20 and that the complex is stable in the absence of a cap structure.

DISCUSSION
Initiation of translation of the vast majority of eukaryotic mRNAs is cap dependent, and the recognition of the cap structure is mediated by the cap-binding protein eIF-4E in the eIF-4F complex, In this report, we characterized the interaction of yeast eIF-4E with the proteins p150 and p20. These proteins are encoded by the genes CAF20 (p20) and TZF4631 (p150). Size fractionation of RSW preparations and immunoadsorption of fractions to a monoclonal anti-yeast eIF-4E antibody revealed that both proteins bind independently of each other to eIF-4E. This was further substantiated by the findings that (i) p20 was bound to eIF-4E in extracts from a TZF4631-disrupted strain and (ii) p150 was complexed to eIF-4E in the absence of p20 thus demonstrating the existence of eIF-4E .p20, eIF-4E .p150, and eIF-4E -p20 .p150 complexes. In addition, we showed that these complexes are stable in the absence of a cap structure.
Previous studies reported the cross-linking of a 150-and a 20-kDa protein to the cap structure (Goyer et aL, 1989): The 150-kDa protein was suggested to be the equivalent of p220 in mammalian eIF-4F. There is strong evidence that this 150-kDa protein and p150 described in this report are identical, since both polypeptides are recognized by the same polyclonal anti-yeast eIF-4F antibody, run identically in SDS-polyacrylamide gels, and coelute with eIF-4E from a cap affinity column. In addition, Goyer et al. (1989) showed that their 150-kDa protein copurifies with eIF-4E through a DEAEcellulose chromatographic step and cosediments with eIF-4E in a sucrose density gradient. Taking all these observations together, we suggest that the yeast eIF-4F complex includes eIF-4E, p150, and p20. However, the existence of additional subunits cannot be excluded.
When the amounts of eIF-4E in wild-type and mutant strains were compared, it was evident that eIF-4E was less abundant in RSW preparations from CAF20-and TIF4631disrupted strains, and very much reduced in RSW preparations from double-disrupted strains. Three explanations seem to be plausible to account for this finding: (i) in CAF20-and/ or TZF4631-disrupted strains, eIF-4E may be less stably associated with the ribosome and therefore lost during purification of ribosomes. However, we have observed a similar reduction of eIF-4E in preribosomal extracts of disrupted strains (results not shown); (ii) in the eIF-4F complex individual subunits are protected from proteolytic degradation. In cells deficient in p150 or p20, eIF-4E would no longer be incorporated into an eIF-4F complex and would therefore be degraded. A similar observation was made in HeLa cells, where depletion of eIF-4E lead to a concomitant and drastic decrease in p220 levels (De Benedetti et aL, 1991); (iii) disruptions of CAF20 and TZF4631 could alter eIF-4E gene expression.
Results from CAF20 and TZF4631 disruption experiments suggest that none of the two proteins is essential for survival of yeast cells. In addition, double mutants were viable as well. The CAF20-disrupted strains behave like wild-type cells in terms of growth rate, mating and sporulation, and in vivo translation (not shown) despite the reduced eIF-4E level suggesting that, at least in 5' . cerevisiue, the amount of this factor in the cell is not limiting under normal conditions for initiation of protein synthesis. TZF4631 and, more pronounced, the double-disrupted strains show a slow growth phenotype, but behave normally in mating and sporulation (not shown). These findings may be interpreted to mean that (i) neither of the two proteins is required for translation; (ii) they play a role in translation only under special conditions, or (iii) there exist other genes in the yeast cell encoding proteins which can take over their biological functions. As judged from high stringency Southern blotting analysis, there is no evidence for the presence of a second gene having homology to CAF20. In the case of the TIF4631 gene, however, our recent results show that a second gene exists whose encoded protein shows remarkable homology in its COOHterminal half with the TZF4631-encoded protein ~1 5 0 .~ Preliminary results revealed that double disruptions of TZF4631 and this second gene are lethal. Thus, it is possible that the function of p150 can be fulfilled by a second gene product. Such a redundancy is known for many other genes encoding translational components. Most ribosomal protein genes are duplicated, and two initiation factors, eIF-4A and eIF-4D, are encoded by duplicate genes. Therefore, it would not be too surprising if a second gene product exists, which can functionally replace p150.
In summary, we have described complexes between eIF-4E, p150, and p20, and proposed that this complex represents yeast eIF-4F.