Schizosaccharomyces pombe has a novel eukaryotic initiation factor 4F complex containing a cap-binding protein with the human eIF4E C-terminal motif KSGST.

Genetic and biochemical analyses were performed on the cytoplasmic cap-binding complex (eukaryotic initiation factor (eIF) 4F) of Schizosaccharomyces pombe. Genomic and cDNA sequencing of the S. pombe gene (tif1) encoding the cap-binding component eIF4E revealed the presence of two introns in a reading frame of 219 codons. The encoded sequence of 218 amino acids shows a greater degree of identity to the mammalian eIF4E sequence than does its counterpart from Saccharomyces cerevisiae. In particular, unlike its S. cerevisiae counterpart, S.pombe eIF4E has a C-terminal Ser209 within the motif KSGST that is a site of phosphorylation in hamster and rabbit eIF4E. Of relevance to its potential regulatory role, eIF4E was found to be encoded by an mRNA with a six-nucleotide leader and to be of low abundance in vivo. Cross-linking experiments identified S. pombe eIF4E as the major cap-binding protein while a further protein, p36, also showed cap-dependent binding. eIF4A was not associated with the cap-binding complex. While S. pombe eIF4E was shown capable of binding S. cerevisiae p20, an equivalent protein was absent from the eIF4F complex isolated from S. pombe cells. S. pombe 4F therefore shows a remarkable combination of structural and functional properties, some of which it shares with its higher and its lower eukaryotic counterparts.

Recognition of the m 7 Gppp 5Ј cap by the cap-binding complex eIF4F 1 (eukaryotic initiation factor 4F) is thought to be a prerequisite of efficient translational initiation on the majority of eukaryotic mRNAs (1). The "core" protein of this complex is eIF4E, which not only binds the cap structure but also interacts with the other eIF4F components (2,3). The overall identifiable structure of eIF4F always contains eIF4E but otherwise varies somewhat from organism to organism (4). The human complex, at least as defined biochemically, comprises eIF4E (25 kDa) together with the DEAD motif protein eIF4A (46 kDa) and eIF4G (154 kDa) and is thought to require eIF4B (69 kDa) to mediate binding of the 43 S preinitiation complex to mRNA (5). One model proposes that eIF4A and eIF4B jointly "unwind" intramolecular structures in the 5Ј-untranslated region of the mRNA, thus smoothing the path of the scanning ribosome toward initiation (6). Moreover, it has been suggested that eIF4G functions as a docking station for eIF4E, eIF4A, and eIF3, thus coordinating their respective activities at the 5Ј end of the mRNA (7,8). eIF4G is also a substrate for viral proteases; picornaviral protease 2A cleaves it into an N-terminal eIF4E-binding domain and a C-terminal eIF4A/eIF3-binding domain (7). The eIF4F complexes of plants (9), Drosophila (10), and Saccharomyces cerevisiae (11) have considerably different structures from that of eIF4F in mammalian cells (4). None of these complexes contain eIF4A, and they show diversity with respect to the other components. In S. cerevisiae, eIF4E can associate with either of two forms of eIF4G (Ref. 12; p150, 107 kDa; p130, 104 kDa) and a third component of unknown function, p20 (18 kDa;13,14). Neither p20 nor eIF4B (49 kDa) are essential in S. cerevisiae (13,15), although disruption of the latter results in a slow growth phenotype (15).
The least variable of the eIF4F components is eIF4E. This essential protein is present in all cases examined so far and generally retains certain conserved features, most notably eight individual tryptophan residues distributed over a central region of the amino acid sequence. Moreover, eIF4E species from different organisms can be at least partly functionally exchangeable. For example, mouse eIF4E can substitute for its S. cerevisiae homologue in vivo (16). The primary evidence that eIF4E is required for translation derives from in vitro experiments, but in fact its exact role in vivo is still unknown. Indeed, there is some suspicion that this factor is involved in processes other than translational initiation. This is suggested by the observation that a fraction of the cellular population of eIF4E in COS cells (17) and S. cerevisiae (18) is located in the nucleus. Moreover, the S. cerevisiae gene encoding eIF4E has been identified as the locus of a cell cycle mutation (cdc33) that arrests the mitotic cycle at the "start" stage (19).
Despite intensive investigation, the question how translational initiation can be regulated via eIF4F remains unclear. One potentially important regulatory mechanism relates to the phosphorylation status of eIF4E. Correlations between the level of eIF4E phosphorylation and the rate of cellular protein synthesis have suggested that phosphorylation of mammalian eIF4E has a positive effect on this factor's activity (20). Moreover, it has been reported that the phosphorylated form has a 3-fold enhanced affinity for the cap (21). After some confusion about the true major site of phosphorylation in the mammalian eIF4E amino acid sequence, there is now agreement that this is * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM (22,23). This residue is found in the motif KSGST in the eIF4E sequences of human, rabbit, and mouse. However, this C-terminal motif is missing in S. cerevisiae eIF4E, which is relatively poorly phosphorylated at two N-terminal sites (Ser 2 and Ser 15 ; 14). A further question relevant to the potential regulatory role of eIF4E that remains unresolved is whether the activity of this factor constitutes a key point of strong kinetic control for the overall process of translational initiation. In particular, there has been uncertainty on the issue whether eIF4E is "limiting" in terms of its abundance in the cell (18, 24 -26).
Looking at the known eIF4F complexes as a whole, it seems likely that they constitute variants on a common theme. However, apart from eIF4E and possibly eIF4G, the truly essential components of the minimal eIF4F complex that is functional in cap-dependent translation remain unknown. Moreover, it is unclear to what extent the different overall structures described to-date represent individual responses to specific functional requirements in the context of translation. For example, the particular features of S. cerevisiae eIF4F raise challenging questions, including whether the observed type of structure is a general feature in lower eukaryotes, and whether eIF4E phosphorylation in yeast is not related to functional regulation via the proposed mammalian type pathway. Given the potential of work in yeast to provide important insight into the structure, function, and regulation of eIF4F, there is a pressing need to understand the significance of these apparent deviations from the mammalian type of system. In the present paper, we describe the analogous complex in Schizosaccharomyces pombe, a fission yeast with a number of characteristics more typical of higher than of lower eukaryotic cells. We report a remarkable combination of features; S. pombe eIF4F has a novel overall structure but retains the mammalian C-terminal KSGST motif in its eIF4E component as well as sharing other properties with its counterpart complex in S. cerevisiae.
General DNA and RNA Methods-S. pombe chromosomal DNA was isolated as described previously (28). Southern blot analysis was performed using Hybond-N membranes (Amersham Corp.). Hybridization was performed at 42°C in the presence of 50% formamide and 6 ϫ SSC according to a standard procedure (29). S. pombe total RNA was prepared as described by Kä ufer et al. (30). Poly(A) ϩ mRNA was isolated from total RNA using the Oligotex TM mRNA kit (Qiagen). Northern blot analysis was performed after RNA gel electrophoresis using the glyoxal denaturation method (29). Colony hybridization of the Escherichia coli amplified S. pombe 3-kilobase DNA bank was performed at 42°C using Hybond-N membranes in the presence of 50% formamide and 6 ϫ SSC. DNA probes were labeled with a [ 32 P]dATP using the Prime-a-Gene kit (Promega). S1 nuclease mapping was performed according to Sambrook et al. (29). Primer extension analysis of the 5Ј end of the mRNA using poly(A) ϩ mRNA was performed according to Leer et al. (31). The primer used was 5Ј-ATCAAAAACCGTCCGCAGACC-3Ј, which corresponds to the region 67-87 of the S. pombe eIF4E ORF. The S. pombe gene encoding eIF4E (sptif1) was cloned directly using chromosomal DNA from a wild-type strain. The DNA sequence of the newly characterized S. pombe DNA as well as of all plasmid constructs was determined using either the Sequenase 2.0 kit (Amersham Corp.) or the ABI PRISM dye terminator cycle sequencing kit (Perkin-Elmer) and the ABI 373 DNA Sequencer.
Plasmid Construction-The complete S. pombe eIF4E cDNA sequence was reassembled in the S. cerevisiae expression vector YCp33Supex2 (32) in three steps. First, the 193-bp HindIII-NcoI fragment from the genomic S. pombe clone (containing the 5Ј end of the gene) and the 800-bp NcoI-BamHI fragment from the sp12217 cDNA (a plasmid from an S. pombe cDNA bank containing the C-terminal coding region) were both ligated into BluescriptSKϩ (Stratagene). Second, an NdeI site was engineered at the AUG start codon using polymerase chain reaction amplification. Third, the resulting NdeI-BamHI 860-bp fragment containing the complete cDNA sequence of the eIF4E gene was cloned into YCpSupex2 between the NdeI and BglII sites, yielding YCpSupex2-SP4E. The same fragment was also used for cloning in pCYTEXP3 (33), generating the construct pCYTEXP3-SP4E. For the two-hybrid system (34), either human, S. cerevisiae, or S. pombe eIF4Eencoding sequences were inserted between the SmaI and BamHI sites of the polylinker of the pGBT9 vector (Clontech) containing the DNAbinding domain of the GAL4 transcriptional activator. The gene sequence encoding the p20 protein from S. cerevisiae was cloned as a SmaI-BamHI fragment into the polylinker of the pGAD424 vector (Clontech) bearing the GAL4 activation domain. pSL301 (35) was used for the cloning of chromosomal fragments.
Protein Methods-eIF-4E was purified from S. pombe cells grown in YPD (27) medium using a 100-liter fermentor. Cells were harvested in mid-exponential phase (A 600 ϭ 0.8), washed with cold water, and resuspended in buffer H (20 mM Hepes (pH 7.4), 100 mM KCl, 15 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Disruption of the cells was achieved using a Braun shaker (B. Braun type 853022/0, Germany) in the presence of glass beads (0.45-0.5-mm diameter). Cellular debris was removed by centrifugation at 30,000 ϫ g for 20 min. The extract was passed through a column packed with a 7-methyl-GDP Sepharose resin (Pharmacia Biotech Inc.). The column was washed with buffer H, and with buffer H containing 0.1 mM GDP. Cap-binding protein was eluted with buffer H in the presence of 0.1 mM 7-methyl-GDP. Internal peptides were generated via cyanogen bromide cleavage and trypsin digestion (36). The tryptic peptides were resolved using reverse-phase high performance liquid chromatography. Microsequencing was performed using an ABI Procise 494 protein sequencer. Recombinant S. pombe eIF4E was purified from E. coli cells grown in TB medium at 30°C and harvested in stationary phase (A 600 ϭ 4.5-5.0). The cells were disrupted by sonication. The cell debris was removed by centrifugation at 30,000 ϫ g for 30 min, and affinity purification was performed according to a standard protocol (37).
Nucleotide Sequence Accession Number-The sequence of S. pombe tif1 (Fig. 2B) has been submitted to the EMBL Nucleotide Sequence Database in Cambridge, UK, and assigned the accession number X99444.
Immunoblotting Analysis-Following SDS-polyacrylamide gel electrophoresis, the transfer of the proteins to Immobilon polyvinylidene difluoride membranes was carried out according to the directions of the supplier (1986, Millipore Corp.) using a trans-blot semi-dry transfer cell (Bio-Rad) at 15 V for 30 min. The membrane was incubated in TBS (20 mM Tris-HCl (pH 8.0), 150 mM NaCl containing 2% bovine serum albumin) for 1 h, incubated with mouse antiserum diluted 1:2000 in TBS for 4 h at 4°C, and further incubated with goat anti-mouse alkaline phosphatase antibody (Promega Corp.) diluted 1:20,000 in TBS for 2 h. Washes of the membrane were done by incubating it in TBST containing 0.1% bovine serum albumin. All the incubations were carried out at room temperature. Color development was performed by following the instructions of the supplier (Promega Corp.). The polyclonal anibodies were raised in mice against recombinant eIF4E from S. cerevisiae (18) and S. pombe (this work). In the eIF4E quantitation experiments, cells of both species were harvested once they had reached A 550 ϭ 0.2 and were counted in a microscope counting chamber.

RESULTS AND DISCUSSION
Cloning and Sequencing of the Chromosomal Gene Encoding eIF4E from S. pombe-We initiated this study of eIF4F in S. pombe by cloning and sequencing the gene encoding the core component of the cap-binding complex, eIF4E. Most of the reading frame encoding S. pombe eIF4E was initially obtained as a cDNA sequence identified via a homology search through a cDNA data base established in the course of a genome sequencing project for S. pombe. We used the identified partial cDNA as a probe in subsequent analysis of S. pombe DNA. Southern analysis of S. pombe total chromosomal DNA restricted with several enzymes showed that the genome contains only one copy of the gene encoding eIF4E, located on a 2.8 -3-kilobase HindIII DNA fragment (Fig. 1A). Genomic DNA fragments in this range were therefore isolated from an agarose gel and used to generate a partial S. pombe DNA bank in pBluescript II SKϩ. Positive clones bearing the chromosomal S. pombe eIF-4E-encoding gene were identified via colony filter hybridization. Sequence analysis revealed that the chromo-somal HindIII restriction fragment extended on the 5Ј side only to position Ϫ61 relative to the eIF4E reading frame. Southern blot analysis of S. pombe genomic DNA using a 193-bp HindIII-NcoI fragment (containing the 5Ј end of the HindIII fragment and part of the reading frame up to position ϩ127) showed that a 3.5-kilobase EcoRI-NcoI fragment contained the sequence contiguous with the HindIII fragment 5Ј of the eIF4E reading frame. This fragment was inserted into pSL301 using the same strategy as used for the HindIII fragment.
Pulsed-field gel electrophoresis of S. pombe genomic DNA followed by Southern blotting revealed that the eIF4E reading frame is located on chromosome 1 of the S. pombe genome (data not shown).
This work generated a complete nucleotide sequence for the chromosomal gene and its flanking regions (Fig. 2). The chromosomal gene has three exons and two introns. Using the same abbreviation as that adopted for S. cerevisiae, we have named this first S. pombe gene characterized that encodes a translation initiation factor tif1. The donor site for the first intron conforms to the standard consensus motif (G/G)UA(A/U)GU (39), whereas the donor site of the second intron deviates from this motif at position ϩ4. The 3Ј splice site of each intron has a YAG motif on the intron side. There are putative branch sites at positions Ϫ16 to Ϫ12 (CUAAC) and at Ϫ15 to Ϫ11 (CUAAU), respectively. The longer second intron contains several poly(U) runs. Examination of the sequence 5Ј of the main ORF reveals the presence of a putative TATA box, whereas in the 3Ј-untranslated region two potential polyadenylation signals are identifiable (compare Ref. 40). The translational start codon has an A at Ϫ3, a feature typical of many initiation sites in S. cerevisiae (41). However, unlike the majority of initiation sites in S. cerevisiae and S. pombe (39), the tif1 start context is The main open reading frame (ORF) of S. pombe eIF4E was deduced from examination of the cDNA and genomic sequences; it encodes a polypeptide of 218 amino acids (Fig. 2). The amino acid sequence predicted on the basis of the DNA sequences was confirmed at the N terminus by direct analysis of the protein isolated from S. pombe. Further partial sequences could be confirmed via amino acid sequencing of tryptic fragments (Fig. 2). Moreover, the cDNA sequence was recloned by means of the polymerase chain reaction into the E. coli expression vector pCYTEXP3 (33), allowing synthesis of the corresponding protein in this bacterial host and its purification via cap-analogue affinity chromatography. Electrophoretic analysis of the resulting protein revealed that the recombinant form showed similar mobility to that of eIF4E isolated directly from S. pombe (Fig. 3). Comparison of the complete predicted amino acid sequence of S. pombe eIF4E with those of its counterpart proteins from other organisms revealed the presence of a number of important features (Fig. 4). The S. pombe eIF4E amino acid sequence shows a higher degree of identity (34%) to the human eIF4E sequence than does the equivalent S. cerevisiae sequence (30.5%). The degree of sequence identity between the S. pombe and S. cerevisiae proteins is 43%.
Characterization of the Encoded mRNA-The results of Northern blotting indicated that the mRNA encoding S. pombe eIF4E is approximately 860 nucleotides long (Fig. 1B). In order to determine the transcription start site(s), a 21-nucleotide long synthetic primer complementary to nucleotides ϩ66 to ϩ86 was annealed to poly(A) ϩ RNA and extended by Moloney murine leukemia virus reverse transcriptase. The results obtained were consistent with the existence of a 5Ј end of the mRNA located 6 nucleotides upstream of the translation initiation site (Fig. 1D). We also checked this result independently by means of S1 nuclease mapping analysis. Poly(A) ϩ mRNA was hybridized with an NcoI-BamHI fragment that is complementary to the promoter region plus 43 codons of the main ORF. The S1 protection experiment yielded only one band. Comparison with the mobilities of sequencing reaction products was used as the basis for the calculation of the position of the 5Ј initiation site, yielding a result of Ϫ6/Ϫ7 (Fig. 1C). This means that the start codon of tif1 is relatively short and thus unlikely to allow optimal translational initiation.
Synthesis and Function of S. pombe eIF4E in S. cerevisiae-In order to test if the newly cloned gene is functionally homologous to its S. cerevisiae counterpart in vivo, the S. pombe tif1 gene was expressed in the S. cerevisiae strain 4-2, in which the chromosomal copy of the gene encoding eIF4E (CDC33) is disrupted by a LEU2 insertion (16). The S. pombe tif1 reading frame was recloned into an expression vector (YCp-SUPEX2; 32) in the form of a polymerase chain reaction copy bearing an NdeI site at the 5Ј end which included the start codon. Overexpression of tif1 using the strong inducible GPF promoter supported functional complementation of the disrupted CDC33 gene in a shuffled S. cerevisiae 4-2 derivative strain when the promoter was fully induced using galactose medium. These results were confirmed by means of tetrad analysis of a suitably crossed heteroallelic diploid strain (Fig.  5), whereby not all of the tetrads showed evidence of complementation. This is not unusual when the complementing gene supports only a partial activity. Indeed, reducing the amount of S. pombe eIF4E synthesized using this promoter by using a combined glucose/galactose medium did not allow growth of the haploid CDC33-disrupted S. cerevisiae strain (data not shown). It is therefore clear that growth of S. cerevisiae is only supported by an amount of S. pombe eIF4E significantly in excess of the normal level of its own homologous factor (compare Ref. 25). Finally, the growth rate of the derivative of S. cerevisiae 4-2 containing only S. pombe eIF4E was slower than that of the 4-2 strain when this contained the wild-type S. cerevisiae CDC33 gene.
Cap and Protein Binding Functions of S. pombe eIF4E-We analyzed the interactions of S. pombe eIF4E in the in vivo environment of S. cerevisiae (Fig. 3). A cap-analogue affinity column was used for the preparation of cap-binding proteins from extracts derived from the derivatives of S. cerevisiae strain 4-2. The S. pombe eIF4E isolated in this way from S. cerevisiae cells was found bound to a protein showing similar electrophoretic mobility to S. cerevisiae p20 (Fig. 3B). In contrast, no protein of equivalent electrophoretic mobility was evident in cap-analogue-binding fractions isolated from S. pombe (Fig. 3B). This suggested that, despite the absence of the p20 type protein in the S. pombe cap-analogue-binding fractions, S. pombe eIF4E may be capable of binding S. cerevisiae p20. We therefore turned to two-hybrid analysis to provide an independent means of testing this potential property of S. pombe eIF4E. The S. cerevisiae strain HF7c was transformed with two types of plasmid, pGBT9 expressing the eIF-4E protein from either S. cerevisiae, S. pombe, or Homo sapiens, and pGAD424, containing the p20-encoding sequence from S. cerevisiae. ␤-Galactosidase activity was measured using either  (14); Ser 209 , which is present in both the human sequence (22,23) and in the S. pombe sequence; Ser 53 , which is missing in the S. pombe sequence and now thought not to be a major site of phosphorylation. FIG. 3. Synthesis of S. pombe eIF4E in E. coli and S. cerevisiae. A 870-bp fragment containing the S. pombe tif1 ORF was inserted between the NdeI and BamHI restriction sites of the pCYTEXP3 expression vector polylinker (Schneppe et al. (33)) and expressed in E. coli  (42), which has a wild-type chromosomal copy of CDC33, and the wild-type strain S. pombe DSM 7057 (S.p.eIF4E wt). In each experiment, the total protein extract derived from the strain indicated was subjected to cap-analogue affinity purification, thus isolating eIF4E and associated proteins. The amounts of extract loaded were adjusted to yield comparable staining levels. This gel was run under conditions designed to clearly separate either of the eIF4E proteins from p20. Both the S. cerevisiae and the S. pombe forms of eIF4E bind p20 when expressed in the S. cerevisiae host strain. In contrast, no direct equivalent to p20 is evident in the S. pombe host strain.
filter assays (Fig. 6) or liquid ␤-galactosidase assays (see legend to Fig. 6). The test was positive for S. pombe eIF4E, confirming the result obtained in the cap-analogue affinity chromatography experiment. In contrast, human eIF4E, which was also able to substitute for its S. cerevisiae homologue as the functional cap-binding protein in vivo (data not shown), was not associated with p20.
Cross-linking of S. pombe eIF4F Proteins to mRNA-Given that isolation of S. pombe eIF4E by means of cap-analogue affinity chromatography may have led to the loss of relatively weakly binding components of the eIF4F complex, we performed further studies using RNA-protein cross-linking (Fig.  7). In initial experiments, we set out to identify the polypeptides that are able to interact in a cap-dependent fashion with mRNA. Protein fractions from S. pombe and, for comparison, S. cerevisiae, were UV-irradiated in the presence of mRNA bearing a 32 P-labeled cap. Two S. pombe polypeptides of apparent molecular masses 30 and 36 kDa, respectively, were found to cross-link in a cap-analogue-sensitive fashion (Fig. 7A). The lower, much stronger band corresponded to the main band observed when cross-linking was performed with S. cerevisiae fractions. With the help of Western blotting we could confirm that this major band contains S. pombe eIF4E (data not shown), whereas the band at 36-kDa probably corresponds to an additional component of eIF4F. We were unable to obtain clear evidence for cross-linking to a larger, eIF4G-type protein. This might be attributable to the inherent instability of the eIF4G protein (compare Ref. 11).
We also performed cross-linking using generally labeled mRNA (Fig. 7B). In this case, only the S. pombe eIF4E band was evident in the 30-kDa range of the gel, and its intensity was only partially cap-analogue-sensitive. However, one fur-ther S. pombe band was apparent, showing a mobility equivalent to approximately 45 kDa. Comparison with the results obtained with S. cerevisiae indicates that this protein could be the S. pombe homologue of eIF4A, whose presence in the S. cerevisiae lanes could be confirmed by Western blotting of the cross-linked proteins (data not shown). The strength of crosslinking of the proteins in the 45-kDa region did not respond to the addition of cap-analogue, as would be expected of proteins whose binding is not cap-specific.
Quantitation of eIF4E Proteins in S. pombe and S. cerevisiae-A key issue related to eIF4E function is the abundance and availability (to ribosomes and mRNA) of this protein in the cell. In order to perform comparative quantitation, we raised polyclonal antibodies against S. pombe eIF4E. We then used Western blotting to assess the relative amounts of the respective eIF4E proteins in total cell extracts from S. pombe and S. cerevisiae (Fig. 8). By means of comparison with the staining intensities obtained with serial dilutions of the recombinant S. pombe (this work) and S. cerevisiae (18) eIF4E proteins, we estimated the relative amount of eIF4E as a function of total cell protein and per cell. The results (Fig. 8) revealed that S. pombe eIF4E has an even lower abundance than its counterpart in S. cerevisiae. Whether calculated on the basis of content per cell or as a function of total cell protein, we estimated that eIF4E is at least five times less abundant in S. pombe. FIG. 6. Two-hybrid analysis confirms the interaction between S. pombe eIF4E and p20. The interaction assay was positive for S. pombe eIF4E:p20 (column 2) and S. cerevisiae eIF4E:p20 (column 3) but not for human eIF4E:p20 (column 1). Typical test results are shown for three sets of three independent double transformants. In further experiments, quantitative ␤-galactosidase assays were performed on extracts from the three hybrid strains, revealing that the specific activities (expressed in Miller units,Ref. 45) for S. cerevisiae eIF4E/p20:S. pombe eIF4E/p20:human eIF4E/p20 were in the ratio 1.0:0.8:0.001 (with the S. cerevisiae value normalized to 1.0).

FIG. 7.
Photochemical cross-linking of yeast fractions to radioactive mRNA. A, ␣-32 P-cap-labeled mRNA was irradiated together with recombinant S. pombe eIF4E (lanes 1 and 2), S. pombe S30 proteins that had been isolated by cap-analogue affinity chromatography (lanes 4 and 5), S. pombe S30 proteins prior to affinity chromatography ( lanes 6 and 8), and S. cerevisiae S30 proteins (lanes 7 and 9). B, capped [␣ 32 P]CTP-labeled mRNA was irradiated together with recombinant S. pombe eIF4E (lanes 1 and 2), S. pombe S30 proteins isolated using cap-analogue affinity chromatography (lanes 4 and 5), S. pombe S30 proteins (lanes 6 and 8), and S. cerevisiae S30 proteins (lanes 7 and 9). Some incubations contained 0.65 mM m 7 GTP (ϩ). The molecular masses of protein standards (panels A and B, in both cases lane 3) are indicated in kilodaltons on the left side. Arrows on the right side indicate the positions of cross-linked bands referred to in the text.
A Novel Type of eIF4F Complex in S. pombe-In conclusion, we have described a new type of eIF4F in S. pombe. The cap-binding component eIF4E shows features typical of the counterpart proteins in man, rabbit, and mouse, yet retains the ability to bind the (S. cerevisae) p20 protein. Moreover, the overall structure of eIF4F in S. pombe is novel; there is no indication of binding to eIF4A, and we detected an additional p36 component. It is uncertain whether there is an eIF4G component. The most striking aspect of the S. pombe eIF4E sequence is the presence of a potential phosphorylation site at position 209 in the C terminus. Apart from the "core motif " KSGST, this region shares a more extensive similarity to the equivalent regions of the mammalian proteins (H⅐D⅐⅐(S/ T)KSGST⅐⅐⅐K⅐R). Given that S. cerevisiae eIF4E lacks this type of motif, it is an intriguing possibility that, of the two yeasts examined, only S. pombe may possess a C-terminal regulatable phosphorylation site. The fact that eIF4E is less abundant in S. pombe cells might allow tighter control to be exercised via this protein.
Vertebrate eIF4E has been shown to interact with binding proteins that regulate its activity in response to changes in insulin levels (43,44). In S. cerevisiae, the only potential candidate for such a regulatory protein reported so far is the small protein found associated with eIF4E generally referred to as p20, which has a molecular weight of 18 kDa. We found that the amount of this protein found bound to eIF4E extracted from cells grown under different conditions varies and that there is some correlation with the level of its phosphorylation (14). However, disruption of the gene encoding p20 has no effect on cell viability (13), and it has yet to be demonstrated that p20 has a regulatory role in vivo. The striking observation that S. pombe eIF4E also binds S. cerevisiae p20 creates a riddle yet to be solved, given the apparent absence of an equivalent protein in the isolated S. pombe eIF4F complex. At the same time, we will need to address the role of the p36 protein identified in the cross-linking experiments described in the present work. Overall, further investigations of eIF4F in S. pombe can therefore be expected to provide new insight into the significance of the composition and function of this complex in eukaryotic translation.