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Previous Article | Next Article 
Molecular and Cellular Biology, August 2002, p. 5434-5442, Vol. 22, No. 15
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.15.5434-5442.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received 31 January 2002/ Returned for modification 8 March 2002/ Accepted 30 April 2002
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the initiator methionyl-tRNA (
) is specifically formylated by the enzyme methionyl-tRNA formyltransferase (MTF) to generate formylmethionyl-tRNA (
). The formyl group acts as a positive determinant for selection of the by the initiation factor IF2 (54) and as a second negative determinant for blocking the binding of the tRNA to the elongation factor. Formylation of the initiator is important for protein synthesis in Escherichia coli, mutant initiator tRNAs defective in formylation are extremely poor in initiation of protein synthesis, and a strain of E. coli carrying disruptions in the MTF gene has severe growth defects (12, 33, 59).
In contrast to the situation with eubacteria, mitochondria, and chloroplasts presented above, in the cytoplasm of eukaryotes and in archaebacteria, there is no formylation of the initiator tRNA (
), and the 
is utilized directly to initiate protein synthesis with methionine (42, 47). However, the cytoplasmic initiator of yeast contains some of the determinants in the tRNA (Fig. 1) important for recognition by E. coli MTF (12a, 23) and can therefore be formylated in vitro by E. coli MTF (43). The resulting 
initiates protein synthesis in cell extracts of both E. coli and rabbit reticulocytes, suggesting that the eukaryotic initiation factor eIF2 can, at least to some extent, also recognize the (14).
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and results in cells that grow much slower. The slow-growth phenotype is not due to reduced rates of protein synthesis in the cytoplasm due to formylation of the initiator Met-tRNA or due to the depletion of cytoplasmic formate pools. The slow-growth phenotype is fully rescued by coexpressing the E. coli polypeptide deformylase (DEF), an enzyme that removes formyl groups from the N terminus of proteins, strongly suggesting that protein synthesis in these cases initiates, at least to some extent, with formylmethionine and that synthesis of N-formylmethionine-containing proteins affects normal cell growth. We discuss the implications of these results, including a possible use of the yeast strain expressing E. coli MTF for the screening in vivo of antibacterial drugs targeted against MTF. |
MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ura3-52 trp
1 imt::TRP1 imt3::TRP1 imt4::TRP1) (4) was from A. S. Bystrom, Umea University, Umea, Sweden; EKY3 (ser1 ura3-52 trp1 leu2 his4 shm1::URA3 shm2::LEU2) (17) was from D. R. Appling, University of Texas at Austin, and JEL1 (MAT leu2 trp1 ura3-52 prb1-1122 pep4 his3::PGAL1-GAL4) (27) was from J. C. Wang, Harvard University, E. coli XL1-Blue (recA1 endA1 gyr96 thi-1 hsdR17 supE44 relA1 lac [F' proAB lacIq ZM15 Tn10 (Tetr)]) from Stratagene was used as the routine strain for cloning. Yeast strains were grown in YPD (1% Bacto yeast extract, 2% Bacto peptone, 2% dextrose) or in synthetic minimal medium SMRG (0.67% yeast nitrogen base without amino acids, 0.5% Casamino Acids, 2% raffinose, and 3% glycerol). For induction, the cells were grown in SMRG supplemented with 2% galactose. Plasmids containing URA3 and LEU2 markers were selected by growing in medium lacking uracil and leucine as required. The strain EKY3 was grown in medium containing 0.7% yeast nitrogen base without amino acids, 2% galactose (or dextrose), 375 mg of serine per liter, 20 mg of tryptophan per liter, 20 mg of histidine per liter, and 40 mg of leucine per liter. When required, potassium formate (pH 5.0) was supplemented to a final concentration of 100 mg/liter. Construction of plasmids. The E. coli MTF gene was cloned in the yeast expression vector YEp352-GAL1 (37). The wild-type and mutant MTF genes were amplified by PCR with the plasmids pQE16FMTp and pQE16FMTpR42L (44, 45) as templates and the oligonucleotides 5'-ATCGGATCCAAAAAAAATGTCAGAATCACTACGTATTATT-3' containing a BamHI site as the forward primer and 5'-CTCAGCTAATTAAGCTTAGTG-3' containing a HindIII site as the reverse primer. The PCR products coding for MTF-six-His fusion proteins were digested with BamHI and HindIII and cloned into the respective sites of YEp352-GAL1 to obtain YEp.MTF WT (wild type) and YEp.MTF R42L. The gene for E. coli DEF was amplified by PCR with genomic DNA isolated from E. coli JM109 as a template and 5'-AGAGCTCGAGAAAAAAAATGTCAGTTTTGCAAGTGTTACATG-3' containing a site for XhoI as the forward primer and 5'-ACTAGCATGCTTAGTGATGGTGATGGTGATGAGCCCGGGCTTTCAGACG-3' coding for the six-His tag and containing a site for SphI as the reverse primer. The PCR product was digested with XhoI and SphI and cloned into the respective sites of plasmid pAS565, a pRS315-based vector (51). A NotI-ApaI fragment from this plasmid containing the GAL1 promoter and the DEF gene was then cloned into the 2µm vector pRS425 (7) at these sites to obtain pRS425.DEF. The E133A mutation in the E. coli DEF gene was introduced into this plasmid by the Quikchange mutagenesis procedure with the Pfu DNA polymerase (Stratagene) to generate pRS425.DEF E133A.
Immunoblot analysis. Cells from 1.5 ml of culture were collected and resuspended in 50 µl of water followed by the addition of an equal volume of lysis buffer (4% sodium dodecyl sulfate [SDS], 10 mM EDTA) and glass beads. The suspension was mixed vigorously for 2 min with a Vortex mixer, kept in boiling water for 5 min, and mixed again for 2 min. After centrifugation, the protein content in the clarified supernatant was estimated by using the Micro BCA (bicinchoninic acid) protein assay reagent kit (Pierce). Proteins (15 µg per lane) were resolved on SDS-12% polyacrylamide gels (22) and transferred onto Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore) with a buffer (pH 8.3) containing 50 mM Tris, 40 mM glycine, 0.04% SDS, and 20% methanol. The membrane was blocked in 5% nonfat dry milk in TBS (20 mM Tris-HCl [pH 7.5], 140 mM NaCl, and 0.05% Tween 20) at room temperature for 60 min. The membrane was then washed three times for 10 min each with TBS and incubated with a monoclonal antibody against the four-His epitope (Qiagen; 1:2,000 dilution) for 60 min at room temperature. The membrane was subsequently washed three times for 10 min each and incubated with peroxidase-conjugated secondary anti-mouse antibody (New England Biolabs; 1:3,000 dilution) for 1 h at room temperature. After three washes for 10 min each, the bound antibodies were detected with enhanced chemiluminescence reagents (New England Biolabs). In some experiments, the membrane was also probed with polyclonal antiserum against MTF as described previously (26).
Isolation of total tRNA.
A single colony of S. cerevisiae CKY473 was grown in 10 ml of YPD to saturation. Five milliliters of this culture was inoculated into 100 ml of YPD and grown for 24 h at 30°C. The cells were harvested and resuspended in 40 ml of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (TE). An equal volume of phenol (equilibrated with TE) was added, and the contents were mixed vigorously in a shaker at room temperature for 30 min. The phases were separated by centrifugation, and the nucleic acids were recovered from the aqueous phase by ethanol precipitation. The pellet was washed with 70% ethanol and dissolved in 2 ml of TE. High-molecular-weight nucleic acids were removed by the addition of 0.5 ml of 5 M NaCl and centrifugation. Total tRNA was recovered from the supernatant by ethanol precipitation. The yield was 
100 A260 units.
Aminoacylation and formylation of cytoplasmic initiator tRNA.
Total tRNA isolated from yeast was aminoacylated with methionine with E. coli MetRS or aminoacylated with methionine and subsequently formylated with E. coli MTF (62). The reaction mixture was extracted with phenol equilibrated with 10 mM sodium acetate (pH 4.5). The tRNA was recovered by precipitation and used for electrophoresis on acid-urea polyacrylamide gels to provide markers of cytoplasmic , , and .
Northern blot analysis of tRNAs. Total tRNAs were isolated from yeast cells (3 ml of culture) under acidic conditions (0.3 M NaOAc [pH 4.5]), and 0.1 A260 unit was subjected to electrophoresis on acid-urea-polyacrylamide gels and electroblotted on to Nytran-Plus membrane (Schleicher & Schuell) as described previously (57). The membrane was washed with 4x SET (1x SET is 0.15 M NaCl, 2 mM EDTA, and 0.03 M Tris-HCl [pH 8.0]) containing 1% SDS, baked at 70°C for 90 min, and prehybridized at 42°C in 4x SET containing 0.1% SDS and 10x Denhardt's solution. tRNAs were detected by hybridization to a 5'-32P labeled oligonucleotide probe complementary to nucleotides 8 to 24 of the yeast cytoplasmic initiator tRNA.
Measurement of protein synthesis rates. Protein synthesis rates were measured essentially as described previously (16, 50). For in vivo labeling, the synthetic medium lacking methionine contained leucine (40 mg/liter), tyrosine (30 mg/liter), isoleucine (30 mg/liter), phenylalanine (50 mg/liter), glutamic acid (100 mg/liter), aspartic acid (100 mg/liter), valine (150 mg/liter), threonine (200 mg/liter), serine (400 mg/liter), histidine (20 mg/liter), lysine (30 mg/liter), tryptophan (20 mg/liter), arginine (20 mg/liter), 0.67% yeast nitrogen base without amino acids, 2% raffinose, 3% glycerol, and 2% galactose. The yeast strains were grown in 200 ml of this medium to the early log phase, and protein synthesis rates were measured over a period of 6 h. At various times, cells corresponding to 2 A600 units were harvested and resuspended in 300 µl of fresh medium. Easy Tag Express protein labeling mix (50 µCi; 1,175 Ci of L-[35S]methionine per mmol; NEN) was added, and the mixture was incubated for 5 min at 30°C. To stop incorporation of [35S]methionine into proteins, 1 ml of a solution containing 1.2 mg of methionine per ml and 0.5 mg of cycloheximide per ml was added, and the culture was immediately frozen on dry ice. Cells were lysed by the addition (150 µl) of a freshly prepared solution containing 2 N NaOH and 12% ß-mercaptoethanol, and proteins were precipitated with trichloroacetic acid (TCA) as described previously (63). The pellet was washed twice with 1.5 ml of acetone and suspended in 300 µl of 1% SDS containing a few acid-washed glass beads (425 to 600 µm in size; Sigma), and the suspension was mixed vigorously for 2 min with a Vortex mixer and boiled for 5 min. The clear supernatant obtained after centrifugation was used for determining the TCA-precipitable radioactivity on filter paper discs. The total protein concentration in these samples was estimated with the BCA reagents (Pierce). The rate of protein synthesis is expressed as the cpm of radioactivity incorporated per minute into 1 µg of TCA-precipitable protein. To ensure that the acid-precipitable 35S radioactivity measured reflected incorporation into protein and not into [35S]Met-tRNA, the extracts were also spotted on filters soaked in 1 N NaOH and then used for TCA precipitation. The counts in these samples were essentially the same as those in samples directly analyzed by TCA precipitation.
Isolation of ribosomes.
Ribosomes were isolated from the following yeast strains as described previously (48): S. cerevisiae CKY473 (grown in YPD for 44 h), S. cerevisiae CKY473/YEp.MTF WT+pRS425 (grown in SMRG+Gal, -Leu for 60 h), and S. cerevisiae CKY473/YEp.MTF WT+pRS425.DEF (grown in SMRG+Gal, -Leu for 48 h). The cells were suspended in 10 ml of cold buffer A (20 mM Tris-HCl [pH 7.5], 16 mM MgCl2, 100 mM KCl, 0.2 mM EDTA, 12 mM ß-mercaptoethanol). Acid-washed glass beads (5 g) were added, and the suspension was mixed vigorously for 1 min with a Vortex mixer followed by chilling on ice for 2 min; the vigorous mixing followed by chilling was repeated six times. The lysate was poured off, and the glass beads were washed twice with buffer A. The lysate and washings were pooled, cell debris was removed by centrifugation at 3,000 x g for 10 min at 4°C, and the supernatant was further clarified by centrifugation at 27,000 x g for 20 min at 4°C. The clear supernatant was subjected to ultracentrifugation at 100,000 x g for 130 min at 4°C. The S100 pellet was overlaid with 5 ml of buffer A and left overnight on ice. The suspension was centrifuged at 27,000 x g for 20 min at 4°C, and the clear supernatant was then subjected to ultracentrifugation as described before.
Isolation and two-dimensional gel electrophoresis of ribosomal proteins. Proteins were isolated from the ribosomes and subjected to two-dimensional electrophoresis by a protocol provided by John Warner, Albert Einstein College of Medicine, Bronx, N.Y. The ribosomal pellet was suspended in 5 ml of a mixture containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 6 mM ß-mercaptoethanol, and 1 mM MgCl2, followed by addition of 0.5 ml of 1 M MgCl2. Glacial acetic acid (final concentration, 67%) was slowly added, and the solution was stirred for 15 min and clarified by centrifugation (17,000 x g, 20 min, 4°C). The supernatant was dialyzed against 1% acetic acid. Two-dimensional gel electrophoresis was performed with the Mini-PROTEAN II 2-D cell apparatus (Bio-Rad). The first-dimension gel was in capillary tubes (1-mm internal diameter by 7.5 cm long) containing 4% (wt/vol) acrylamide, 0.1% (wt/vol) bisacrylamide, 8 M urea, 0.057 M Bis-Tris (adjusted to pH 5.0 with acetic acid). The ribosomal proteins were lyophilized and dissolved in sample buffer containing 8 M urea, 10 µM ß-mercaptoethanol, 10% glycerol, 1% acetic acid, and 0.01% basic fuchsin. The electrophoresis buffer contained 10 mM Bis-Tris adjusted to pH 4.0 with acetic acid (anode) and 0.179 M potassium acetate (pH 5.0) (cathode). The second dimension was on SDS-15% polyacrylamide gels.
N-terminal sequence analysis of RPL3, a yeast large ribosomal subunit protein. Proteins were electroblotted onto Immobilon membranes (Millipore) as described previously (29). After transfer, the membrane was washed three times (10 min each) in MilliQ water, stained with 0.1% Coomassie blue in 50% MeOH for 2 min, and destained in 50% MeOH-10% acetic acid and washed repeatedly with MilliQ water and air dried. The RPL3 protein on membrane was then subjected to Edman degradation at the Biopolymers Laboratory, Massachusetts Institute of Technology.
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RESULTS |
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in vivo, we expressed the E. coli MTF in the cytoplasm of yeast under the control of a galactose-inducible promoter with a high-copy-number (2µm) plasmid. For this purpose, the genes coding for the wild-type MTF and an inactive R42L mutant of MTF were cloned as C-terminal six-His fusions in the yeast expression vector YEp352-GAL1. The expression of the enzyme in total protein extracts was analyzed by immunoblotting with a monoclonal antibody recognizing the four-His epitope (Fig. 2A). A signal corresponding to the size of purified MTF is detected in extracts of yeast cells containing the recombinant plasmids upon induction with galactose. Both wild-type and the R42L mutant MTFs are expressed, and they accumulate to similar levels (lanes 3 and 5). There was no detectable expression in the uninduced state (lanes 2 and 4). Similar results were obtained with polyclonal antibodies raised against E. coli MTF (data not shown). Taken together, the results indicate the expression and stable accumulation of both the wild-type and R42L mutant MTFs in the cytoplasm of yeast without significant proteolysis.
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in eubacteria and as in archaebacteria and in the cytoplasm of eukaryotes (42, 47). We investigated the in vivo state of the cytoplasmic yeast initiator tRNA in cells expressing the six-His-tagged wild-type MTF and the R42L mutant MTF. Previous studies had shown that introduction of the polyhistidine tag does not affect the activity of MTF (44). The R42L mutant MTF is 1,400-fold less active in formylation of the E. coli initiator tRNA (45, 46) and is used as a negative control here.
Total tRNAs isolated from cells under acidic conditions (pH 4.5) were separated by electrophoresis on an acid-urea-polyacrylamide gel and subjected to Northern blot analysis with a probe specific to the yeast cytoplasmic initiator tRNA (Fig. 2B). For use as markers, total tRNA (lane 1) isolated from S. cerevisiae CKY473 under basic conditions (pH 8.0) was aminoacylated in vitro with E. coli MetRS (lane 2) or aminoacylated with MetRS and subsequently formylated with E. coli MTF (lane 3) and subjected to electrophoresis on the same gel. As for the E. coli initiator tRNA (57), all three forms of the yeast cytoplasmic initiator tRNA, the , the , and the are separated clearly from one another (Fig. 2B, lanes 1, 2, and 3). The mobility under acidic conditions of S. cerevisiae initiator tRNA isolated from the cells expressing the wild-type MTF (Fig. 2B, lane 5) shows that up to 70% of the is formylated. There was no detectable accumulation of any in uninduced cells (lane 4) or in cells expressing the inactive R42L mutant of MTF (lane 7). In these cells, the initiator tRNA is present mostly (>70%) in the aminoacylated form.
Effect of formylation of the yeast cytoplasmic initiator tRNA on growth rates. The growth of S. cerevisiae CKY473 strain containing the YEp352-GAL1 vector alone or with the wild-type or the R42L mutant MTF genes was studied on agar plates in the absence or presence of the inducer galactose (Fig. 3). In the absence of galactose (uninduced), there was no detectable difference in growth (left). In the presence of galactose (induced), cells expressing the wild-type MTF grow significantly more slowly than cells expressing the R42L mutant MTF (right). The cells expressing R42L mutant MTF do not show any slow-growth phenotype compared to cells containing the empty vector YEp352-GAL1. In liquid minimal medium also (see Materials and Methods for SMRG medium), the cells expressing the wild-type MTF enzyme grow much more slowly, with a doubling time of 300 min compared to the doubling time of 120 min for cells containing vector alone or cells expressing the R42L mutant MTF (Fig. 4). Similar effects of expression of wild-type MTF enzyme on growth were seen in S. cerevisiae UMY543 (4), which contains a single functional copy of the cytoplasmic initiator tRNA gene instead of the normal four or five copies in most laboratory strains, and in S. cerevisiae JEL1 (27), which expresses larger amounts of MTF due to the presence of higher levels of the GAL4 activator protein (data not shown).
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Attempts to detect formylmethionine at the N terminus of proteins. Several approaches were used to detect formylmethionine at the N terminus of proteins in CKY473 transformants expressing the E. coli MTF.
(i) Comparison of two-dimensional polyacrylamide gel electrophoresis patterns of ribosomal proteins. Many, but not all, of the ribosomal proteins in yeast contain an N-terminal acetyl group as a result of posttranslational N-terminal acetylation often following the removal of the initiating methionine (2). Mutant proteins that lack these N-terminal acetyl groups because of a defect in one of the N-acetyltransferases (NAT1p) migrate differently from the wild-type protein on two-dimensional gels (55). It was expected, therefore, that if some of the ribosomal proteins were to carry a formylmethionine at the N terminus instead of methionine or another amino acid, these proteins would also migrate differently on two-dimensional gels. To investigate this, we isolated ribosomes from CKY473 cells transformed with either the empty vector or the vector carrying the E. coli MTF gene and grown in the presence of galactose (inducing conditions). The ribosomal proteins were subjected to two-dimensional gel electrophoresis and visualized with Coomassie blue or silver stain. There was essentially no difference in the relative mobilities of the proteins isolated from the two sets of ribosomes (data not shown). Thus, if there is any formylmethionine at the N terminus of the yeast ribosomal proteins in cells expressing the E. coli MTF, it is below the detection level of the staining techniques.
(ii) N-terminal sequence analysis of RPL3, a yeast large ribosomal subunit protein. A second approach used N-terminal sequence analysis to investigate whether there was a formyl group blocking the N terminus of a protein known to normally have a free amino group. The protein of choice was RPL3, a yeast large subunit ribosomal protein, which is known to have a free amino group at its N terminus and which is well separated from other proteins on two-dimensional gels (18, 55). Ribosomal proteins from CKY473 were separated on two-dimensional gels, transferred to PVDF membranes by electroblotting, and visualized by staining with Coomassie blue. The tailing part of the spot corresponding to RPL3 in the first dimension, which is most likely to contain a blocked N terminus, was excised and used for N-terminal sequence analysis by the Edman degradation procedure. The sequences of RPL3 isolated from the CKY473 cells transformed with the empty vector and with the vector containing the E. coli MTF gene were both the same, S-H-R-K-Y-E. Importantly, there was little methionine released in the second cycle of sequencing, as would have been expected if there was a significant fraction of the RPL3 protein that had been initiated with formylmethionine.
The attempts to identify formylmethionine at the N terminus of proteins described above have involved analyses of only the ribosomal proteins. The possibility that proteins that carry formylmethionine at the N terminus will not be assembled into ribosomes has not been ruled out, but is extremely unlikely. Many of the ribosomal proteins in yeast are acetylated at the N terminus. Mutant proteins that lack the N-terminal acetyl group because of mutations in the N-terminal acetyltransferases are still assembled into ribosomes (2, 55). Thus, the presence or absence of an N-terminal acetyl group has no effect on assembly of a protein on the ribosome. It is, therefore, extremely unlikely that the presence or absence of a formyl group at the N terminus will have an effect on assembly of a protein on the ribosome.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Attempts at identifying formylmethionine at the N terminus of proteins in cells expressing the E. coli MTF have, however, not been successful so far. This is most likely because only a small fraction of protein chains, perhaps 5 to 10%, initiate with formylmethionine, and this amount is below the limits of detection of the methods used. It is known that the initiation factor eIF2, which binds to the initiator and transports it to the ribosome, has a significantly reduced affinity for compared to (61). Therefore, is likely to be less active in initiation of protein synthesis in the cytoplasm of yeast than . Whether the function of recently identified yIF2 protein, the yeast homologue of the bacterial IF2 protein, now called eIF5B (6, 39), is also affected by formylation of the cytoplasmic initiator tRNA is not known. Our conclusion that only a small fraction of protein chains initiates with formylmethionine in yeast cells expressing E. coli MTF is also consistent with the fact that these cells display a slow-growth phenotype and not a lethal phenotype. In E. coli, in which all protein chains are normally initiated with formylmethionine, the formyl group is removed from all but a few proteins (35, 60) and DEF is an essential gene. Since yeast does not have a gene for DEF (10), if most of the protein chains in yeast expressing the E. coli MTF were to initiate with formylmethionine, one might have expected a lethal phenotype.
Given the possibility that is likely to be less active in initiation of protein synthesis than , the finding that formylation of up to 70% of the cytoplasmic initiator tRNA does not have a significant effect on overall rates of protein synthesis is quite surprising. Previous studies with yeast have shown that depletion of cytoplasmic initiator tRNA levels down to approximately 40% of normal levels (4, 9) leads to a significant reduction in growth rate. This has been commonly assumed to be due to an effect on rates of initiation of protein synthesis. Hinnebusch and coworkers (8, 13) have shown that derepression of GCN4 mRNA translation can be a sensitive measure of the 
. GTP ternary complex levels in yeast. It would, therefore, be interesting to see whether formylation of the yeast cytoplasmic initiator tRNA leads to derepression of GCN4 mRNA translation.
Why does initiation of protein chains with formylmethionine in yeast lead to slow growth? The answer to this question is likely to lie in the posttranslational N-terminal processing and, in some cases, further N-terminal modifications that occur on newly synthesized proteins (3). Many proteins in their mature form typically lack the N-terminal methionine in eukaryotes and formylmethionine in eubacteria. The enzyme methionine aminopeptidase (MAP), which removes the N-terminal methionine from proteins (36), is essential in both S. cerevisiae (24) and E. coli (5). In eubacteria, the formyl group in formylmethionine at the N terminus of proteins has to be removed by DEF prior to the action of MAP, as suggested by biochemical data showing that MAP is inactive on peptides containing formylmethionine (53). Therefore, initiation of proteins with formylmethionine in S. cerevisiae is likely to block the action of the yeast MAP(s) on proteins, and this could be one of the major reasons for the observed slow-growth phenotype of these strains. It is known that loss of one or the other of the MAP activities can have a significant effect on eukaryotic cells. For example, drugs that inhibit MAP2 affect endothelial cell proliferation and block angiogenesis (11, 52). Although only a fraction of the yeast protein chains are likely to be initiated with formylmethionine, the slow-growth phenotype could be due to the cumulative effect of this on activity of several proteins important for various purposes in the cell.
Following the posttranslational removal of the N-terminal methionine in yeast, many of the proteins are subsequently modified by N-terminal acetylation (40) and a few are modified by N-terminal myristoylation (15). The myristoyl-group in particular is thought to play an important role in the localization of some proteins, such as adenylcyclase, in the membrane and in activity of the protein. Therefore, another reason for the slow-growth phenotype of cells expressing E. coli MTF could be due to an effect on further posttranslational N-terminal modifications following removal of the initiating methionine.
It is also possible that the presence of a formyl group at the N terminus of some proteins is, in itself, partly responsible for the slow-growth phenotype of yeast cells expressing E. coli MTF. The presence of a formyl group at the N terminus could affect the activity and/or the stability of some key proteins. It is known that the activity of some yeast proteins can be negatively affected by N-terminal acetylation (1). Also, the synthesis and degradation of some proteins are very tightly regulated in yeast and other eukaryotic cells. Examples of these are cyclins and other proteins, which are involved in regulation of the cell cycle (19). Therefore, if the presence of formylmethionine to the extent of 5 to 10% of a yeast cyclin or other proteins results in their stabilization or destabilization, this could interfere with progression of the cell cycle.
Finally, because protein synthesis in eubacteria is initiated with formylmethionine, and formylation of the initiator tRNA is important for initiation of protein synthesis in most eubacteria (38, 58), MTF is considered to be a potential target for antibacterial drugs. One concern has been the fact that mitochondria also initiate protein synthesis with formylmethionine, and, therefore, drugs targeted against MTF could be toxic to humans. Recent results suggest, however, that this may not be a serious concern. First, we have shown that formylation of initiator tRNA is not essential for yeast mitochondrial protein synthesis (25). If this situation also holds for mammalian mitochondrial protein synthesis, MTF would then be an excellent target for antibacterial drugs. Second, there are important differences between the eubacterial MTFs and the mammalian mitochondrial MTFs (56). The eubacterial MTFs have a highly conserved arginine residue present in a flexible loop that is critical for the activity of MTF (45, 46, 49), whereas mammalian mitochondrial MTF does not have the arginine residue. Therefore, it is quite possible that drugs targeted against eubacterial MTFs will not have any effect on mammalian mitochondrial MTFs. The findings reported in this paper that yeast cells expressing E. coli MTF grow very slowly could be used to screen for drugs targeted against eubacterial MTF. Such drugs should rescue the slow-growth phenotype of these cells. Since the screening procedure relies on selection for faster growth of a eukaryotic cell, rather than inhibition of growth or killing of a bacterial cell, a further advantage is offered: any drugs that have detrimental effects on growth of eukaryotic cells would be automatically screened out.
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ACKNOWLEDGMENTS |
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This work was supported by grant R37GM17151 from the National Institutes of Health.
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FOOTNOTES |
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), YEp.MTF WT (
), and YEp.MTF R42L (
), respectively, in SMRG minimal medium containing 2% galactose.
