Jürgen H. Nett, Jacques Kessl, Tina Wenz and Bernard L. Trumpower EXPERIMENTAL PROCEDURES - PDF

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Eur. J. Biochem. 268, (2001) q FEBS 2001 The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstream

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Eur. J. Biochem. 268, (2001) q FEBS 2001 The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstream codons Jürgen H. Nett, Jacques Kessl, Tina Wenz and Bernard L. Trumpower Department of Biochemistry, Dartmouth Medical School, Hanover, NH, USA The selection of the site for initiation of translation for the Saccharomyces cerevisiae NFS1 gene was examined using mutated AUG1, AUG2 and AUG3 codons. When AUG1 of the yeast NFS1 gene was mutated to UUG and the resulting mrna was translated in vitro using a reticulocyte system, initiation from the mutated codon was abolished and occurred instead at downstream codons at increased rates. When the same mrna was translated using a yeast extract, translation initiated at the mutated codon, albeit at a reduced rate, and there was no increased translation at downstream AUG codons. The NFS1 gene in which AUG1 was replaced by UUG was also able to substitute for the wild-type gene in vivo in yeast. Western blots confirmed that the encoded protein was the same size as that encoded by the wild-type gene and that both the wild-type and mutated proteins localized to mitochondria. This is apparently the first example of a yeast protein where mutagenesis of AUG1 does not lead to alternate use of a downstream AUG. Keywords: initiation codon; translation; ribosomal scanning; NFS1 gene. The AUG codon nearest the 5 0 end of the mrna is used to initiate translation of proteins in virtually all eukaryotic organisms. Studies on the CYC1 gene and the HIS4 locus in Saccharomyces cerevisiae have concluded that initiation at the first AUG precludes initiation at subsequent AUG codons and that loss of the first AUG codon by mutation results in initiation at the next AUG codon [1,2]. In higher eukaryotes, mutations in the sequence surrounding the first AUG can lead to inefficient initiation at the first AUG and subsequent initiation at a downstream AUG. An optimal sequence context or the introduction of a stem-loop structure at a specific distance downstream of the start codon can lead to initiation of translation at a non-aug codon in higher eukaryotes [3 6]. In contrast to this, the sequence context in yeast has only a minor role in modulating AUG recognition [7]. This is probably the reason why yeast seems to be unable to efficiently use non-aug codons as start sites of translation initiation [2,8,9]. The Nifs gene, initially identified in the nitrogen-fixing bacteria Azotobacter vinelandii, codes for a cysteine desulfurase that is able to catalyze the de novo assembly of iron sulfur clusters in vitro [10 12]. Recently it was shown by Land & Rouault that the differential utilization of in frame AUGs in the transcript of the human Nifs gene leads to translation of proteins of varying lengths and the localization of the proteins in different subcellular compartments Correspondence to B. L. Trumpower, Department of Biochemistry, Dartmouth Medical School, Department of Biochemistry, 7200 Vail, Hanover, NH 03755, USA. Fax: , Tel.:: , Abbreviations: YPD, yeast extract/peptone/dextrose; YPEG, yeast extract/peptone/ethanol/glycerol; SD, synthetic dropout; MPP, matrix processing peptidase. (Received 5 June 2001, revised 13 August 2001, accepted 14 August 2001) [13]. The yeast orthologue NFS1 encodes a mitochondrial protein that is required for the synthesis of mitochondrial and cytosolic iron sulfur proteins [14]. We analyzed the possibility of differential AUG utilization in yeast NFS1 translation by site directed mutagenesis of AUG1, AUG2 or AUG3. Surprisingly, when the first AUG was replaced by the triplet UUG and the product was translated in vitro using yeast lysate, initiation of translation from AUG2 was not increased as expected. Instead, the newly generated UUG was used as the start codon for transcriptional initiation, albeit with decreased efficiency. These results suggest that in yeast mutation of the first AUG triplet after the start of the mrna does not always result in increased initiation of translation at the next AUG codon. EXPERIMENTAL PROCEDURES Construction of plasmids The plasmids used are listed in Table 1. The NFS1 reading frame and it s 5 0 and 3 0 untranslated regions were amplified by PCR with yeast genomic DNA as template. The amplified DNA was then subcloned into prs316 and prs315 [15] to generate plasmids pjn104 and pjn106, respectively. Site directed mutagenesis was performed on plasmid pjn115, which is pbluescriptks containing the NFS1 gene from pjn106. The codons for AUG1, AUG2 and AUG3 of the NFS1 gene were mutated to UUG by inverse PCR [16] of pjn115. The mutagenized fragments were then excised with Xho I and Avr II and subcloned back into pjn106. For in vitro transcription/translation the NFS1 reading frame and 50 nucleotides of upstream sequence were amplified from genomic DNA by PCR and subcloned into pgem3. This plasmid was named pjn102 and used as template for inverse PCR to change the codons for AUG1 and AUG2 to UUG generating plasmids pjn130 and pjn139, respectively. All 5210 J. H Nett et al. (Eur. J. Biochem. 268) q FEBS 2001 Table 1. Plasmids and yeast strains used in this study. Plasmid Markers Mutation in NFS1 gene pjn104 URA3, Amp, CEN, NFS1 None pjn106 LEU2, Amp, CEN, NFS1 None pjn126 LEU2, Amp, CEN, NFS1 ATG1!TTG pjn127 LEU2, Amp, CEN, NFS1 ATG2!TTG pjn129 LEU2, Amp, CEN, NFS1 ATG1!TTG, ATG2!TTG pjn133 LEU2, Amp, CEN, NFS1 ATG3!TTG pjn150 LEU2, Amp, CEN, NFS1 ATG1!TTG, ATG3!TTG pjn151 LEU2, Amp, CEN, NFS1 ATG2!TTG, ATG3!TTG pjn152 LEU2, Amp, CEN, NFS1 ATG1!TTG, ATG2!TTG, ATG3!TTG pjn102 Amp, Sp6,T7 promoter, NFS1 None pjn130 Amp, Sp6,T7 promoter, NFS1 ATG1!TTG pjn139 Amp, Sp6,T7 promoter, NFS1 ATG2!TTG Yeast Strain YPH500 X 499 JN49 JN56 JN74 JN78 JN80 JN82 JN83 JN98 JN99 JN100 Genotype a/a ura3-52 lys2-801 ade2-101 trp1-d63 his3-d200 leu2-d1 a/a YPH markers, NFS1/nfs1D1::TRP1 a YPH markers, nfs1d1::trp1, pjn104 a YPH markers, nfs1d1::trp1, pjn106 a YPH markers, nfs1d1::trp1, pjn126 a YPH markers, nfs1d1::trp1, pjn127 a YPH markers, nfs1d1::trp1, pjn129 a YPH markers, nfs1d1::trp1, pjn133 a YPH markers, nfs1d1::trp1, pjn150 a YPH markers, nfs1d1::trp1, pjn151 a YPH markers, nfs1d1::trp1, pjn152 of the introduced mutations were verified by sequencing the relevant coding regions. S. cerevisiae strains and growth conditions The yeast strains used are listed in Table 1. Yeast were grown in yeast extract/peptone/dextrose (YPD), yeast extract/peptone/ethanol/glycerol (YPEG) or synthetic dropout (SD) medium in which the relevant nutritional supplements were omitted. A null allele of NFS1 was made in the isogenic diploid YPH499xYPH500 [15] by targeted deletion of the NFS1 open reading frame [17] with the TRP1 gene. Trp 1 transformants were selected and screened by colony PCR to generate strain JN49. Proper integration of the TRP1 gene in the NFS1 locus was confirmed by PCR genotyping such that predicted unique fragments were generated by internal TRP1 primers and external NFS1 primers. Strain JN56 in which plasmid pjn104 (NFS1, CEN, URA3 ) contains the NFS1 gene covering the chromosomal NFS1 deletion (nfs1d1::trp1 ), was made by isolating a Ura 1,Trp 1 spore derived from diploid strain JN49 transformed with pjn104. Plasmids containing the mutagenized NFS1 gene were introduced into JN56 by plasmid shuffling as described below. Plasmid shuffling The LEU2 marked plasmids containing the mutagenized NFS1 genes were individually transformed into JN56 and Leu 1 transformants were selected. The primary Leu 1 transformants were replica plated to synthetic media lacking leucine but supplemented with 5-fluoroorotic acid (1 g per 800 ml) to select for loss of the URA3 plasmid. Duplicate replica plates were incubated for 3 days at 30 8C and colonies were picked and restreaked on synthetic media lacking either leucine or uracil to confirm loss of the URA3 plasmid. To confirm that the Leu2 marked plasmids had not reverted to wild-type, they were recovered from the yeast strains and sequenced. In vitro transcription and translation In vitro transcription and translation using the TnTwcoupled reticulocyte lysate system were performed according to supplier recommendations. Before use in the import experiment, polyribosomes were removed by centrifugation at g for 20 min. In vitro transcription using phage SP6 polymerase was performed according to supplier recommendations. In vitro translation using a yeast extract was performed according to Hansen and coworkers [18] with minor modifications. The extract was prepared by freezing the yeast cells in liquid nitrogen and breaking them in a stainless steel Waring blender. This treatment disrupts the mitochondria and releases MPP from the mitochondrial matrix. The RNA cap structure analogs (purchased from NEB) used were m 7 G(5 0 )ppp(5 0 )G for the reticulocyte lysate system and G(5 0 )ppp(5 0 )G for the yeast lysate system. To visualize the translated proteins, 0.5 ml of the reticulocyte lysate translation mixture or 3 ml of the yeast translation mixture were analyzed by SDS/PAGE and fluorography of the dried gels. q FEBS 2001 Substitution of the AUG start codon in yeast (Eur. J. Biochem. 268) 5211 Other methods Isolation of mitochondria for Western blotting and import of in vitro translated proteins into mitochondria were performed as described previously [19]. Growth rates of the yeast strains were determined in liquid YPEG medium at 30 8C. RESULTS An NFS1 gene that has AUG1 changed to UUG can substitute for wild-type NFS1 in vivo We have been investigating the import and assembly of the Rieske iron sulfur protein into mitochondria in yeast [19]. As part of these studies we were interested in identifying the enzyme that inserted the iron sulfur cluster into this protein. To explore the possibility that the Nfs protein might perform this function in yeast mitochondria, we wanted to test whether the targeting of the yeast NFS1-encoded protein was regulated through alternative AUG utilization in the NFS gene, similar to what has been reported for the human Nifs protein. To this end we constructed yeast strains in which the first three AUG codons of the reading frame had been individually and cumulatively replaced by UUG. If targeting of the NFS1-encoded protein was indeed regulated by such a mechanism, the AUG1 mutation should have prohibited the Nfs1 protein from entering the mitochondria and led to a nonviable or at least nonrespiratory competent phenotype, as the Nfs1 protein is believed to be involved in the assembly of iron sulfur clusters into multiple enzymes of cell respiration [14]. However, we found that the strains with the mutated AUG codons were viable and also competent for respiration. When we recovered the plasmids carrying the altered NFS1 genes and sequenced them we found that the NFS1 genes still carried the expected mutations. Strains carrying the mutation of AUG1 to UUG grew at a slower rate, reflected as an increase in doubling time from 3.6 to 4.9 h on nonfermentable carbon sources (Table 2), suggesting that the amount of Nfs1 protein produced was now a rate limiting step for the growth of the yeast cells. On the other hand the mutation of AUG2 or AUG3 to UUG did not have a significant effect on growth. Different translational start sites are used when mutated NFS1 mrna is translated in vitro in reticulocyte lysate or yeast lysate When mrna from the wild-type NFS1 gene is translated in a reticulocyte lysate system the majority of protein is derived from initiation of translation at AUG1 (Fig. 1). To a lesser extent AUG2, AUG3 and AUG4 are used as start sites, and initiation from AUG5 is barely detectable. When AUG1 is changed to UUG, no protein derived from initiation at the mutated codon is detectable. Instead, translation initiation from AUG2, AUG3 and AUG4 increases as expected. There is also a new species of protein that results from initiation at a site between UUG1 and AUG2 (Fig. 1). We do not know exactly at which site this initiation occurs, but there are 11 potential codons between AUG1 and AUG2 that differ in only one base from AUG, as indicated by the underlined codons in Fig. 2. When AUG2 is changed to UUG and the resulting mrna is translated in the reticulocyte system, the protein distribution is very similar to that from the wild-type gene. The only difference is the lack of protein derived from initiation at that missing AUG2, as expected. When we performed the same experiments using a yeast lysate system, we obtained dramatically different results. When mrna from wild-type NSF1 or from the NSF1 with the AUG2!UUG mutation was used as template, only AUG1 was selected as a start site for translation (Fig. 1). Most notably, in contrast to what was seen for the reticulocyte system, mutagenesis of AUG1 to UUG did not lead to increased translation initiation from a downstream AUG as would be expected according to the ribosomal scanning mechanism. Instead, the UUG codon introduced by the mutagenesis was used for initiation. Although the amount of protein derived from the UUG start codon was reduced, it is still easily detectable (Fig. 1). Another difference observed with the yeast lysate system is that the precursor Nfs1 protein is processed during the in vitro translation reaction to its mature form. This processing step is performed by matrix processing peptidase (MPP) that is released from the mitochondria during preparation of the yeast lysate, and that immediately cleaves the majority of translated protein. We showed this by importing the translated proteins from the reticulocyte system (Fig. 3A) and the yeast lysate system (Fig. 3B) into Table 2. Doubling times in YPEG medium of yeast strains containing NFS1 genes with mutated AUG codons. The negative control was the NFS1 null strain transformed with prs315, the positive control was the NFS1 null strain transformed with prs315 containing the wild-type NFS1 gene. Mutated codon Growth on 5FOA Growth on YPEG Doubling time (h) Negative Control Positive Control AUG AUG AUG AUG AUG AUG AUG 5212 J. H Nett et al. (Eur. J. Biochem. 268) q FEBS 2001 Fig. 2. Nucleotide and deduced protein sequence of S. cerevisiae NFS1. Only the first 156 codons and amino acids of the 497 total are shown. The positions of AUGs 1 5 are marked above the nucleotide sequence. The 11 potential start sites that only differ in one base from AUG between AUG1 and AUG2 are underlined. The MPP processing site is marked by an arrow (b). Fig. 1. In vitro translation of mutated NFS1 mrna in rabbit reticulocyte lysate and yeast lysate. For the reticulocyte lysate system the migration positions of proteins derived from initiation at AUG1 through AUG5 are indicated. (?) indicates a protein derived from an unknown start site between AUG1 and AUG2. For the yeast lysate system the migration positions of precursor (p-nfs1) and mature (m-nfs1) NFS1-encoded proteins are indicated. yeast mitochondria in vitro. When the mrna with the AUG2!UUG mutation was translated in the reticulocyte system there was a wild-type level of import of the resulting protein, but when the same mrna was translated in the yeast lysate system, only the fraction of the protein that still contained the presequence was import competent and protected from externally added proteinase K. Additionally, when AUG1 was changed to UUG and the translated proteins from the reticulocyte or yeast systems were imported, only the small fraction of protein that was derived from initiation at the mutated codon contained the presequence and was imported in vitro. The control experiment, showing that almost all of the product of the yeast translation system is the same size as mature Nfs1p after import into mitochondria is shown in Fig. 3C. To determine the amounts of protein encoded by the mutated gene present in the yeast and to confirm that the protein is targeted to mitochondria we performed Western blots of subcellular fractions from wild-type yeast and the mutant in which the AUG1 was changed to UUG. As shown in Fig. 4, the Nfs1 protein resulting from the altered initiation codon is the same size as that resulting from the wild-type gene. The protein from both yeast strains is recovered in the mitochondria. As expected from the in vitro translation experiments, the amount of protein resulting from the UUG initiation codon is markedly less than that from the wild-type gene. This accounts for the decreased growth rate (Table 2) of the mutant. Although we detected small amounts of Nfs1 protein in the nuclear and postmitochondrial supernatant fractions, these were due to contamination of those fractions with mitochondria, as indicated by immunoblotting of the fractions with antibodies to two mitochondrial marker proteins, cytochrome c 1 and Rieske iron sulfur protein (data not shown). The Nfs1 protein detected in the nuclear and postmitochondrial fractions was the same size as that detected in mitochondria. We were unable to detect the unprocessed form of Nfs1p in any of the fractions. DISCUSSION The ribosomal scanning mechanism [20] is a wellestablished model of how eukaryotic ribosomes select the site for initiation of translation. The 40S ribosomal subunit binds first near the 5 0 end of a mrna and then advances until it encounters the first AUG codon. At this point, the q FEBS 2001 Substitution of the AUG start codon in yeast (Eur. J. Biochem. 268) 5213 Fig. 4. Western blots of NFS1-encoded proteins derived from genes containing AUG and UUG initiation codons. Mitochondria were isolated by differential centrifugation [19] from the two yeast strains and resolved by SDS/PAGE. Western blots of the mitochondrial proteins were probed with antibodies to Nfs1 protein and two marker proteins of the inner mitochondrial membrane, cytochrome c 1 and Rieske iron sulfur protein [21] to demonstrate equal amounts of mitochondria loaded into the gel lanes. Fig. 3. Import into mitochondria of NFS1-encoded proteins derived from genes containing mutated AUG codons. NFS1 mrna in which AUG1 or AUG2 had been replaced by UUG was translated in rabbit reticulocyte lysate (A) or yeast lysate (B) and the resulting proteins imported into yeast mitochondria for the indicated times. After import half of each sample was treated with proteinase K. In (B) the panel for mutant AUG1!UUG is shown at high contrast and after 10 times longer exposure time of the gel to allow better visualization of the band for the mature Nfs1 protein (m-nfs1). Panel (C) shows the translation products when wild-type NFS1 mrna or NFS1 mrna in which AUG1 had been replaced by UUG was translated in the yeast lysate in lanes 1 and 2. The remaining four lanes show the product obtained from translation of the wild-type NFS1 mrna in the rabbit reticulocyte lysate before and after import into yeast mitochondria. When import was blocked by eliminating the membrane potential, there was no change in size of the protein and it was completely degraded by proteinase K. After 20 min of import most of the Nfs1p was imported and converted to its mature size and the mature protein was resistant to proteinase K. 40S ribosome halts and the 60S subunit joins. Studies in S. cerevisiae have shown that the loss of the first AUG by mutation leads to initiation at the next AUG codon [1,2]. Our results suggest, however, that at least in S. cerevisiae there are exceptions to this rule. When we mutated the first AUG codon in the NFS1 gene to UUG and translated the corresponding mrna in a yeast lysate translation system the rate of initiation of translation from downstream AUG codons did not increase. Instead, the rate of initiation at the mutated codon was reduced. When the same experiments were performed in a reticulocyte lysate system, however, translation from the mutated codon was almost completely abolished and initiation occurred at downstream AUG codons at an increased rate. The fact that the same mrna supports initiation of tran
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