P-type ATPase spf1 mutants show a novel resistance mechanism for the killer toxin SMKT

SMKT, a killer toxin produced by the halotolerant yeast Pichia farinosa KK1, consists of alpha and beta subunits with folding remarkably similar to that of the fungal killer toxin KP4, a Ca2+ channel inhibitor. The budding yeast Saccharomyces cerevisiae is sensitive to SMKT. To understand the killing mechanism of SMKT, we isolated SMKT-resistant mutants of S. cerevisiae and characterized them. Five spf mutants (sensitivity to the P. farinosa killer toxin) fell into a single genetic complementation group, designated spf1. The SPF1 gene was cloned by complementation of the mutant phenotype. The SPF1 gene encodes a putative P-type ATPase of 1215 amino acid residues that contains 12 membrane-spanning regions. Gene disruption revealed that the SPF1 gene is not essential for viability but is required for the sensitivity to SMKT. The spf1 disruptant showed some phenotypes characteristic of glycosylation-defective mutants and secreted underglycosylated invertase. Fluorescence-activated cell-sorting analysis and indirect immunofluorescence microscopy showed that SMKT interacts with the cell surface of the resistant cells but not with that of sensitive cells, suggesting a novel resistance mechanism for this toxin. The glycosylation-defective phenotype and possible killer-resistant mechanisms are discussed in comparison with the Golgi Ca2+ pump Pmr1p.     

Lethal effect of the expression of a killer gene SMK1 in Saccharomyces cerevisiae

Expression of the SMK1 gene which encodes the yeast killer toxin SMKT is lethal in Saccharomyces cerevisiae. Effects of deletion and site-directed mutagenesis of SMK1 on the lethality and the secretion of the gene products were examined. Deletion of the interstitial gamma peptide or the C-terminal loop from Ala208 to the C-terminal Asp222 had no effect on the lethality. Those SMK1 products that lacked either the gamma peptide or the C-terminal loop were expressed in the cells but were not secreted into the culture medium, suggesting that these peptides may have a role in secretion or in protein stability. On the other hand, deletion of the signal sequence resulted in complete loss of the lethal activity. Entering the secretory pathway may be critical for the lethality. Further, deletion of the region from the C-terminus to Leu207 resulted in loss of the lethal activity. Leu207 is located at the C-terminus of the central strand of the beta-sheet structure of SMKT and its side chain is thrust into a hydrophobic environment between the beta-sheet and the alpha-helices. The result obtained upon substitutions of Ala, Ser or Glu for Leu207 suggested that the side chain of Leu207 stabilizes the hydrophobic environment that contributes to the overall structure of the SMK1 product.     

Immunochemical and mutational analyses of P-type ATPase Spf1p involved in the yeast secretory pathway.

The yeast SPF1 gene encodes a novel P-type ATPase, the substrate of which specificity has not been identified. It is required for sensitivity to SMKT, a killer toxin produced by the halotolerant yeast Pichia farinosa. To investigate the function of Spf1p, Asp487, the putative phosphorylation site of Spf1p, was replaced by Asn. Expression of the altered SPF1, with Asp487 replaced by Asn, did not suppress the SMKT-resistant phenotype of spf1 mutants, suggesting that the catalytic activity of this ATPase is required for acquisition of sensitivity to SMKT. Subcellular fractionation experiments indicated that the fractionation pattern of Spf1p was similar to that of an early Golgi protein, Och1p. Cells lacking Spf1p had an abnormal fractionation pattern of Sec12p. The spf1 disruptant also showed increased expression of Kar2p and sensitivity to tunicamycin. The glycosylation-defective phenotype and possible role of Spf1p in the secretory pathway are discussed.     

A Method for the Rapid Determination of Formic and Acetic Acids in Tobacco

The yeast SPF1 gene encodes a novel P-type ATPase, the substrate of which specificity has not been identified. It is required for sensitivity to SMKT, a killer toxin produced by the halotolerant yeast Pichia farinosa. To investigate the function of Spf1p, Asp487, the putative phosphorylation site of Spf1p, was replaced by Asn. Expression of the altered SPF1, with Asp487 replaced by Asn, did not suppress the SMKT-resistant phenotype of spf1 mutants, suggesting that the catalytic activity of this ATPase is required for acquisition of sensitivity to SMKT. Subcellular fractionation experiments indicated that the fractionation pattern of Spf1p was similar to that of an early Golgi protein, Och1p. Cells lacking Spf1p had an abnormal fractionation pattern of Sec12p. The spf1 disruptant also showed increased expression of Kar2p and sensitivity to tunicamycin. The glycosylation-defective phenotype and possible role of Spf1p in the secretory pathway are discussed.     

The CUG codon is decoded in vivo as serine and not leucine in Candida albicans.

Previous studies have shown that the yeast Candida albicans encodes a unique seryl-tRNA(CAG) that should decode the leucine codon CUG as serine. However, in vitro translation of several different CUG-containing mRNAs in the presence of this unusual seryl-tRNA(CAG) result in an apparent increase in the molecular weight of the encoded polypeptides as judged by SDS-PAGE even though the molecular weight of serine is lower than that of leucine. A possible explanation for this altered electrophoretic mobility is that the CUG codon is decoded as modified serine in vitro. To elucidate the nature of CUG decoding in vivo, a reporter system based on the C. albicans gene (RBP1) encoding rapamycin-binding protein (RBP), coupled to the promoter of the C. albicans TEF3 gene, was utilized. Sequencing and mass-spectrometry analysis of the recombinant RBP expressed in C. albicans demonstrated that the CUG codon was decoded exclusively as serine while the related CUU codon was translated as leucine. A database search revealed that 32 out of the 65 C. albicans gene sequences available have CUG codons in their open reading frames. The CUG-containing genes do not belong to any particular gene family. Thus the amino acid specified by the CUG codon has been reassigned within the mRNAs of C. albicans. We argue here that this unique genetic code change in cellular mRNAs cannot be explained by the 'Codon Reassignment Theory'.     

Development of a transformation system for gene knock-out in the flavinogenic yeast Pichia guilliermondii

Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5'-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib(-) Ura(+) phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3-12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3' end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8-0.9 kb sequences homologous to the target gene.     

Non-standard translational events in Candida albicans mediated by an unusual seryl-tRNA with a 5'-CAG-3' (leucine) anticodon.

From in vitro translation studies we have previously demonstrated the existence of an apparent efficient UAG (amber) suppressor tRNA in the dimorphic fungus Candida albicans (Santos et al., 1990). Using an in vitro assay for termination codon readthrough the tRNA responsible was purified to homogeneity from C.albicans cells. The determined sequence of the purified tRNA predicts a 5'-CAG-3' anticodon that should decode the leucine codon CUG and not the UAG termination codon as originally hypothesized. However, the tRNA(CAG) sequence shows greater nucleotide homology with seryl-tRNAs from the closely related yeast Saccharomyces cerevisiae than with leucyl-tRNAs from the same species. In vitro tRNA-charging studies demonstrated that the purified tRNA(CAG) is charged with Ser. The gene encoding the tRNA was cloned from C.albicans by a PCR-based strategy and DNA sequence analysis confirmed both the structure of the tRNA(CAG) and the absence of any introns in the tRNA gene. The copy number of the tRNA(CAG) gene (1-2 genes per haploid genome) is in agreement with the relatively low abundance (< 0.5% total tRNA) of this tRNA. In vitro translation studies revealed that the purified tRNA(CAG) could induce apparent translational bypass of all three termination codons. However, peptide mapping of in vitro translation products demonstrated that the tRNA(CAG) induces translational misreading in the amino-terminal region of two RNA templates employed, namely the rabbit alpha- and beta-globin mRNAs. These results suggest that the C.albicans tRNA(CAG) is not an 'omnipotent' suppressor tRNA but rather may mediate a novel non-standard translational event in vitro during the translation of the CUG codon. The possible nature of this non-standard translation event is discussed in the context of both the unusual structural features of the tRNA(CAG) and its in vitro behaviour.     

Transfer RNA structural change is a key element in the reassignment of the CUG codon in Candida albicans.

The human pathogenic yeast Candida albicans and a number of other Candida species translate the standard leucine CUG codon as serine. This is the latest addition to an increasing number of alterations to the standard genetic code which invalidate the theory that the code is frozen and universal. The unexpected finding that some organisms evolved alternative genetic codes raises two important questions: how have these alternative codes evolved and what evolutionary advantages could they create to allow for their selection? To address these questions in the context of serine CUG translation in C.albicans, we have searched for unique structural features in seryl-tRNA(CAG), which translates the leucine CUG codon as serine, and attempted to reconstruct the early stages of this genetic code switch in the closely related yeast species Saccharomyces cerevisiae. We show that a purine at position 33 (G33) in the C.albicans Ser-tRNA(CAG) anticodon loop, which replaces a conserved pyrimidine found in all other tRNAs, is a key structural element in the reassignment of the CUG codon from leucine to serine in that it decreases the decoding efficiency of the tRNA, thereby allowing cells to survive low level serine CUG translation. Expression of this tRNA in S.cerevisiae induces the stress response which allows cells to acquire thermotolerance. We argue that acquisition of thermotolerance may represent a positive selection for this genetic code change by allowing yeasts to adapt to sudden changes in environmental conditions and therefore colonize new ecological niches.     

The genetic code of the fungal CTG clade

Genetic code alterations discovered over the last 40 years in bacteria and eukaryotes invalidate the hypothesis that the code is universal and frozen. Mitochondria of various yeast species translate the UGA stop codon as tryptophan (Trp) and leucine (Leu) CUN codons (N = any nucleotide) as threonine (Thr) and fungal CTG clade species reassigned Leu CUG codons to serine and translate them ambiguously in their cytoplasms. This unique sense-to-sense genetic code alteration is mediated by a Ser-tRNA containing a Leu 5'-CAG-3'anticodon (ser-tRNA(CAG)), which is recognized and charged with Ser (~97%) by the seryl-tRNA synthetase (SerRS) and with Leu (~3%) by the leucyl-tRNA synthetase (LeuRS). This unusual tRNA appeared 272 ± 25 million years ago and had a profound impact on the evolution of the CTG clade species. Here, we review the most recent results and concepts arising from the study of this codon reassignment and we highlight how its study is changing our views of the evolution of the genetic code.     

Characterization of the solution properties of Pichia farinosa killer toxin using PGSE NMR diffusion measurements

The solution behaviour with respect to pH and NaCl concentration of the tertiary structure and propensity for aggregation of salt- mediated killer toxin (SMKT) from Pichia farinosa was examined using pulsed-gradient spin-echo NMR diffusion measurements. It was found that in 0.15m NaCl the tertiary structure of SMKT was constant below pH 5.0, with the native SMKT existing as an unaggregated heterodimer containing the -subunit in a compactly folded form. However, above pH 5.0 the -subunit dissociated and lost its compact structure, becoming a random coil with an 37% increase in effective hydrodynamic radius. To determine the effects of NaCl concentration on the tertiary structure of SMKT, diffusion measurements were performed at pH 3.5 and NaCl concentrations up to 2M. Both the tertiary structure and aggregation state of SMKT were found to be insensitive to the salt concentration which indicates that the activity of the toxin is not a direct result of salt–protein interactions.     

Functional expression of the rat organic anion transporter 1 (rOAT1) in Saccharomyces cerevisiae

Organic anion transporter 1 (OAT1) is localized in the basolateral membrane of the proximal tubule in the kidney and plays an essential role in eliminating a wide range of organic anions, preventing their toxic effects on the body. Structural and functional studies of the transporter would be greatly assisted by inexpensive and rapid expression in the yeast Saccharomyces cerevisiae. The gene encoding rat OAT1 (rOAT1) contains many yeast non-preferred codons at the N-terminus and so was modified by fusion of the favored codon sequence of a hemagglutinin (HA) epitope preceding the start codon. The modified gene was cloned into several yeast expression plasmids, both integrative and multicopy, with either ADH1 promoter or GAL1 promoter in order to find a suitable expression system. Compared with the wild type gene, a substantial increase in rOAT1 expression was achieved by modification in the translational initiation region, suggesting that the codon chosen at the N-terminus influenced its expression. The highest inducible expression of rOAT1 was obtained under GAL1 promoter in 2 mu plasmid. A large fraction of rOAT1 was glycosylated in yeast, unaffected by growth temperature. The recombinant yeast expressing rOAT1 showed an increase in the uptake of p-aminohippurate (PAH) and this showed a positive correlation with rOAT1 expression level. Location of rOAT1 predominantly in the yeast plasma membrane confirmed correct processing. The importance of glycosylation for rOAT1 targeting was also shown. To our knowledge, this is the first successful functional expression of rOAT1 in the yeast S. cerevisiae.     

The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe

We have cloned and sequenced the alcohol dehydrogenase gene of the fission yeast Schizosaccharomyces pombe. The gene was isolated by transformation and complementation of a Saccharomyces cerevisiae strain which lacked functional alcohol dehydrogenase with an S. pombe gene bank constructed in the autonomously replicating yeast plasmid YEp13. Southern hybridization analysis indicates that S. pombe contains only one alcohol dehydrogenase gene. The structural region of the gene is 50% homologous to the alcohol dehydrogenase encoding genes of the budding yeast S. cerevisiae. The gene exhibits a very strong codon usage bias; with the set of predominantly used codons generally resembling that which S. cerevisiae employs preferentially. All of the differences in codon usage bias between S. pombe and S. cerevisiae are in the direction of greater G + C content in S. pombe codons. It is argued that this observation supports the hypothesis that selection toward uniform codon-anticodon binding energies contributes to codon usage bias and that the optimum binding energy is, on the average, higher in S. pombe than S. cerevisiae.     

First position wobble in codon-anticodon pairing: amber suppression by a yeast glutamine tRNA

A 2.4-kb fragment of DNA isolated from the Saccharomyces cerevisiae genome was found to suppress amber mutations when its carrier plasmid was present in high copy number. A 1.2-kb subclone of this fragment was sufficient to confer suppressor activity. Sequencing has established that this fragment carries a normal glutamine tRNA gene. Deletion of this tRNA gene from the subclone resulted in the loss of suppressor activity. The tRNAGln has the anticodon CUG that normally recognizes the glutamine codon CAG. We propose that suppression occurs via an inefficient readthrough of the UAG amber stop codons during translation. Such readthrough requires wobble in the first position of the codon.     

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.     

The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstream codons.

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.     

Initiation of herpes simplex virus thymidine kinase polypeptides.

When employed as a transgene reporter, the herpes simplex type 1 virus (HSV1) thymidine kinase gene (tk) is ectopically expressed in mouse testis. The principal testicular mRNA lacks the 5'-end of the tk reading frame. As a result the principal translation products, P2 and P3, are N-terminally truncated. These co-migrate in SDS-PAGE with polypeptides synthesised during HSV1 infection that were previously thought to be initiated at methionine codons ATG46 and ATG60. Prompted by these observations we generated modified tk genes each carrying only one of the first three ATG codons. Transfected cells expressed both full-length enzyme (P1) and P2 when only ATG1 was unmodified, P2 and P3 when only ATG46 was unmodified or P2 and a fourth polypeptide (P4) when only ATG60 was unmodified. Our observations indicate that P3 is initiated at ATG46 rather than ATG60, while P2 is initiated at a non-ATG codon rather than ATG46 and P4 is initiated at ATG60. When either of two putative non-ATG initiation codons was modified P2 was no longer produced. Cells mainly expressing either P1 or P3 exhibited the same sensitivity to Ganciclovir as cells transfected with the unaltered tk gene. P1 and P3 both have TK activity while P4 probably has none.