The adenylate kinase family in yeast: identification of URA6 as a multicopy suppressor of deficiency in major AMP kinase.

The gene URA6 encoding uridylate kinase (UK) from Saccharomyces cerevisiae was isolated as a multicopy suppressor of the respiratory-deficient phenotype of an S. cerevisiae mutant defective in the gene AKY2 encoding AMP kinase (AK). The URA6 gene also restored temperature resistance to two different temperature-sensitive mutations in the gene encoding Escherichia coli AK. By contrast, the gene encoding UK of Dictyostelium discoideum on a multicopy yeast shuttle plasmid, expressed under control of the constitutive yeast AKY2 promoter, failed to complement the deficiency in yeast, although such transformants expressed high UK activity. We show that yeast UK exerts significant AK activity which is responsible for the complementation and is absent in the analogous enzyme from D. discoideum. Since UK also significantly phosphorylates CMP (but not GMP), it must be considered an unspecific short-form nucleoside monophosphate kinase. Wild-type mitochondria lack UK activity, but import AKY2. Since multicopy transformation with URA6 heals the Pet- phenotype of AKY2 disruption mutants, the presence of AKY2 in the mitochondrial intermembrane space is not required to maintain respiratory competence. However, furnishing UK with the bipartite intermembrane space-targeting presequence of cytochrome c1 improves the growth rates of AKY2 mutants with nonfermentable substrates, suggesting that AK activity in mitochondria is helpful, though not essential for oxidative growth.     

Constraints on amino acid substitutions in the N-terminal helix of cytochrome c explored by random mutagenesis.

The interaction of the N- and C-terminal helices is a hallmark of the cytochrome c family. Oligodeoxyribonucleotide-directed random mutagenesis within the gene encoding the C102T protein variant of Saccharomyces cerevisiae iso-1-cytochrome c was used to generate a library of mutations at the evolutionary invariant residues Gly-6 and Phe-10 in the N-terminal helix. Transformation of this library (contained on a low-copy-number yeast shuttle phagemid) into a yeast strain lacking a functional cytochrome c, followed by selection for cytochrome c function, reveals that 4-10% of the 400 possible amino acid substitutions are compatible with function. DNA sequence analysis of phagemids isolated from transformants exhibiting the functional phenotype elucidates the requirements for a stable helical interface. Basic residues are not tolerated at position 6 or 10. There is a broad volume constraint for amino acids at position 6. The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effect alone is sufficient to promote helical association. There are severe constraints that limit the combinations consistent with function, but the number of functionally consistent combinations observed exemplifies the plasticity of proteins.     

Mutants with temperature-sensitive defects in the Escherichia coli mismatch repair system: sensitivity to mispairs generated in vivo

We have used direct selections to generate large numbers of mutants of Escherichia coli defective in the mismatch repair system and have screened these to identify mutants with temperature-sensitive defects. We detected and sequenced mutations that give rise to temperature-sensitive MutS, MutL, and MutH proteins. One mutation, mutS60, results in almost normal levels of spontaneous mutations at 37 degrees C but above this temperature gives rise to higher and higher levels of mutations, reaching the level of null mutations in mutS at 43 degrees C. However, at 37 degrees C the MutS60 protein can be much more easily titrated by mispairs than the wild-type MutS, as evidenced by the impaired ability to block homologous recombination in interspecies crosses and the increased levels of mutations from weak mutator alleles of mutD (dnaQ), mutC, and ndk. Strains with mutS60 can detect mispairs generated during replication that lead to mutation with much greater sensitivity than wild-type strains. The findings with ndk, lacking nucleotide diphosphate kinase, are striking. An ndk mutS60 strain yields four to five times the level of mutations seen in a full knockout of mutS. These results pose the question of whether similar altered Msh2 proteins result from presumed polymorphisms detected in tumor lines. The role of allele interactions in human disease susceptibility is discussed.     

KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth.

Suppressors of a temperature-sensitive RNA polymerase II mutation were isolated to identify proteins that interact with RNA polymerase II in yeast cells. Ten independently isolated extragenic mutations that suppressed the temperature-sensitive mutation rpb1-1 and produced a cold-sensitive phenotype were all found to be alleles of a single gene, SRB1. An SRB1 partial deletion mutant was further investigated and found to exhibit several pleiotropic phenotypes. These included suppression of numerous temperature-sensitive RNA polymerase II mutations, alteration of the temperature growth range of cells containing wild-type RNA polymerase, and sterility of cells of alpha mating type. The ability of SRB1 mutations to suppress the temperature-sensitive phenotype of RNA polymerase II mutants did not extend to other temperature-sensitive mutants investigated. Isolation of the SRB1 gene revealed that SRB1 is KEX2. These results indicate that the KEX2 protease, whose only known substrates are hormone precursors, can have an important influence on RNA polymerase II and the temperature-dependent growth properties of yeast cells.     

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: evidence for a single, catalytically active, cytochrome c binding domain

Forty-six charge-reversal mutants of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutants include the conversion of all 20 glutamate residues and 24 of the 25 aspartate residues in CcP, one at a time, to lysine residues. In addition, two positive-to-negative charge-reversal mutants, R31E and K149D, are included in the study. The mutants have been characterized by absorption spectroscopy and hydrogen peroxide reactivity at pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c (C102T) as substrate at pH 7.5. Many of the charge-reversal mutations cause detectable changes in the absorption spectrum of the enzyme reflecting increased amounts of hexacoordinate heme compared to wild-type CcP. The increase in hexacoordinate heme in the mutant enzymes correlates with an increase in H 2O 2-inactive enzyme. The maximum velocity of the mutants decreases with increasing hexacoordination of the heme group. Steady-state velocity studies indicate that 5 of the 46 mutations (R31E, D34K, D37K, E118K, and E290K) cause large increases in the Michaelis constant indicating a reduced affinity for cytochrome c. Four of the mutations occur within the cytochrome c binding site identified in the crystal structure of the 1:1 complex of yeast cytochrome c and CcP [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] while the fifth mutation site lies outside, but near, the crystallographic site. These data support the hypothesis that the CcP has a single, catalytically active cytochrome c binding domain, that observed in the crystal structures of the cytochrome c/CcP complex.     

Characterization of Saccharomycopsis lipolytica mutants that express temperature-sensitive synthesis of isocitrate lyase

Four mutants specifically deficient in the activity of isocitrate lyase were independently isolated in the alkane yeast Saccharomycopsis lipolytica. Genetic analysis by means of protoplast fusion and mitotic haploidization revealed that the mutations were recessive and non-complementary at a single genetic locus, icl. icl is a structural gene for isocitrate lyase, because some revertants from icl-1 and icl-3 mutants produced thermolabile isocitrate lyase in comparison with the wild-type enzyme, and also because the gene dosage effect was observed on the specific activity of isocitrate lyase in icl+/icl-1 and icl+/icl-3 heterozygotes. The icl-3 mutation also gave rise to temperature-sensitive revertants that could grow on acetate at 23 degrees C but not at 33 degrees C, exhibiting temperature-sensitive synthesis as well as thermostable activity of isocitrate lyase. Studies on purified isocitrate lyase showed that this enzyme is tetrameric and that the enzyme synthesized at 23 degrees C by a temperature-sensitive synthesis mutant was indistinguishable from the wild-type enzyme with respect to the subunit molecular weight (59,000), the isoelectric pH (5.3), the thermostability, and the Km value for threo-Ds-isocitrate (0.2 mM). When induced by acetate at 33 degrees C, the temperature-sensitive synthesis mutant did not express isocitrate lyase activity but did synthesize polypeptides whose electrophoretic mobilities were equal to that of the purified mutant enzyme. Hence, the temperature-sensitive mutation assumed in the structural gene for isocitrate lyase might have prevented the maturation of the polypeptide chains synthesized at the restrictive temperature.     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. Mitochondrial protein synthesis and analysis of the polypeptide composition of cytochrome c oxidase.

Three previously isolated mutants of Neurospora crassa, temperature-sensitive for the production of cytochrome aa3, have been further analyzed. These mutants have a slightly reduced capacity for mitochondrial protein synthesis when grown at 41 degrees C, although this relative deficiency appeared to be no greater than the deficiency in other cytochrome-aa3-deficient mutants. Thermolability studies revealed that the cytochrome c oxidase purified from each of the mutants grown at 23 degrees C is no more sensitive to heat inactivation than the enzyme isolated from wild-type cells. Sodium dodecylsulfate gel electrophoresis of immunoprecipitates obtained from the mitochondria of each of the mutants grown at 23 degrees C, using antiserum directed against holocytochrome c oxidase, indicated that all the subunits of cytochrome c oxidase were present in relative amounts similar to those found in mitochondria from wild-type cultures. However, when the mitochondria from mutant cultures grown at 41 degrees C were examined in the above fashion, only subunits 5 and 6 of the oxidase were detected. Nonetheless, the mitochondrially synthesized subunit 1, 2 and 3 polypeptides could be immunoprecipitated from mitochondria isolated from mutant cells grown at 41 degrees C and labelled with [3H]leucine in medium containing cycloheximide. Although subunits 4 and 7 could not be detected, because a suitable antibody was not available, the fact that five of the seven subunits were present, but not associated with each other, suggested that the genetic defects in these mutants may affect the process of cytochrome c oxidase assembly.     

The consequences of base-pair substitution mutations in AT- and GC-rich bacteria

The likely consequences, in terms of premature stop codons, detectable missense mutants, silent missense mutants, and degenerate codon changes, have been determined for all 12 individual base substitution changes. This has been done for the full, 61 sense codon, genetic code and also for the much more limited codon availabilities of AT- or GC-rich DNA. The specificities and outcomes of individual base substitutions are likely to be rather different at AT- or GC-rich extremes, and also from the situation at an intermediate DNA base-ratio where all 61 sense codons are available. In particular, at DNA base-ratio extremes many mutations will be to non-utilized codons, which may well act as nonsense mutants. These in turn will give novel classes of suppressor-containing revertants. Even in bacteria with intermediate DNA base-ratios, particular codons for a given amino acid may be favoured, over alternatives, because their use maximizes, or minimizes, the mutational consequences of one, or more, base substitution changes.     

Rescue of growth defects of yeast cdc48 mutants by pathogenic IBMPFD-VCPs

VCP/p97/Cdc48 is a hexameric ring-shaped AAA ATPase that participates in a wide variety of cellular functions. VCP is a very abundant protein in essentially all types of cells and is highly conserved among eukaryotes. To date, 19 different single amino acid-substitutions in VCP have been reported to cause IBMPFD (inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia), an autosomal dominant inherited human disease. Moreover, several similar single amino acid substitutions have been proposed to associate with a rare subclass of familial ALS. The mechanisms by which these mutations contribute to the pathogenesis are unclear. To elucidate potential functional differences between wild-type and pathogenic VCPs, we expressed both VCPs in yeast cdc48 mutants. We observed that all tested pathogenic VCPs suppressed the temperature-sensitive phenotype of cdc48 mutants more efficiently than wild-type VCP. In addition, pathogenic VCPs, but not wild-type VCP, were able to rescue a lethal cdc48 disruption. In yeast, pathogenic VCPs, but not wild-type VCP, formed apparent cytoplasmic foci, and these foci were transported to budding sites by the Myo2/actin-mediated transport machinery. The foci formation of pathogenic VCPs appeared to be associated with their suppression of the temperature-sensitive phenotype of cdc48 mutants. These results support the idea that the pathogenic VCP mutations create dominant gain-of-functions rather than a simple loss of functional VCP. Their unique properties in yeast could provide a convenient drug-screening system for the treatment of these diseases.     

Identification, cloning, and characterization of the Escherichia coli sohA gene, a suppressor of the htrA (degP) null phenotype.

Two extragenic suppressors which allow temperature-sensitive htrA mutant Escherichia coli bacteria to grow at 42 degrees C and simultaneously acquire a cold-sensitive phenotype at 30 degrees C were isolated. The cold-sensitive phenotype exhibited by one of the mutants was used to clone the corresponding wild-type copy of the suppressor gene. This was done through complementation with a mini-mu plasmid E. coli DNA library, by selection for colonies which were no longer cold sensitive, at 30 degrees C. The cloned suppressor gene was shown to complement the cold-sensitive phenotype of both suppressor mutations. It was mapped to 68 min on the E. coli chromosome through hybridization to the Kohara library of overlapping lambda transducing bacteriophages, which covers the entire E. coli chromosome. The complementing gene was further subcloned on an 830-base-pair (bp) DNA fragment. DNA sequencing revealed the presence of an open reading frame (ORF) of 333 bp which could encode a protein of 12,359 Mr. Subcloning of various DNA fragments from within this 830-bp DNA fragment suggests that this ORF is most likely responsible for suppression of the cold-sensitive phenotype of the htrA suppressor bacteria. By using a T7 polymerase system to overproduce plasmid-encoded proteins, a protein of approximately 12,000 Mr was produced by this cloned DNA fragment. This ORF defines a previously undiscovered gene in E. coli, called sohA (suppressor of htrA).     

Primary structures of mutationally altered ribosomal protein L7/L12 and their effects on cellular growth and translational accuracy

The amino acid sequences of mutationally altered ribosomal protein L7/L12 from four different rplL mutants of Escherichia coli were determined and correlated with some features of the mutant ribosomes. Two of the rplL mutations are deletions around position 40, which give rise to a shortened hinge region between the two domains of L7/L12. The other two mutants harbor point mutations at position 74 (Gly----Asp) or at position 82 (Glu----Lys), which are in or close to an evolutionarily conserved sequence in the C-terminal domain. The two latter mutations are associated with decreased rates of growth and translational elongation. All four mutants show increased nonsense codon read-through in vivo. Ribosomes from one of the deletion mutants show clearly increased missense error rates in vitro.     

The dominant temperature-sensitive lethal DTS7 of Drosophila melanogaster encodes an altered 20S proteasome beta-type subunit.

Proteasomes are multicatalytic complexes that function as the major proteolytic machinery in regulated protein degradation. The eukaryotic 20S proteasome proteolytic core structure comprises 14 different subunits: 7 alpha-type and 7 beta-type. DTS7 is a dominant temperature-sensitive (DTS) lethal mutation at 29 degrees that also acts as a recessive lethal at ambient temperatures. DTS7 maps to cytological position 71AB. Molecular characterization of DTS7 reveals that this is caused by a missense mutation in a beta-type subunit gene, beta2. A previously characterized DTS mutant, l(3)73Ai1, results from a missense mutation in another beta-type subunit gene, beta6. These two mutants share a very similar phenotype, show a strong allele-specific genetic interaction, and are rescued by the same extragenic suppressor, Su(DTS)-1. We propose that these mutants might act as "poison subunits," disrupting proteasome function in a dosage-dependent manner, and suggest how they may interact on the basis of the structure of the yeast 20S proteasome.     

Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein

In vivo selections were used to isolate 43 temperature-sensitive gene V mutants of the bacteriophage f1 from a collection of mutants constructed by saturation mutagenesis of the gene. The sites of temperature-sensitive substitutions are found in both the beta-sheets and the turns of the protein, and some sites are exposed to the solvent while others are not. Thirteen of the variant proteins were purified and characterized to evaluate their free energy changes upon unfolding and their affinities for single-stranded DNA, and eight were tested for their tendencies to aggregate at 42 degrees C. Each of the three temperature-sensitive mutants at buried sites and six of ten at surface sites had free energy changes of unfolding substantially lower (less stabilizing) than the wild-type at 25 degrees C. A seventh mutant at a surface site had a substantially altered unfolding transition and its free energy of unfolding was not estimated. The affinities of the mutant proteins for single-stranded DNA varied considerably, but two mutants at a surface site, Lys69, had much weaker binding to single-stranded DNA than any of the other mutants, while two mutants at another surface site, Glu30, had the highest DNA-binding affinities. The wild-type gene V protein is stable at 42 degrees C, but six of the eight mutants tested aggregated within a few minutes and the remaining two aggregated within 30 minutes at this temperature. Overall, each of the temperature-sensitive proteins tested had a tendency to aggregate at 42 degrees C, and most also had either a low free energy of unfolding (at 25 degrees C), or weak DNA binding. We suggest that any of these properties can lead to a temperature-sensitive gene V phenotype.     

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.     

Mapping of functional sites on the primary structure of the contractile tail sheath protein of bacteriophage T4 by mutation analysis

In order to determine the functional roles of amino acid residues in gp18 (gp: gene product), the contractile tail sheath protein of bacteriophage T4, the mutation sites and amino acid replacements of available and newly created missense mutants with distinct phenotypes were determined. Amber mutants were also utilized for amino acid insertion by host amber suppressor cell strains. It was found that mutants that gave rise to a particular phenotype were mapped in a particular region along the polypeptide chain. Namely, all amino acid replacements in the cold-sensitive mutants (cs, which grows at 37 degrees C, but not at 25 degrees C) and the heat-sensitive mutant (hs, lose viability by incubation at 55 degrees C for 30 min) except for one hs mutant were mapped in a limited region in the C-terminal domain. On the other hand, all the temperature-sensitive mutants (ts, grow at 30 degrees C, but not at 42 degrees C) and carbowax mutants (CBW, can adsorb to the host bacterium in the presence of high concentrations of polyethylene glycol, where wild-type phage cannot) were mapped in the N-terminal protease-resistant domain, except for one ts mutant. The results suggested that the C-terminal region of gp18 is important for contraction and assembly, whereas the N-terminal protease-resistant domain constitutes the protruding part of the tail sheath.     

Human MutL homolog (MLH1) function in DNA mismatch repair: a prospective screen for missense mutations in the ATPase domain

Germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1 are responsible for the majority of hereditary non-polyposis colorectal cancer (HNPCC), an autosomal-dominant early-onset cancer syndrome. Genetic testing of both MSH2 and MLH1 from individuals suspected of HNPCC has revealed a considerable number of missense codons, which are difficult to classify as either pathogenic mutations or silent polymorphisms. To identify novel MLH1 missense codons that impair MMR activity, a prospective genetic screen in the yeast Saccharomyces cerevisiae was developed. The screen utilized hybrid human-yeast MLH1 genes that encode proteins having regions of the yeast ATPase domain replaced by homologous regions from the human protein. These hybrid MLH1 proteins are functional in MMR in vivo in yeast. Mutagenized MLH1 fragments of the human coding region were synthesized by error-prone PCR and cloned directly in yeast by in vivo gap repair. The resulting yeast colonies, which constitute a library of hybrid MLH1 gene variants, were initially screened by semi-quantitative in vivo MMR assays. The hybrid MLH1 genes were recovered from yeast clones that exhibited a MMR defect and sequenced to identify alterations in the mutagenized region. This investigation identified 117 missense codons that conferred a 2-fold or greater decreased efficiency of MMR in subsequent quantitative MMR assays. Notably, 10 of the identified missense codons were equivalent to codon changes previously observed in the human population and implicated in HNPCC. To investigate the effect of all possible codon alterations at single residues, a comprehensive mutational analysis of human MLH1 codons 43 (lysine-43) and 44 (serine-44) was performed. Several amino acid replacements at each residue were silent, but the majority of substitutions at lysine-43 (14/19) and serine-44 (18/19) reduced the efficiency of MMR. The assembled data identifies amino acid substitutions that disrupt MLH1 structure and/or function, and should assist the interpretation of MLH1 genetic tests.     

Proton titration curve of yeast iso-1-ferricytochrome c. Electrostatic and conformational effects of point mutations.

The proton titration curves of yeast iso-1-ferricytochrome c and selected point mutants of this protein have been determined between pH 3 and 11 at 10 and 25 degrees C with a computer-controlled titration system. Initial titration of the wild-type protein to acidic pH followed by subsequent titrations to alkaline and then acidic pH demonstrates hysteresis, with one more group (28.7) titrating between pH 11 and 3 than originally titrated (27.7) between pH 3 and 11. Initial titration to alkaline pH, however, resulted in observation of the same number of groups in both directions of titration (28.7 vs 28.6). At 10 degrees C, 7.5 fewer groups were found to titrate over the same range of pH. Titration curves obtained for six cytochrome c mutants modified at Arg-38, Phe-82, Tyr-48, and Tyr-67 were analyzed by subtraction of the corresponding titration curve for the wild-type protein to produce difference titration curves. In most cases, the effects of these mutations as revealed in the difference titration curves could be accounted for as either the result of introduction of an additional group titrating within this pH range, the result of a change in the pK of a titrating residue, and/or the result of a change in the pK for either the first acidic or the first alkaline protein conformational transition. In addition to demonstration of the electrostatic consequences of the mutations in cytochrome c studied here, this study establishes the general usefulness of precise proton titration curve analysis in the characterization of variant proteins produced through recombinant genetic techniques.     

The stationary-phase-exit defect of cydC (surB) mutants is due to the lack of a functional terminal cytochrome oxidase.

The surB gene was identified as a gene product required for Escherichia coli cells to exit stationary phase at 37 degrees C under aerobic conditions. surB was shown to be the same as cydC, whose product is required for the proper assembly and activity of cytochrome d oxidase. Cytochrome d oxidase, encoded by the cydAB operon, is one of two alternate terminal cytochrome oxidases that function during aerobic electron transport in E. coli. Mutations inactivating the cydAB operon also cause a temperature-sensitive defect in exiting stationary phase, but the phenotype is not as severe as it is for surB mutants. In this study, we examined the phenotypes of surB1 delta(cydAB) double mutants and the ability of overexpression of cytochrome o oxidase to suppress the temperature-sensitive stationary-phase-exit defect of surB1 and delta(cydAB) mutants and analyzed spontaneous suppressors of surB1. Our results indicate that the severe temperature-sensitive defect in exiting stationary phase of surB1 mutants is due both to the absence of terminal cytochrome oxidase activity and to the presence of a defective cytochrome d oxidase. Membrane vesicles prepared from wild-type, surB1, and delta(cydAB) strains produced superoxide radicals at the same rate in vitro. Therefore, the aerobic growth defects of the surB1 and delta(cydAB) strains are not due to enhanced superoxide production resulting from the block in aerobic electron transport.