Contact-shifted resonances in the 1H NMR spectra of cytochrome b5. Resonance identification and spin density distribution in the heme group.

This paper describes the identification of some of the contact-shifted resonances in the 1H NMR spectrum of low spin ferric cytochrome b5. In these experiments comparison with cytochrome b5 which had been reconstituted with deuteroheme IX played an important role. NMR techniques used include double resonance experiments, line width analyses, and studies of the pH-dependence of the 1H NMR chemical shifts. The electronic heme structure derived from these resonance assignments is characterized by a highly anisotropic spin density distribution. This anisotropy is most strikingly manifested in the resonances of the vinyl and propionic acid substituents of the protoheme IX. The experiments described in this paper further revealed the coexistence in aqueous solutions of two different molecular species of cytochrome b5, which can be simultaneously observed in the regions of the 1H NMR spectrum which contain the largely contact-shifted resonances.

NAM7 nuclear gene encodes a novel member of a family of helicases with a Zn-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae.

The yeast nuclear gene NAM7 was previously isolated within a genomic fragment of 7.7 kb (1 kb = 10(3) bases or base-pairs), having the ability to suppress mitochondrial intronic mutations defective in RNA splicing. We have identified and sequenced the region on the insert corresponding to the NAM7 gene. A long open reading frame has been revealed which could code for a protein of 971 amino acids. Comparison of the NAM7 putative protein with data libraries did not reveal any strong similarity with known proteins. However, the NAM7 protein contains several motifs typical for proteins interacting with nucleic acids: (1) five motifs diagnostic for a superfamily of helicases appear in the same order and with similar distances; (2) the N-terminal portion possesses potential Zn-ligand structures belonging to the C chi superfamily. Deletion of the chromosomal copy of NAM7 gene leads to a partial impairment in respiratory growth that is particularly striking at low temperature. Southern blot analysis of DNA extracted from a nam7 :: URA3 deleted strain revealed the presence of a second gene whose sequence is related to that of the NAM7 gene and which could participate in the same process.

Homology between bakers' yeast cytochrome b2 and liver microsomal cytochrome b5.

The amino-acid sequence of the hemebinding region of bakers' yeast cytochrome b(2) [L-(+)-lactate dehydrogenase, EC 1.1.2.3] has been determined. It shows a strong similarity with the sequence of microsomal cytochrome b(5), and appears to be compatible with the same kind of peptide-chain folding, in agreement with data obtained previously by various physiochemical methods. The comparison shows that the fifth and sixth heme ligands must be histidine residues, thus substantiating previous conclusions drawn in particular from photooxidation experiments and nuclear magnetic resonance studies. The data reported in this paper suggest a common origin for the two proteins. Implications for their biochemical evolution are presented.

Long range control circuits within mitochondria and between nucleus and mitochondria. I. Methodology and phenomenology of suppressors.

To uncover the functional circuitry both within the mitochondrial genome and between the mitochondrial and the nuclear genome, we have developed a general method for selecting and characterizing genetically suppressor mutations that restore the respiratory capacity of mit- mitochondrial mutants. Several hundreds of pseudo-wild type revertants due to a second unlinked mutation which suppresses a target mit- mutation were isolated. The suppressor mutations were found located either in the nuclear (abbreviated NAM for 'nuclear accommodation of mitochondria') or in the mitochondrial genome (abbreviated MIM for 'mitochondrial-mitochondrial interaction'). The specificity of action of various suppressors upon some 250 different mit- mutations located in several genes was tested. According to this specificity of action, suppressors were subdivided into two major classes: allele specific or gene specific suppressors. Because the cob-box mitochondrial gene has a mosaic organization, we were able to find a novel third class of extragenic suppressors specific for mit- mutations within the introns of this gene. Four examples of suppressors showing various specificities of action illustrate our approach. (1) a nuclear gene controlling specific alleles of different mitochondrial genes; (2) a nuclear gene controlling selectively one intron of a split mitochondrial gene; (3) a mitochondrial gene controlling specific alleles of different mitochondrial genes; (4) a region in one complex mitochondrial gene which controls selectively one intron of another split mitochondrial gene. Different mechanisms of suppression are discussed stressing the alleviation of splicing deficiencies of intron mutations.

Two adjacent nuclear genes, ISF1 and NAM7/UPF1, cooperatively participate in mitochondrial functions in Saccharomyces cerevisiae.

We previously isolated a nuclear 5.7 kb genomic fragment carrying the NAM7/UPF1 gene, which is able to suppress mitochondrial splicing deficiency when present in multiple copies. We show here that an immediately adjacent gene ISF1 (Increasing Suppression Factor) increases the efficiency of the NAM7/UPF1 suppressor activity. The ISF1 gene has been independently isolated as the MBR3 gene and comparison of the ISF1 predicted protein sequence with data libraries revealed a significant similarity with the MBR1 yeast protein. The ISF1 and NAM7 genes are transcribed in the same direction, and RNase mapping allowed the precise location of their termini within the intergenic region to be determined. The ISF1 gene is not essential for cell viability or respiratory growth. However as for many mitochondrial genes, ISF1 expression is sensitive to fermentative repression; in contrast expression of the NAM7 gene is unaffected by glucose. We propose that ISF1 could influence the NAM7/UPF1 function, possibly at the level of mRNA turnover, thus modulating the expression of nuclear genes involved in mitochondrial biogenesis.

Long range control circuits within mitochondria and between nucleus and mitochondria. II. Genetic and biochemical analyses of suppressors which selectively alleviate the mitochondrial intron mutations.

In the preceding paper of this series (Dujardin et al. 1980 a) we described general methods of selecting and genetically characterizing suppressor mutations that restore the respiratory capacity of mit- mitochondrial mutations. Two dominant nuclear (NAM1-1 and NAM2-1) and one mitochondrial (mim2-1) suppressors are more extensively studied in this paper. We have analysed the action spectrum of these suppressors on 433 mit- mutations located in various mitochondrial genes and found that they preferentially alleviate the effects of mutations located within intron open reading frames of the cob-box gene. We conclude that these suppressors permit the maturation of cytochrome b mRNA by restoring the synthesis of intron encoded protein(s) catalytically involved in splicing i.e. mRNA-maturase(s) (cf. Lazowska et al. 1980). NAM1-1 is allele specific and gene non-specific; it suppresses mutations located within different introns. NAM2-1 and mim2-1 are intron-specific: they suppress mutations all located in the same (box7) intron of the cob-box gene. Analyses of cytochrome absorption spectra and mitochondrial translation products of cells in which the suppressors are associated with various other mit- mutations show that the suppressors restore cytochrome b and/or cytochrome oxidase (cox I) synthesis, as expected from their growth phenotype. This suppression is, however, only partial: some new polypeptides characteristic of the mit- mutations can be still detected in the presence of suppressor. Interestingly enough when box7 specific suppressors NAM2-1 and mim2-1 are associated with a complete cob-box deletion (leading to a total deficiency of cytochrome b and oxidase) partial restoration of cox I synthesis is observed while cytochrome b is still totally absent. These results show that in strains carrying NAM2-1 or mim2-1 the presence of cytochrome b gene is no longer required for the expression of the oxi3 gene pointing out to the possibility of a mutational switch-on of silent genes, whether mitochondrial, mim2-1, or nuclear, NAM2-1. This switch-on would permit the synthesis of an active maturase acting as a substitute for the box7 maturase in order to splice the cytochrome b and oxidase mRNAs.

The respiratory gene OXA1 has two fission yeast orthologues which together encode a function essential for cellular viability.

The Saccharomyces cerevisiae nuclear gene OXA1, which is conserved from prokaryotes to human, was shown to be essential for cytochrome c oxidase and F1F0-ATP synthase biogenesis. We have searched for an orthologue of OXA1 in Schizosaccharomyces pombe, another yeast that is highly diverged from S. cerevisiae and which could more closely model higher eukaryotes. In particular, S. pombe exhibits a limited growth under anaerobic conditions and is petite negative, that is it does not tolerate large deletions of its mitochondrial DNA. Surprisingly, two S. pombe cDNAs able to complement an S. cerevisiae oxa1 mutation were isolated. The corresponding genes have different chromosomal locations and intron contents. They encode distinct proteins, both sharing a weak sequence identity one with the other and with Oxa1p. A phenotypic analysis of both single inactivations demonstrates that only one gene is essential for respiration in S. pombe. However, the double inactivation is lethal. This work gives new insight into the dependence of S. pombe viability upon oxa1 function, providing evidence of a connection between petite negativity, a functional respiratory chain and F1F0-ATP synthase complex in S. pombe.

The NAM1/MTF2 nuclear gene product is selectively required for the stability and/or processing of mitochondrial transcripts of the atp6 and of the mosaic, cox1 and cytb genes in Saccharomyces cerevisiae.

The NAM1/MTF2 gene was firstly isolated as a multicopy suppressor of mitochondrial splicing deficiencies and independently as a gene of which a thermosensitive allele affects mitochondrial transcription in organello. To determine which step in mitochondrial RNA metabolism is controlled in vivo by the NAM1 gene, mitochondrial transcripts of seven transcription units from strains carrying an inactive nam1::URA3 gene disruption in various mitochondrial genetic backgrounds were analysed by Northern blot hybridisations. In a strain carrying an intron-containing mitochondrial genome, the inactivation of the NAM1 gene led to a strong decrease in (or total absence of) the mosaic cytb and cox1 mRNAs and in transcripts of the atp6-rf3/ens2 genes, which are co-transcribed with cox1. Neither the accumulation of unspliced cytb or cox1 pre-mRNAs, nor that of excised circular intron molecules of ai1 or ai2 were observed, but the abundance of the bi1 and ai7 lariats was comparable to that observed in the wild-type strain, thus demonstrating that transcription of the cytb and cox1 genes does occur. In strains carrying the intron-less mitochondrial genome with or without the rf3/ens2 sequence, wild-type amounts of cytb and cox1 mRNAs were detected while the amount of the atp6 mRNA was always strongly decreased. The abundance of transcripts from five other genes was either slightly (21S rRNA) or not at all (cox2, cox3, atp9 and 15S rRNA) affected by the nam1 inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of a human gene involved in cytochrome oxidase assembly by functional complementation of an oxa1- mutation in Saccharomyces cerevisiae.

The yeast nuclear gene OXA1 is essential for cytochrome oxidase assembly, so that a null mutation in the OXA1 gene leads to complete respiratory deficiency. We have cloned by genetic selection a human OXA1 (OXA1Hs) cDNA that complements the respiratory defect of yeast oxa1 mutants. The deduced sequence of the human protein shares 33% identity with the yeast OXA1 protein. The OXA1Hs cDNA corresponds to a single and relatively highly expressed gene. Oxygen consumption measurements and cytochrome absorption spectra show that replacement of the yeast protein with the human homolog leads to the correct assembly of cytochrome oxidase, suggesting that the proteins play essentially the same role in both organisms.

The NAM1 protein (NAM1p), which is selectively required for cox1, cytb and atp6 transcript processing/stabilisation, is located in the yeast mitochondrial matrix.

The NAM1 nuclear gene was shown to control the stability and/or processing of mitochondrial transcripts of the cytochrome b, cytochrome oxidase subunit I and ATP synthase subunit VI genes [Groudinsky O., Bousquet I., Wallis M. G., Slonimski, P. P. & Dujardin G. (1993) Mol. Gen. Genet. 240, 419-427]. In order to better understand the mode of action of the NAM1 gene product, we have examined its intracellular fate. A fusion plasmid enabling bacterial over-expression of the corresponding protein-A-NAM1 cognate was constructed and subsequently employed as an antigen to raise polyclonal antibodies. These antibodies specifically recognise a 50-kDa protein which purifies along with the mitochondria and corresponds to NAM1p. Submitochondrial localisation experiments show that NAM1p is a soluble protein, located interior to the mitoplasts. Matricial location is a strong argument in favour of a direct interaction of NAM1p with particular mitochondrial transcripts and leads us to propose a model in which NAM1p could be an RNA-convoying protein stabilising and directing mitochondrial transcripts towards the inner face of the inner membrane where translation and assembly seem to occur.

An mRNA maturase is encoded by the first intron of the mitochondrial gene for the subunit I of cytochrome oxidase in S. cerevisiae.

We have localized ten oxi3- mutations in the first, al1, intron of the coxl gene. All are splicing deficient, being unable to excise the intron. Complementation experiments disclose several domains in the intron al1: the 5'-proximal and 3'-proximal domains harbor cis-dominant mutations, while trans-recessive ones are located in the intron's open reading frame. Comprehensive analyses of allele-specific polypeptides accumulating in mutants show that they result from the translation of the intron's ORF. We conclude that a specific mRNA maturase involved in splicing of oxidase mRNA is encoded by the intron al1 in a manner similar to the cytochrome b mRNA maturase.

The cytochrome oxidase subunit I split gene in Saccharomyces cerevisiae: genetic and physical studies of the mtDNA segment encompassing the 'cytochrome b-homologous' intron.

We have constructed a refined genetic and physical map of 38 oxi3 mutations. With the help of the rho- clones derived from 'short' and 'long' genes, pairwise crosses between mutants, estimations of their reversion frequencies and analyses of mitochondrially synthesized proteins, we have characterized and localized several mutants in the exon A4 and in the intron aI4. We present genetic and physical evidence that in the 'long' gene the exon A5 is split into at least three quite distinct exons, A5-1, A5-2 and A5-3 where numerous mutations are localized. We suggest that a novel 56 Kd polypeptide, which accumulates in some cis-dominant oxi3- mutants results from the translation of the upstream exons and the downstream aI4 intron.

Novel class of nuclear genes involved in both mRNA splicing and protein synthesis in Saccharomyces cerevisiae mitochondria.

We have cloned three distinct nuclear genes, NAM1, NAM7, and NAM8, which alleviate mitochondrial intron mutations of the cytochrome b and COXI (subunit I of cytochrome oxidase) genes when present on multicopy plasmids. These nuclear genes show no sequence homology to each other and are localized on different chromosomes: NAM1 on chromosome IV, NAM7 on chromosome XIII and NAM8 on chromosome VIII. Sequence analysis of the NAM1 gene shows that it encodes a protein of 440 amino acids with a typical presequence that would target the protein to the mitochondrial matrix. Inactivation of the NAM1 gene by gene transplacement leads to a dramatic reduction of the overall synthesis of mitochondrial protein, and a complete absence of the COXI protein which is the result of a specific block in COXI pre-mRNA splicing. The possible mechanisms by which the NAM1 gene product may function are discussed.

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

We have characterized the nuclear gene NAM8 in Saccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wild-type strains, its overexpression is a prerequisite for the suppressor action. Inactivation of the NAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. The NAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.

Heat shock protein HSP60 can alleviate the phenotype of mitochondrial RNA-deficient temperature-sensitive mna2 pet mutants.

mna2, which belongs to the class I temperature-sensitive pet mutants that lose mitochondrial (mt)RNA at restrictive temperature, was shown by complementation and sequence determination to correspond to the gene coding for HSP60. Both mna2-1 and mna2-2, the two available alleles of mna2, have conservative single amino acid substitutions in the HSP60 gene. Valine substitutes for an alanine (position 47) in mna2-1, and an isoleucine substitutes for a valine (position 77) in mna2-2. These substitutions result in defects in respiration and in steady-state mtRNA accumulation. Wild-type hsp60 alleviates the mtRNA phenotype completely, while partially relieving the respiratory deficiency.

PetCR46, a gene which is essential for respiration and integrity of the mitochondrial genome.

In the frame of the European Pilot Project for the functional analysis of newly discovered open reading frames (ORFs) from Saccharomyces cerevisiae chromosome III, we have deleted entirely the YCR46C ORF by a one-step polymerase chain reaction method and replaced it by the HIS3 marker in the strain W303. The deletion has been checked by meiotic segregation and Southern blot analyses. Characterization of the deleted strain indicates that YCR46C is essential for respiration and maintenance of the mitochondrial genome since its deletion leads to the appearance of 100% of cytoplasmic petites. Hybridization with molecular probes from mtDNA of individual clones of such petites showed that about 50% did hybridize (rho- clones) while others did not (possibly rho degrees clones). The wild-type gene has been cloned and shown to complement the deletion. The gene, which probably codes for a mitochondrial ribosomal protein, has been called petCR46.

Large-scale phenotypic analysis--the pilot project on yeast chromosome III.

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome.