Nicola Altamura

THE SACCHAROMYCES CEREVISIAE OXA1 GENE IS REQUIRED FOR THE CORRECT ASSEMBLY OF CYTOCHROME C OXIDASE AND OLIGOMYCIN-SENSITIVE ATP SYNTHASE

The nuclear gene OXA1 was first isolated in Saccharomyces cerevisiae and found to be required at a post‐translational step in cytochrome c oxidase biogenesis, probably at the level of assembly. Mutations in OXA1 lead to a complete respiratory deficiency. The protein Oxa1p is conserved through evolution and a human homolog has been isolated by functional complementation of a yeast oxa1− mutant. In order to further our understanding of the role of Oxa1p, we have constructed two yeast strains in which the OXA1 open reading frame was almost totally deleted. Cytochrome spectra and enzymatic activity measurements show the absence of heme aa 3 and of a cytochrome c oxido‐reductase activity and dramatic decrease of the oligomycin sensitive ATPase activity. Analysis of the respiratory complexes in non‐denaturing gels reveals that Oxa1p is necessary for the correct assembly of the cytochrome c oxidase and the ATP synthase complex.

Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana

Most cellular ATP is produced within the mitochondria from ADP and Pi which are delivered across the inner‐membrane by specific nuclearly encoded polytopic carriers. In Saccharomyces cerevisiae, some of these carriers and in particular the ADP/ATP carrier, are represented by several related isoforms that are distinct in their pattern of expression. Until now, only one mitochondrial Pi carrier (mPic) form, encoded by the MIR1 gene in S. cerevisiae, has been described. Here we show that the gene product encoded by the YER053C ORF also participates in the delivery of phosphate to the mitochondria. We have called this gene PIC2 for Pi carrier isoform 2. Overexpression of PIC2 compensates for the mitochondrial defect of the double mutant Δmir1 Δpic2 and restores phosphate transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPic‐like proteins are also able to complement the double mutant Δmir1 Δpic2. Finally, we demonstrate that Pic2p is a mitochondrial protein and that its steady state level increases at high temperature. We propose that Pic2p is a minor form of mPic which plays a role under specific stress conditions.

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

SummaryWe 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 H+/O stoicheiometry of mitochondrial respiration.

Mitochondrial respiration is compulsorily linked to proton ejection [ 1,2]. The mechanism by which transmembraneAcH+isgeneratedis, however,unknown [3]. Essential for exploring this issue is knowledge of the quantitative relationship between proton translocation and electron transport. Numerous determinations in mitochondria and bacteria produced a stoicheiometry of 2 H’ translocated for 2 etraversing an effective protonmotive redox loop, or energy conserving site of respiratory chain (reviewed [1,2,4]). Recently, however, the H’/2 eand H’/ATP stoicheiometries have been the subject of much controversy [2,5-91. Thus contrasting results have been reported, which indicate that in mitochondria the H+/O quotient for succinate or quinolrespiration(sites2 t 3) is either 4 [1,7,10-141, or 6 [6,8,15],or 8 [5,9,16,17]. We present here accurate spectrophotometric determination of the rate of respiration with hemoglobin and potentiometric determination of the acidification of the extramitochondrial space, which unequivocally show that the H’/O quotient for oxidation of succinate in mitochondria is, at neutral pH, 4.

MitBASE: a comprehensive and integrated mitochondrial DNA database

MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects all available information from different organisms and from intraspecie variants and mutants. Research institutions from different countries are involved, each in charge of developing, collecting and annotating data for the organisms they are specialised in. The design of the actual structure of the database and its implementation in a user-friendly format are the care of the European Bioinformatics Institute. The database can be accessed on the Web at the following address: http://www.ebi.ac. uk/htbin/Mitbase/mitbase.pl. The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecie diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme.

Overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance

In yeast the UPF1, UPF2 and UPF3 genes encode three interacting factors involved in translation termination and nonsense‐mediated mRNA decay (NMD). UPF1 plays a central role in both processes. In addition, UPF1 was originally isolated as a multicopy suppressor of mitochondrial splicing deficiency, and its deletion leads to an impairment in respiratory growth. Here, we provide evidence that inactivation of UPF2 or  UPF3,  like  that  of  UPF1,  leads  to  an  impairment in respiratory competence, suggesting that their products, Upf1p, Upf2p and Upf3p, are equivalently involved in mitochondrial biogenesis. In addition, however, we show that only Upf1p acts as a multicopy suppressor of mitochondrial splicing deficiency, and its activity does not require either Upf2p or Upf3p. Mutations in the conserved cysteine‐ and histidine‐rich regions and ATPase and helicase motifs of Upf1p separate the ability of Upf1p to complement the respiratory impairment of a Δupf1 strain from its ability to act as a multicopy suppressor of mitochondrial splicing deficiency, indicating that distinct pathways express these phenotypes. In addition, we show that, when overexpressed, Upf1p is not detected within mitochondria, suggesting that its role as multicopy suppressor of mitochondrial splicing deficiency is indirect. Furthermore, we provide evidence that cells overexpressing certain upf1 alleles accumulate a phosphorylated isoform of Upf1p. Altogether, these results indicate that overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance, which relies on Upf1p–Upf2p–Upf3p functional interplay.

MitBASE : a comprehensive and integrated mitochondrial DNA database. The present status

MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects, under a single interface, databases for Plant, Vertebrate, Invertebrate, Human, Protist and Fungal mtDNA and a Pilot database on nuclear genes involved in mitochondrial biogenesis in Saccharomyces cerevisiae. MitBASE reports all available information from different organisms and from intraspecies variants and mutants. Data have been drawn from the primary databases and from the literature; value adding information has been structured, e.g., editing information on protist mtDNA genomes, pathological information for human mtDNA variants, etc. The different databases, some of which are structured using commercial packages (Microsoft Access, File Maker Pro) while others use a flat-file format, have been integrated under ORACLE. Ad hoc retrieval systems have been devised for some of the above listed databases keeping into account their peculiarities. The database is resident at the EBI and is available at the following site: http://www3.ebi.ac.uk/Research/Mitbase/mitbas e.pl. The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecies diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme.

Spectrophotometric determination with hemoglobin of the rate of oxygen consumption in mitochondria

The study of respiratory processes and energy transduction in mitochondria or other respiring preparations often requires analysis of the kinetics of oxygen consulnption and of the quantitative relationship between the rates of oxygen utilization, ATP synthesis and ion transport [l-3]. Accurate determination of the rate of oxygen consurllption presents technical dif~culties when dealing with rapid, transient changes of the respiratory activity, as induced by addition to the respiring material of oxygen, reductants, ADP. Pi and cations. In fact the polarographic method which is generally used to measure oxygen consumption may be inadequate to measure accurately rapid changes of respiratory rates [2,4], as judged from its intrinsic response-time characteristics [S&j. This paper reports the application and suitability of a spectrophotometric method with hemoglobin [7,X] for accurate determination of initial rates of oxygen consunlption during rapid functional transitions of r-espiratory systems. Oxyhemoglobin is used both as oxygen donor and as indicator of respiration, taking advantage of the specific absorbance changes which it undergoes upon deoxygenation.

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 MBRI 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.

Tobramycin is a suppressor of premature termination codons

Premature translation terminations (PTCs) constitute the molecular basis of many genetic diseases, including cystic fibrosis, as they lead to the synthesis of truncated non-functional or partially functional protein. Suppression of translation terminations at PTCs (read-through) has been developed as a therapeutic strategy to restore full-length protein in several genetic diseases. Phenotypic consequences of PTCs can be exacerbated by the nonsense-mediated mRNA decay (NMD) pathway that detects and degrades mRNA containing PTC. Modulation of NMD, therefore, is also of interest as a potential target for the suppression therapy. Tobramycin is an aminoglycoside antibiotic, normally used to treat Pseudomonas aeruginosa pulmonary infection in CF patients. In the present study, by using yeast as a genetic system, we have examined the ability of Tobramycin to suppress PTCs as a function of the presence or absence of NMD. Results demonstrate that Tobramycin exhibits read-through ability on PTCs and preferentially in absence of NMD.