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L A Grivell

Publications and source records attributed to L A Grivell.

At least 19 recordsLinked to original sources
PubMed1992

The yeast nuclear gene MRF1 encodes a mitochondrial peptide chain release factor and cures several mitochondrial RNA splicing defects.

We report the molecular cloning, sequencing and genetic characterization of the first gene encoding an organellar polypeptide chain release factor, the MRF1 gene of the yeast Saccharomyces cerevisiae. The MRF1 gene was cloned by genetic complementation of a respiratory deficient mutant disturbed in the expression of the mitochondrial genes encoding cytochrome c oxidase subunit 1 and 2, COX1 and COX2. For COX1 this defect has been attributed to an impaired processing of several introns. Sequence analysis of the MRF1 gene revealed that it encodes a protein highly similar to prokaryotic peptide chain release factors, especially RF-1. Disruption of the gene results in a high instability of the mitochondrial genome, a hallmark for a strict lesion in mitochondrial protein synthesis. The respiratory negative phenotype of mrf1 mutants lacking all known mitochondrial introns and the reduced synthesis of mitochondrial translation products encoded by unsplit genes confirm a primary defect in mitochondrial protein synthesis. Over-expression of the MRF1 gene in a mitochondrial nonsense suppressor strain reduces suppression in a dosage-dependent manner, shedding new light on the role of the '530 region' of 16S-like ribosomal RNA in translational fidelity.

Amino Acid Sequence↗
PubMed1992

Sequence comparison of new prokaryotic and mitochondrial members of the polypeptide chain release factor family predicts a five-domain model for release factor structure.

We have recently reported the cloning and sequencing of the gene for the mitochondrial release factor mRF-1. mRF-1 displays high sequence similarity to the bacterial release factors RF-1 and RF-2. A database search for proteins resembling these three factors revealed high similarities to two amino acid sequences deduced from unassigned genomic reading frames in Escherichia coli and Bacillus subtilis. The amino acid sequence derived from the Bacillus reading frame is 47% identical to E.coli and Salmonella typhimurium RF-2, strongly suggesting that it represents B.subtilis RF-2. Our comparison suggests that the expression of the B.subtilis gene is, like that of the E.coli and S. typhimurium RF-2 genes, autoregulated by a stop codon dependent +1 frameshift. A comparison of prokaryotic and mitochondrial release factor sequences, including the putative B.subtilis RF-2, leads us to propose a five-domain model for release factor structure. Possible functions of the various domains are discussed.

Amino Acid Sequence↗
PubMed1992

Determinants for binding of a 40 kDa protein to the leaders of yeast mitochondrial mRNAs.

An abundant yeast mitochondrial 40 kDa protein (p40) binds with high specificity to the 5'-untranslated region of cytochrome c oxidase subunit II (COX2) mRNA. Using mobility shift and competition assays, we show here that purified p40 complexes with the leaders of all eight mitochondrial mRNAs of Saccharomyces cerevisiae. The location of the protein binding site on the different leaders is not conserved with respect to the AUG start codon. In vitro RNA footprint and deletion experiments have been used to define the p40-binding site on the leaders of COX1 and ATP9 mRNAs. Nucleotides at, and near, a single stranded region are protected or exposed for DEPC modification by binding of p40 to these leaders. Removal of this region from the COX1 messenger shows that it is essential for the protein-RNA interaction. While no obvious sequence similarity can be detected between the single stranded regions in different leaders, a nearby helical segment is conserved. A consensus model for p40-RNA interactions is presented and the possible biological function of p40 is discussed.

Base Sequence↗
PubMed1992

Primary structure and import pathway of the rotenone-insensitive NADH-ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae.

The gene encoding the yeast mitochondrial rotenone-insensitive internal NADH: ubiquinone-6 oxidoreductase has been sequenced. The DNA sequence indicates the presence of an open reading frame of 1539 bp predicted to encode a protein of 513 amino acid residues (57.2 kDa). The NADH dehydrogenase is synthesized as a precursor protein containing a signal sequence of 26 residues. In vitro import experiments show that the precursor NADH dehydrogenase is cleaved to the mature size by the matrix processing peptidase. Both cleavage and translocation across the mitochondrial membrane(s) are dependent on the membrane potential component of the proton-motive force. Comparison of the protein sequence of the yeast NADH dehydrogenase with the data bank indicates that the enzyme from yeast is homologous to the NADH dehydrogenase of Escherichia coli (22.2% identical residues). Both NADH dehydrogenases contain in the central part of the protein a sequence predicted to fold into a beta alpha beta structure involved in the binding of NADH or FAD(H2). Various aspects of the protein structure are discussed.

Amino Acid Sequence↗
PubMed1992

MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria.

To identify components of the mitochondrial protein import pathway in yeast, we have adopted a positive selection procedure for isolating mutants disturbed in protein import. We have cloned and sequenced a gene, termed MPI1, that can rescue the genetic defect of one group of these mutants. MPI1 encodes a hydrophilic 48.8 kDa protein that is essential for cell viability. Mpi1p is a low abundance and constitutively expressed mitochondrial protein. Mpi1p is synthesized with a characteristic mitochondrial targeting sequence at its amino-terminus, which is most probably proteolytically removed during import. It is a membrane protein, oriented with its carboxy-terminus facing the intermembrane space. In cells depleted of Mpi1p activity, import of the precursor proteins that we tested thus far, is arrested. We speculate that the Mpi1 protein is a component of a proteinaceous import channel for translocation of precursor proteins across the mitochondrial inner membrane.

Amino Acid Sequence↗
PubMed1992

The identification of 18 nuclear genes required for the expression of the yeast mitochondrial gene encoding cytochrome c oxidase subunit 1.

Eighteen nuclear mutants of the yeast Saccharomyces cerevisae, each disturbed in the biosynthesis of the mitochondrially encoded cytochrome c oxidase subunit 1 (cox1) and each representing a distinct complementation group, have been examined to identify the level at which COX1 expression is affected. RNA blotting revealed that most have a defect in the processing of COX1 precursor-mRNA; only a few are defective in COX1 transcription and/or pre-mRNA stability. In most RNA-processing mutants, the absence of the COX1 messenger results from a defect in the splicing of one or more COX1 introns. In turn, this defect can be ascribed to a mutation in a nuclear gene which is either directly involved in splicing or else acts indirectly by impairing COX1 translation.

Blotting, Northern↗
PubMed1992

Genetic approaches to the study of mitochondrial biogenesis in yeast.

In contrast to most other organisms, the yeast Saccharomyces cerevisiae can survive without functional mitochondria. This ability has been exploited in genetic approaches to the study of mitochondrial biogenesis. In the last two decades, mitochondrial genetics have made major contributions to the identification of genes on the mitochondrial genome, the mapping of these genes and the establishment of structure-function relationships in the products they encode. In parallel, more than 200 complementation groups, corresponding to as many nuclear genes necessary for mitochondrial function or biogenesis have been described. Many of the latter are required for post-transcriptional events in mitochondrial gene expression, including the processing of mitochondrial pre-RNAs, the translation of mitochondrial mRNAs, or the assembly of mitochondrial translation products into the membrane. The aim of this review is to describe the genetic approaches used to unravel the intricacies of mitochondrial biogenesis and to summarize recent insights gained from their application.

Cell Nucleus↗
PubMed1992

Global regulation of mitochondrial biogenesis in Saccharomyces cerevisiae: ABF1 and CPF1 play opposite roles in regulating expression of the QCR8 gene, which encodes subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase.

The multifunctional DNA-binding proteins ABF1 and CPF1 bind in a mutually exclusive manner to the promoter region of the QCR8 gene, which encodes 11-kDa subunit VIII of the Saccharomyces cerevisiae mitochondrial ubiquinol-cytochrome c oxidoreductase (QCR). We investigated the roles that the two factors play in transcriptional regulation of this gene. To this end, the overlapping binding sites for ABF1 and CPF1 were mutated and placed in the chromosomal context of the QCR8 promoter. The effects on transcription of the QCR8 gene were analyzed both under steady-state conditions and during nutritional shifts. We found that ABF1 is required for repressed and derepressed transcription levels and for efficient induction of transcription upon escape from catabolite repression, independently of DNA replication. CPF1 acts as a negative regulator, modulating the overall induction response. Alleviation of repression through CPF1 requires passage through the S phase. Implications of these findings for the roles played by ABF1 and CPF1 in global regulation of mitochondrial biogenesis are discussed.

Base Sequence↗
PubMed1991

Isolation and inactivation of the nuclear gene encoding the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae.

We have recently described the isolation of a mitochondrial rotenone-insensitive NADH:ubiquinone oxidoreductase from Saccharomyces cerevisiae [de Vries, S. & Grivell, L. A. (1988) Eur. J. Biochem. 176, 377-384]. We now report the isolation of the nuclear gene encoding this single-subunit enzyme. Null mutants have been constructed by means of one-step gene disruption. Oxygen-uptake experiments, performed with mitochondria isolated from the mutant cells, showed that this NADH dehydrogenase catalyzes the oxidation of NADH generated inside the mitochondrion. Inactivation of this NADH dehydrogenase does not affect growth on glucose and ethanol, but growth on lactate, pyruvate and acetate is impaired or absent. This phenotype is discussed in terms of the interplay between different metabolic pathways in yeast.

Amino Acid Sequence↗
PubMed1991

Properties of an abundant RNA-binding protein in yeast mitochondria.

We have previously identified a protein with Mr approximately 40,000 (p40) that binds with high specificity and affinity to the 5'-untranslated leaders of mitochondrial mRNAs in yeast. Here we show that this protein is abundant, comprising about 0.4% of total mitochondrial protein. p40 is present in a cytoplasmic (rho degree) petite mutant that lacks mitochondrial protein synthesis and is therefore nuclear encoded. p40 can be detected by immunological techniques in cell lysates of several different pet mutants, specifically disturbed in the translation of individual mitochondrial mRNAs. It is thus not one of the translation factors defined by any of these mutations. In the case of a pet111 mutant, which is specifically blocked in the translation of COX2 mRNA, extracts still display COX2 mRNA binding activity, indicating that p40 complex formation in vitro is not dependent on the presence of PET111.

Binding Sites↗
PubMed1990

Yeast general transcription factor GFI: sequence requirements for binding to DNA and evolutionary conservation.

GFI is an abundant DNA binding protein in the yeast S. cerevisiae. The protein binds to specific sequences in both ARS elements and the upstream regions of a large number of genes and is likely to play an important role in yeast cell growth. To get insight into the relative strength of the various GFI-DNA binding sites within the yeast genome, we have determined dissociation rates for several GFI-DNA complexes and found them to vary over a 70-fold range. Strong binding sites for GFI are present in the upstream activating sequences of the gene encoding the 40 kDa subunit II of the QH2:cytochrome c reductase, the gene encoding ribosomal protein S33 and in the intron of the actin gene. The binding site in the ARS1-TRP1 region is of intermediate strength. All strong binding sites conform to the sequence 5' RTCRYYYNNNACG-3'. Modification interference experiments and studies with mutant binding sites indicate that critical bases for GFI recognition are within the two elements of the consensus DNA recognition sequence. Proteins with the DNA binding specificities of GFI and GFII can also be detected in the yeast K. lactis, suggesting evolutionary conservation of at least the respective DNA-binding domains in both yeasts.

Base Sequence↗
PubMed1990

A 40 kd protein binds specifically to the 5'-untranslated regions of yeast mitochondrial mRNAs.

Using a gel mobility shift assay we show that a 40 kd protein (p40), present in extracts of yeast mitochondria, binds specifically to the 5'-untranslated leader of cytochrome c oxidase subunit II mRNA. Binding of p40 to coxII RNA protects an 8-10 nucleotide segment from diethylpyocarbonate modification, indicating that the protein interacts with only a restricted region of the 5'-leader. This segment is located at position -12 with respect to the initiation AUG. Deletion of 10 nucleotides encompassing this site completely abolishes protein binding. Nevertheless, Bal31 deletion analysis within the coxII leader shows that a major part of the leader is essential for p40 binding, suggesting that binding of the protein is also dependent on secondary structural features. p40 binds to other mitochondrial leader mRNAs including those for coxI, coxIII and cyt b. p40 is present in a cytoplasmic (rho0) petite mutant lacking mitochondrial protein synthesis. It is therefore presumably nuclear encoded. The possible biological function of the protein is discussed.

Base Sequence↗
PubMed1990

Expression of the gene encoding subunit II of yeast QH2: cytochrome c oxidoreductase is regulated by multiple factors.

In Saccharomyces cerevisiae, the COR2 gene codes for the 40 kDa subunit II of the QH2: cytochrome c oxidoreductase, an enzyme of the mitochondrial respiratory chain. Regions in the 5' flank of this gene important for regulated expression were identified by assaying beta-galactosidase activities in cells carrying different COR2-lacZ fusion genes. Sequences downstream of position -201 relative to the translational initiation codon are sufficient to confer regulation by carbon source, whereas sequences downstream of position -153 do not give rise to significant expression. A binding site for the abundant general transcription factor GFI is present in the region between -201 and -153 just upstream from sequences which resemble the consensus DNA recognition sequence of the regulatory protein complex HAP2/HAP3: 5'-TNATTGGT-3'. By quantitating RNA levels and assaying beta-galactosidase activities we show that synthesis of COR2, which is not a hemoprotein, is regulated by HAP1, HAP2/HAP3 and heme.

Base Sequence↗
PubMed1990

Structural analysis of a group II intron by chemical modifications and minimal energy calculations.

Folding of the yeast mitochondrial group II intron aI5c has been analysed by chemical modification of the in vitro synthesised RNA with dimethylsulfate and diethylpyrocarbonate. Computer calculations of the intron secondary structure through minimization of free energy were also performed in order to study thermodynamic properties of the intron and to relate these to data obtained from chemical modification. Comparison of the two sets of data with the current phylogenetic model structure of the intron aI5 reveals close agreement, thus lending strong support for the existence of a typical group II intron core structure comprising six neighbouring stem-loop domains. Local discrepancies between the experimental data and the model structures have been analyzed by reference to thermodynamic properties of the structure. This shows that use of the latest refined set of free energy values improves the structure calculation significantly.

Base Sequence↗
PubMed1989

An ARS/silencer binding factor also activates two ribosomal protein genes in yeast.

GFI is an abundant yeast DNA-binding protein, capable of binding to both ARS sequences and to the upstream regions of a number of nuclear genes coding for mitochondrial proteins (Dorsman et al., Nucl. Acids Res., 16 [1988] 7287-7301). GFI binding sites conform to the consensus RTCRYN5ACG, an element also present in the binding sites of factors designated SUF and TAF. These factors act as trans-activators of the constitutive transcription of the genes for ribosomal proteins S33 and L3 respectively. We now present evidence that GFI, TAF and SUF are probably the same protein. We speculate that one of the functions of GFI is the adjustment of the expression of a number of gene families to cell growth rate.

Base Sequence↗