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L A Grivell

Publications and source records attributed to L A Grivell.

At least 55 records · Page 3Linked to original sources
PubMed1995

Nucleo-mitochondrial interactions in mitochondrial gene expression.

All proteins encoded by mitochondrial DNA (mtDNA) are dependent on proteins encoded by nuclear genes for their synthesis and function. Recent developments in the identification of these genes and the elucidation of the roles their products play at various stages of mitochondrial gene expression are covered in this review, which focuses mainly on work with the yeast Saccharomyces cerevisiae. The high degree of evolutionary conservation of many cellular processes between this yeast and higher eukaryotes, the ease with which mitochondrial biogenesis can be manipulated both genetically and physiologically, and the fact that it will be the first organism for which a complete genomic sequence will be available within the next 2 to 3 years makes it the organism of choice for drawing up an inventory of all nuclear genes involved in mitochondrial biogenesis and for the identification of their counterparts in other organisms.

Amino Acid Sequence↗
PubMed1994

Complete DNA sequence of yeast chromosome II.

In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

Base Composition↗
PubMed1994

Isolation and characterisation of the linked genes, FPS1 and QCR8, coding for farnesyl-diphosphate synthase and the 11 kDa subunit VIII of the mitochondrial bc1-complex in the yeast Kluyveromyces lactis.

The KlQCR8 gene of the yeast Kluyveromyces lactis encoding subunit VIII of the mitochondrial bc1 complex is 70.2% identical to its counterpart in Saccharomyces cerevisiae (ScQCR8). As in S. cerevisiae, chromosomal linkage between the K. lactis QCR8 and FPS1 genes is conserved, the two genes being separated by only 292 bp. Disruption of the KlQCR8 gene results in a respiratory-deficient phenotype. Compared with S. cerevisiae, expression of the KlQCR8 gene in glucose-grown cells is relatively high, yet is significantly induced when the cells are grown on non-fermentable carbon sources. The QCR8 promoters regions of the two yeasts lack overall DNA sequence similarity, but share DNA-binding sites for the transcription factors ABF1, CPF1 and HAP2/3/4. Deletion from the KlQCR8 promoter of a 93 bp region containing these sites significantly lowers mRNA levels during growth on either glucose or ethanol/glycerol, with a consequent reduction of growth rate on ethanol/glycerol.

Alkyl and Aryl Transferases↗
PubMed1994

Isolation and characterisation of the linked genes APA2 and QCR7, coding for Ap4A phosphorylase II and the 14 kDa subunit VII of the mitochondrial bc1-complex in the yeast Kluyveromyces lactis.

We report the isolation and characterization of the KlQCR7 gene encoding subunit VII of the mitochondrial bc1 complex of the yeast Kluyveromyces lactis. The coding region is 69.3% identical to its counterpart in Saccharomyces cerevisiae (ScQCR7). Like the KlQCR8 gene (Mulder et al., accompanying paper) expression of the KlQCR7 gene during growth on glucose is high and can be further induced when cells are grown on non-fermentable carbon sources. The chromosomal linkage of the APA2 and QCR7 genes is conserved between S. cerevisiae and K. lactis. The intergenic regions containing the QCR7 promoters of the two yeasts, differ significantly in length and lack overall DNA sequence similarity, but they do share a binding site for the transcription factor complex HAP2/3/4. The KlQCR7 promoter contains, in addition, a CPF1 consensus binding site which is absent from ScQCR7. Deletion of a 35 bp region containing these two sites severely lowers the mRNA expression during growth on both glucose and ethanol/glycerol, but growth rate on both carbon sources is only mildly affected. Interestingly, in respect to the KlQCR7 gene, KlCPF1 seems to act as an important transcriptional activator, thus contrasting the proposed repressor function of ScCPF1 for the ScQCR8 gene of S. cerevisiae.

Amino Acid Sequence↗
PubMed1994

Sequence of the HAP3 transcription factor of Kluyveromyces lactis predicts the presence of a novel 4-cysteine zinc-finger motif.

The Kluyveromyces lactis homologue of the Saccharomyces cerevisiae HAP3 gene was isolated by functional complementation of the respiratory-deficient phenotype of the S. cerevisiae hap3::HIS4 strain SHY40. The KlHAP3 gene encodes a protein of 205 amino acids, of which the central B-domain of 90 residues is highly homologous to HAP3 counterparts of S. cerevisiae and higher eukaryotes. The protein contains a novel 4-cysteine zinc-finger motif and we propose by analogy that all other homologous HAP3 proteins contain the same motif, with the position containing the third cysteine being occupied by a serine residue. In contrast to the situation in S. cerevisiae, disruption of the KlHAP3 gene in K. lactis does not result in a respiratory-deficient phenotype and the growth of the null strain is indistinguishable from wild type. There is also no effect on the expression of the carbon source-regulated KlCYC1 gene, suggesting either a different role for the HAP2/3/4 complex, or the existence of a different mechanism of carbon source regulation. Sequence verification of the S. cerevisiae HAP3 locus reveals that, just as in K. lactis, a long open reading frame (ORF) is present upstream of the HAP3 gene. These highly homologous ORFs are predicted to have at least eight membrane-spanning fragments, but do not show significant homology to any known sequence present in databases. The ScORFX gene is transcribed in the opposite direction to ScHAP3, but, in contrast to an earlier report by Hahn et al. (1988), the transcripts of the two genes do not overlap. The model proposed by these authors, in which the ScHAP3 gene is regulated by an anti-sense non-coding mRNA, is therefore not correct.

Amino Acid Sequence↗
PubMed1994

The aromatic nature of residue 66 of the 11-kDa subunit of ubiquinol-cytochrome c oxidoreductase of the yeast Saccharomyces cerevisiae is important for the assembly of a functional enzyme.

Transformation of multi- and single-copy plasmids carrying a mutated version (LTN2, region 66-YWYWW-70 replaced by SASAA) of QCR8, the gene encoding the 11-kDa subunit ubiquinol-cytochrome c oxidoreductase of Saccharomyces cerevisiae, to a QCR8(0) strain indicated the importance of this aromatic region for the assembly of a functional enzyme. Sequencing of plasmids giving spontaneous restoration of growth to some colonies among the single-copy LTN2 transformants showed that changing the sequence SASAA into the sequence FASAA could, to a large extent, overcome the observed assembly defect, indicating the importance of the aromatic nature of residue 66.

Amino Acid Sequence↗
PubMed1994

The C-terminus of the 14-kDa subunit of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae is involved in the assembly of a functional enzyme.

Disruption of QCR7, the gene encoding the 14-kDa subunit of ubiquinol-cytochrome-c oxido-reductase of the yeast Saccharomyces cerevisiae, results in an inactive enzyme which lacks holo-cytochrome b and has severely reduced levels of apo-cytochrome b, the Rieske Fe-S protein and the 11-kDa subunit [Schoppink, P. J., Berden, J. A. & Grivell, L. A. (1989) Eur. J. Biochem. 181, 475-483]. An episomal system was developed to study the effect on complex III of transformation of in vitro mutagenised QCR7 genes to a QCR7(0) mutant. Transformation of a gene (TNT1) in which the 12 C-terminal residues are replaced by 3 amino acids encoded by an oligonucleotide containing a stop codon in all three reading frames (STOP-oligonucleotide), only leads to partial complementation of the respiratory capacity of the yeast strain. The amounts of apo-cytochrome b, the Rieske Fe-S protein and the 11-kDa subunit are reduced and enzymic activity, together with the amount of holo-cytochrome b, is lowered to about 40% of that of the wild type, indicating a normal turnover number of the mutant enzyme. Transformation of the QCR7(0) mutant with another gene (TNT2) encoding the first 96 residues of the 14-kDa subunit fused to 9 amino acids encoded by the STOP-oligonucleotide, leads to a phenotype almost indistinguishable from that of the QCR7(0) mutant. The role of the charged C-terminus of the 14-kDa (and the 11-kDa) subunit in the assembly of a functional complex III is discussed.

Amino Acid Sequence↗
PubMed1994

Intron mobility. Invasive introns.

The evidence that introns in fungal and plant organellar DNAs are mobile genetic elements has strengthened significantly; like Shaw's cockney heroine, Eliza Doolittle, they cannot help but betray their origins.

DNA, Fungal↗
PubMed1994

The complete sequence of a 33 kb fragment on the right arm of chromosome II from Saccharomyces cerevisiae reveals 16 open reading frames, including ten new open reading frames, five previously identified genes and a homologue of the SCO1 gene.

We report here the sequence of a 33,117 bp DNA fragment located approximately 30 kb from the centromere on the right arm of Saccharomyces cerevisiae chromosome II. We have detected 16 open reading frames (ORFs) longer than 450 bp, provisionally called YBR0301 to YBR0322, covering 70.4% of the entire sequence. The ORFs YBR0301, YBR0302, YBR0303, YBR0305 and YBR0315 correspond to previously sequenced S. cerevisiae genes GAL10, GAL1, FUR4, CAL1 and L2B, respectively. Translation products of two other ORFs, YBR0308 and YBR0312 exhibit similarity to previously known S. cerevisiae proteins: the mitochondrially associated protein SCO1 and the protein kinase YKR2. The predicted protein product of the ORF YBR0321 shows a 41.6% identity score with the Escherichia coli pyroxamine 5'-phosphate oxidase. The nine other ORFs show no significant homology to known proteins.

Amino Acid Sequence↗
PubMed1994

Sequence of the AFG3 gene encoding a new member of the FtsH/Yme1/Tma subfamily of the AAA-protein family.

A nuclear gene from Saccharomyces cerevisiae was cloned by genetic complementation of a temperature-sensitive respiratory-deficient mutant. DNA sequence analysis reveals that it encodes a protein with homology to Yme1, FtsH and Tma, proteins which belong to the AAA-protein family (ATPases associated with diverse cellular activities). The members of this family are involved in very different biological processes. Yme1p, a yeast mitochondrial protein, affects the rate of DNA escape from mitochondria to the nucleus and the Escherichia coli FtsH protein is apparently involved in the post-translational processing of PBP3, a protein necessary for septation during cell division. This newly sequenced gene, which we have designated AFG3 for ATPase family gene 3, encodes a putative mitochondrial protein of 760 amino acid residues that is closely related to FtsH, Tma (protein from Lactococcus lactis) and Yme1p with 58, 55 and 46% identity respectively.

Adenosine Triphosphatases↗
PubMed1994

Centromere promoter factors (CPF1) of the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are functionally exchangeable, despite low overall homology.

The KlCPF1 gene, coding for the centromere and promoter factor CPF1 from Kluyveromyces lactis, has been cloned by functional complementation of the methionine auxotrophic phenotype of a Saccharomyces cerevisiae mutant lacking ScCPF1. The amino-acid sequences of both CPF1 proteins show a relatively-low overall identity (31%), but a highly-homologous C-terminal domain (86%). This region constitutes the DNA-binding domain with basic-helix-loop-helix and leucine-zipper motifs, features common to the myc-related transcription factor family. The N-terminal two-thirds of the CPF1 proteins show no significant similarity, although the presence of acidic regions is a shared feature. In KlCPF1, the acidic region is a prominent stretch of approximately 40 consecutive aspartate and glutamate residues, suggesting that this part might be involved in transcriptional activation. In-vitro mobility-shift experiments were used to establish that both CPF1 proteins bind to the consensus binding site RTCACRTG (CDEI element). In contrast to S. cerevisiae, CPF1 gene-disruption is lethal in K. lactis. The homologous CPF1 genes were transformed to both S. cerevisiae and K. lactis cpf1-null strains. Indistinguishable phenotypes were observed, indicating that, not withstanding the long nonconserved N-terminal region, the proteins are sufficiently homologous to overcome the phenotypes associated with cpf1 gene-disruption.

Amino Acid Sequence↗
PubMed1993

Single point mutations in domain II of the yeast mitochondrial release factor mRF-1 affect ribosome binding.

We have recently described two yeast strains that are mutated in the MRF1 gene encoding the mitochondrial release factor mRF-1. Both mutants provoke gene-specific defects in mitochondrial translational termination. In the present study we report the cloning, sequencing, as well as an analysis of residual activities of both mutant mrf1 alleles. Each allele specifies a different single amino acid substitution located one amino acid apart. The amino acid changes do not affect the level or cellular localization of the mutant proteins, since equal amounts of wild type and mutant mRF-1 were detected in the mitochondrial compartment. Over-expression of the mutant alleles in wild type and mrf1 mutant yeast strains produces a phenotype consistent with a reduced affinity of the mutant release factors for the ribosome, indicating that the mutations map in a release factor domain involved in ribosome binding. We also demonstrate that nonsense suppression caused by a mutation in the mitochondrial homolog of the E. coli small ribosomal protein S4 can be reversed by a slight over-expression of the MRF1 gene.

Alleles↗
PubMed1993

Yeast mitochondrial NAD(+)-dependent isocitrate dehydrogenase is an RNA-binding protein.

We have previously described the characterisation of an abundant mitochondrial protein (p40) that binds specifically to 5'-untranslated leaders of mitochondrial mRNAs in yeast. p40 consists of two polypeptides with M(r) of 40 and 39 kDa. Limited sequence analysis of p40 identifies it as the Krebs cycle enzyme NAD(+)-dependent isocitrate dehydrogenase (Idh). Both enzyme and RNA-binding activities are specifically lost in cells containing disruptions in either IDH1 or IDH2, the nuclear genes encoding the two subunits of the enzyme, thus conclusively identifying p40 as Idh and showing that both activities are dependent on the simultaneous presence of both subunits. Although we still must ascertain whether and how either function of Idh is regulated and whether the two functions are compatible or mutually exclusive, this combination of dehydrogenase activity and RNA-binding in a single protein may be part of a general regulatory circuit linking the need for mitochondrial function to mitochondrial biogenesis.

Amino Acid Sequence↗
PubMed1993

A region of the C-terminal part of the 11-kDa subunit of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae contributes to the structure of the Qout reaction domain.

QCR8, the gene encoding the 11-kDa subunit of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae has been resequenced in the course of a search for mutants disturbed in subunit function. Resequencing shows that the previously published sequence [Maarse A.C. & Grivell L.A. (1987) Eur. J. Biochem 155, 419-425] lacks a C at position 185 of the coding sequence. As a result of this extra nucleotide, the reading frame now contains 285 base pairs and it codes for a protein of 94 amino acids with a calculated molecular mass of 11.0 kDa. Despite the altered C-terminus, similarity to the corresponding beef heart subunit is not significantly altered. One mutant (LTN1), arising from hydroxylamine mutagenesis, has been studied in detail: Assembly of the enzyme appears to be normal, as judged from the levels of the subunits observed in Western blots, while spectral analysis showed that only holo-cytochrome b was lowered to 70% of that of the wildtype. Measurement of the specific activity and calculation of the turnover number of the enzyme showed that these were 45% and 56% of that of the wild type, respectively. Further analysis of the mutant showed that the affinity for the inhibitor myxothiazol was decreased, that the 11-kDa subunit stabilises the enzyme once assembly has occurred, and that the reduction of cytochrome b via the Qout site is impaired. Sequence analysis showed that this mutant carries a deletion of 12 nucleotides at position 206-217 of the coding sequence, resulting in the replacement of residues 69-73 (WWKNG) by a cysteine. These results are discussed in terms of the 11-kDa subunit contributing to the conformation of the Qout binding domain.

Amino Acid Sequence↗
PubMed1993

The multifunctional regulatory proteins ABF1 and CPF1 are involved in the formation of a nuclease-hypersensitive region in the promoter of the QCR8 gene.

The abundant DNA-binding proteins ABF1 and CPF1 are members of a family of global regulators with diverse chromosomal functions in the yeast Saccharomyces cerevisiae. Recent evidence suggests that these protein factors may be involved in establishing and maintaining well-defined chromatin in promoter regions and other genetic elements. We have investigated the involvement of ABF1 and CPF1 in chromatin organization at the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase. The promoter region of the QCR8 gene contains overlapping binding sites for ABF1 and CPF1. Nucleosome positioning studies indicate that the QCR8 gene is associated with a phased array of nucleosomes under both catabolite-repressed and derepressed growth conditions. Analysis of binding site mutants reveals that both ABF1 and CPF1 are involved in maintaining a nuclease-hypersensitive region in the QCR8 promoter. The chromatin structure at QCR8 during steady-state growth is, however, mainly dependent on binding of ABF1 to the promoter region. Implications of these findings for the role played by ABF1 and CPF1 in the regulation of mitochondrial biogenesis and other processes important for cell growth and division will be discussed.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1993

In-vitro translation of mitochondrial mRNAs by yeast mitochondrial ribosomes is hampered by the lack of start-codon recognition.

In an attempt to reconstitute an homologous in-vitro translation system for yeast mitochondrial mRNAs, we have isolated ribosomes, supernatant factors, and tRNAs from mitochondria of Saccharomyces carlsbergensis. While poly(U) is translated faithfully in this system, no translation of in-vitro synthesised cytochrome c oxidase subunit II (COX2) mRNA could be detected. Formation of formylmethionyl-puromycin on mitochondrial ribosomes is stimulated by ApUpG, but not by COX2 mRNA, although mitochondrial small ribosomal subunits bind to this mRNA in vitro, even without added tRNA and initiation factors. We conclude, therefore, that the inability to faithfully translate mitochondrial mRNAs in vitro may be the result of an inability of mitochondrial ribosomes to recognize the initiation codon.

Codon↗