Leslie A. Grivell

The mitochondrial genome of yeast

mtDNA is the simplest DNA in nature which carries genes for the three classes of RNA-rRNAs, tRNAs, and mRNAs. In yeast, this genetic system has a number of unusual features that may even be of interest to molecular biologists who do not particularly care about mitochondrial biogenesis: -The gene for the large rRNA is split and the intervening sequence varies in different strains. There is suggestive evidence that the structural genes for cytochrome b and subunit I of cytochrome oxidase are split as well. -The mtDNA has a low G + C content (16%) and about half of it consists of sections with a G + C content of <5%. -The mtDNAs of related yeast strains show hypervariability in sequence although gene order is conserved. -The yeast mitochondrial translation system is unusual in several respects: mitochondrial mRNAs are not properly translated in any nonmitochondrial system; the genes for the two major rRNAs are far apart on the DNA; secondary modification of rRNAs is marginal; no authentic 5s RNA has been found; and the system may operate with less than 32 tRNAs, the minimum number required according to the “wobble” hypothesis. -Transmission of genetic markers in one region of the mitochondrial genome shows polarity, and this has been correlated with the presence/absence of a 1000 base pair (bp) insertion. -Yeast mtDNA mutants have been isolated in which the resistance of mitochondrial ribosomes to erythromycin or chloramphenicol is probably due to an alteration in the nucleotide sequence of one of the rRNAs. -Yeast mtDNA frequently suffers massive deletions that remove 20-99.9% of its sequence; in the resulting ppetite mutants, the remaining mtDNA segment amplifies by tandem duplication. The reiterated nonfunctional petite mtDNA is faithfully replicated and retained at the same cellular concentration as wild-type mtDNA, irrespective of the size of the DNA segment retained. This review concentrates on some of these unusual features and on the possible use of the genetic system of yeast mitochondria as a model for studies in gene organization, expression, recombinaReview

Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro

Excised group II introns in yeast mitochondria appear as covalently closed circles under the electron microscope. We show that these circular molecules are branched and resemble the lariats arising through splicing of nuclear pre-mRNAs in yeast and higher eukaryotes. One member of this intron class (aI5c in the gene for cytochrome c oxidase subunit I) is capable of self-splicing in vitro, giving correct exon-exon ligation and resulting in the appearance of both linear and lariat forms of the excised intron. Nuclease digestion of the latter molecules reveals the presence of a complex oligonucleotide with the probable structure AGU, which thus resembles the branch point formed in the spliceosome-dependent reactions undergone by nuclear pre-mRNAs. Unlike group I introns, this group II intron is not demonstrably dependent on GTP for self-splicing and circularization of the isolated, linear intron is not observed. A model accounting for these observations is presented.

ATP-dependent proteases that also chaperone protein biogenesis

The ATP-dependent proteases Clp and FtsH from bacteria, as well as mitochondrial homologs of FtsH and Lon from yeast, may act as chaperones; they mediate not only proteolysis, but also the insertion of proteins into membranes and the disassembly or oligomerization of protein complexes. The coordination of such processes with selective proteolysis may function in the quality control of protein biogenesis.

Two intron sequences in yeast mitochondrial COX1 gene: Homology among URF-containing introns and strain-dependent variation in flanking exons

The DNA sequences of two optional introns in the gene for subunit I of cytochome c oxidase in yeast mitochondrial DNA have been determined. Both contain long unassigned reading frames (URFs). These display regions of amino acid homology with six other URFs, two of which encode proteins involved in mitochondrial RNA splicing. Such conserved regions may thus define functionally important domains of proteins involved in RNA processing. This homology also implies that these URFs had a common ancestral sequence, which has been duplicated and dispersed around the genome. Comparison of the flanking exons in the long strain KL14-4A with their unsplit counterpart in D273-10B reveals clustered sequence differences, which lead in D273-10B to codons rarely used in exons. These differences may be linked to the loss or absence of one of the optional introns.

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.

The role of protein degradation in mitochondrial function and biogenesis

Abstract It has been known for a long time that mitochondria contain their own protein-degradation systems. Only recently, however, have genes for mitochondrial proteases been identified and the powerful techniques of molecular biology been applied to gain insight into the role of protein degradation in mitochondrial biogenesis. It is now clear that the mitochondrial proteases that are involved in the initial stages of degradation are similar to prokaryotic ATP-dependent proteases, and that a division of labour exists between soluble and membrane-bound systems. These systems are essential for the biogenesis of fully functional mitochondria. Their natural targets are currently being identified, and their co-operation with chaperones and possible dual functions as chaperones/proteases are being investigated.

Import of proteins into mitochondria: a 70 kilodalton outer membrane protein with a large carboxy-terminal deletion is still transported to the outer membrane.

The yeast mitochondrial outer membrane contains a major 70‐kd protein which is coded by a nuclear gene. Two forms of this gene were isolated from a yeast genomic clone bank: the intact gene, and a truncated gene which had lost a large part of its 3′ end during the cloning procedure. Upon transformation into yeast, both the intact and the truncated gene are expressed; the truncated gene generates a shortened protein missing 203 amino acids from the carboxy‐terminus. This truncated polypeptide reacts with a monoclonal antibody against the authentic 70‐kd protein and is transported to the mitochondrial outer membrane. By integrative transformation, we have constructed a yeast mutant which lacks the 70‐kd protein and is unable to adapt to growth on a nonfermentable carbon source at 37 degrees C. This phenotypic lesion can be corrected by transforming the mutant with the intact, but not the truncated gene. The carboxy‐terminal sequence of 203 amino acids is thus necessary for the function of the protein, but not for its targeting to the mitochondrion.

SOME YEAST MITOCHONDRIAL RNAS ARE CIRCULAR

11S and 18S fractions of yeast mitochondrial RNAs, isolated by electrophoresis through agarose gels, have been found by electron microscopy to contain approximately 50% circular molecules. Circles in the 11S fraction have a contour length of 0.36 +/- 0.02 micron, which is approximately equal to the length of the majority of linear molecules also present. Circles in the 18S fraction have an average length of 0.78 +/- 0.11 micron. The size distribution is broader than for the 11S fraction, and we cannot exclude the possibility that more than one size class may be present. The 11S circular RNA forms circular R loops and RNA-DNA hybrids with DNA fragments of the oxi 3 region of mtDNA, which contains the structural gene for subunit 1 of cytochrome oxidase. As judged from the electron micrographs, the complete RNA participates in hybrid formation and the sequences coding for it appear to be continuous. Both 11S and 18S circles withstand treatment with DNAase and pronase. They are not eliminated by treatment with 1 M glyoxal in 50% formamide for 1 hr at 50 degrees C. We conclude that they are covalently closed. The function of the circular RNAs is unknown. They may be active as mRNAs, storage forms, or arise in a cut-and-splice process which generates mRNAs from longer transcripts.

Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome X (745 442 bp) reveals a total of 379 open reading frames (ORFs), the coding region covering approximately 75% of the entire sequence. One hundred and eighteen ORFs (31%) correspond to genes previously identified in S. cerevisiae. All other ORFs represent novel putative yeast genes, whose function will have to be determined experimentally. However, 57 of the latter subset (another 15% of the total) encode proteins that show significant analogy to proteins of known function from yeast or other organisms. The remaining ORFs, exhibiting no significant similarity to any known sequence, amount to 54% of the total. General features of chromosome X are also reported, with emphasis on the nucleotide frequency distribution in the environment of the ATG and stop codons, the possible coding capacity of at least some of the small ORFs (<100 codons) and the significance of 46 non‐canonical or unpaired nucleotides in the stems of some of the 24 tRNA genes recognized on this chromosome.

Redirection of the Respiro-Fermentative Flux Distribution in Saccharomyces cerevisiae by Overexpression of the Transcription Factor Hap4p

ABSTRACT Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of Saccharomyces cerevisiae. Glucose control of oxidative metabolism in bakers yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex. To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p. Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration. Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield. Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms.