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Survival of bloodstream form Trypanosoma brucei, the agent of African sleeping sickness, normally requires mitochondrial gene expression, despite the absence of oxidative phosphorylation in this stage of the parasite's life cycle. Here we report that silencing expression of the alpha subunit of the mitochondrial F(1)-ATP synthase complex is lethal for bloodstream stage T. brucei as well as for T. evansi, a closely related species that lacks mitochondrial protein coding genes (i.e. is dyskinetoplastic). Our results suggest that the lethal effect is due to collapse of the mitochondrial membrane potential, which is required for mitochondrial function and biogenesis. We also identified a mutation in the gamma subunit of F(1) that is likely to be involved in circumventing the requirement for mitochondrial gene expression in another dyskinetoplastic form. Our data reveal that the mitochondrial ATP synthase complex functions in the bloodstream stage opposite to that in the insect stage and in most other eukaryotes, namely using ATP hydrolysis to generate the mitochondrial membrane potential.

We have shown previously that mutations in nuclear genes, termed MGI, for mitochondrial genome integrity, can convert the petite-negative yeast Kluyveromyces lactis into a petite-positive form with the ability to produce mitochondrial genome deletion mutants (Chen and Clark-Walker, Genetics, 133, 517-525, 1993). Here we describe that two genes, MGI2 and MGI5, encode the alpha- and gamma-subunits of mitochondrial F1-ATPase. Specific mutations, Phe443-->Ser and Ala333-->Val in MGI2, and Thr275-->Ala in MGI5, confer on cells the ability to produce petite mutants spontaneously with deletions in mitochondrial (mt) DNA and the capacity to lose their mitochondrial genomes upon treatment with ethidium bromide. Structural integrity of the F1 complex seems to be needed for expression of the Mgi- phenotype as null mutations in MGI2 and MGI5 remove the ability to form mtDNA deletions. It is suggested that mgi mutations allow petites to survive because an aberrant F1 complex prevents collapse of the mitochondrial inner membrane potential that normally occurs on loss of mtDNA-encoded F0 subunits.

The conformation of mitochondrial DNA (mtDNA) from yeasts has been examined by pulsed field gel electrophoresis and electron microscopy. The majority of mtDNA from Candida (Torulopsis) glabrata (mtDNA unit size, 19 kb) exists as linear molecules ranging in size from 50 to 150 kb or 2-7 genome units. A small proportion of mtDNA is present as supercoiled or relaxed circular molecules. Additional components, detected by electron microscopy, are circular molecules with either single- or double-stranded tails (lariats). The presence of lariats, together with the observation that the majority of mtDNA is linear and 2-7 genome units in length, suggests that replication occurs by a rolling circle mechanism. Replication of mtDNA in other yeasts is thought to occur by the same mechanism. For Saccharomyces cerevisiae, the majority of mtDNA is linear and of heterogeneous length. Furthermore, linear DNA is the chief component of a plasmid, pMK2, when it is located in the mitochondrion of baker's yeast, although only circular DNA is detected when this plasmid occurs in the nucleus. The implications of long linear mtDNA for hypotheses concerning the ploidy paradox and the mechanism of the petite mutation are discussed.

Unique transcripts for cytochrome b, ATPase subunits 6 and 9, cytochrome oxidase subunits 2 and 3 and S and L rRNA have been mapped by the S1 protection technique to the circular 19-kbp mitochondrial DNA (mtDNA) of the yeast Torulopsis glabrata. In contrast, a number of transcripts have been detected for the mosaic cytochrome oxidase subunit 1 gene with the largest being approximately 5000 nucleotides and the mature message having a length of 1760 nucleotides. Despite the presence in T. glabrata mtDNA of a sequence that hybridizes to the variant 1 gene of Saccharomyces cerevisiae mtDNA we have not detected a transcript of this region. Neither have we detected co-transcripts of adjacent genes in RNA from either glucose-repressed or derepressed cells. However, by comparison of RNA species from the two growth conditions, we have found that the ATPase subunit 6 transcript is lower in amount relative to other species in preparations from glucose-repressed cells. This information, together with the observation of separate transcripts and the knowledge that there are several species of mitochondrial RNA which can be capped by the guanylyl transferase catalysed addition of GMP, suggests that each of the genes investigated in the present study is separately transcribed.

Determination of sequences from the nine regions separating the large genes in the 19-kbp mitochondrial DNA from Torulopsis glabrata has led to the identification of 23 tRNA genes and to the recognition of two types of short repeated sequence implicated in mitochondrial genome expression. The two short repeated sequences are a nonanucleotide motif, 5'-TATAAGTAA-3' and a dodecanucleotide motif, 5'-TATAATATTCTT-3'. By RNA sequence determination it has been found that primary transcripts of the small and large rRNAs commence at the 3' penultimate adenine of the nonanucleotide sequence. This motif has also been found in the DNA sequence upstream from f-methionine, phenylalanine, leucine, tyrosine and glycine tRNAs, cytochrome oxidase subunit 2 and ATPase subunit 9. The dodecanucleotide sequence is found at least once in each of the nine regions between the large genes. Determination of the 3' ends of the small and large rRNAs has shown their location to be 8 and 23 nucleotides downstream from the dodecanucleotide sequence. This motif is thought to be involved in signalling processing of polycistronic transcripts. Such transcripts are invoked to account for the production of mRNAs for cytochrome b, cytochrome oxidase subunits 1 and 3, and the joint mRNA for ATPase subunits 8 and 6 genes that lack an adjacent upstream nonanucleotide transcription initiation signal sequence. Processing of polycistronic transcripts at tRNA sequences is also implicated in the formation of mature mRNAs. From the position of tRNA genes relative to the nonanucleotide motif it appears that clusters of these genes are co-transcribed with downstream sequences for cytochrome oxidase subunits 1 and 3.