The yeast nuclear gene YMEJ was one of six genes recently identified in a screen for mutations that elevate the rate at which DNA escapes from mitochondria and migrates to the nucleus. ymel mutations, including a deletion, cause four known recessive phenotypes: an elevation in the rate at which copies of TRPI and ARSI, integrated into the mitochondrial genome, escape to the nucleus; a heat-sensitive respiratory-growth defect; a cold-sensitive growth defect on rich glucose medium; and synthetic lethality in rho-(cytoplasmic petite) cells. (43,44). This phenomenon is of interest since it has probably played a role in the transfer of genetic information from mitochondrial to nuclear genomes that has occurred during eucaryotic evolution (10,13,19,25,31,47). Equally if not more importantly, an examination of the mechanism by which DNA escapes from mitochondria should provide a novel avenue for identifying and studying factors involved in maintaining the integrity of mitochondria during the dynamic processes of cell division and fusion.To study genetically the mechanism by which DNA escapes mitochondria, we devised a screen for mutations that elevate the rate at which escape occurs (44) generating respiration-competent (at temperatures of 300 or below) petite-negative strains of S. cerevisiae. We also describe here the isolation and nucleotide sequence of YME1 and the detection of its product, Ymelp, in mitochondria. Ymelp has significant homology to a family of putative ATPases whose members are involved in processes ranging from organelle biogenesis to gene expression to cell division. Ymelp appears to be most related to the Escherichia coli FtsH protein, an essential protein involved in septum formation during cell division (46). MATERIALS AND METHODSStrains, strain constructions, and genetic methods. The E. coli strain used for preparation and manipulation of DNA was DH5a [F-endA41 hsdR17(r -iK+) supE44 thi-l X recA gyrA96 relAI A(argF-lacZYA) U169 +80 lacZAM15].The genotypes of the S. cerevisiae strains used in this work are listed in Table 1. Standard genetic techniques were used to construct and analyze the various yeast strains (38).Media. E. coli containing plasmids was grown in LB (10 g of Bacto Tryptone, 10 g of NaCl, and 5 g of yeast extract per liter) plus 125 ,ug of ampicillin per ml. Yeast strains were grown in complete glucose medium (YPD medium), complete ethanol-and-glycerol medium (YPEG), or minimal glucose medium plus the indicated nutrients (SD medium) (44). Ampicillin and nutrients were obtained from Sigma.Isolation of ymel-complementing plasmids. The temperature-sensitive respiratory-growth phenotype caused by the ymel-I mutation was used as the basis for cloning YMEL.PTY62 was transformed (18) with 70 ,ug of DNA prepared from a YCp50-based S. cerevisiae genomic DNA bank (34). Twelve thousand Ura+ colonies grew on minimal SD medium after incubation for 4 days at 30°C. The transformants were prewarmed at 37°C for 1 h and then replica plated onto YPEG plates and incubated at 37°C. Three transform...

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The absence of functional Yme1p, a putative ATP and zinc-dependent protease localized to mitochondria of yeast, results in abnormal mitochondrial function and morphology. Yeast lacking Yme1p lose DNA from mitochondria at an accelerated rate, fail to grow on nonfermentable carbon sources at 37 degrees C, and have severely deficient growth if mitochondrial DNA suffers large deletions or is completely lost. In place of the normal reticulated mitochondrial network, strains lacking Yme1p have punctate mitochondria with some grossly swollen compartments. The growth phenotypes and morphological alterations evident in these mutant yeast can be compensated by a mutation in YNT1, an essential gene in yeast. The sequence of the YNT1 gene product indicates that it is one of a number of related regulatory subunits of the 26S protease. This proteolytic activity is necessary for progression through the cell cycle and has been implicated in the regulation of transcription. Ynt1p is more distantly related to Yme1p.

The transfer of organelle nucleic acid to the nucleus has been observed in both plants and animals. Using a unique assay to monitor mitochondrial DNA escape to the nucleus in the yeast Saccharomyces cerevisiae, we previously showed that mutations in several nuclear genes, collectively called yme mutants, cause a high rate of mitochondrial DNA escape to the nucleus. Here we demonstrate that mtDNA escape occurs via an intracellular mechanism that is dependent on the composition of the growth medium and the genetic state of the mitochondrial genome, and is independent of an RNA intermediate. Isolation of several unique second-site suppressors of the high rate of mitochondrial DNA-escape phenotype of yme mutants suggests that there are multiple independent pathways by which this nucleic acid transfer occurs. We also demonstrate that the presence of centromeric plasmids in the nucleus can reduce the perceived rate of DNA escape from the mitochondria. We propose that mitochondrial DNA-escape events are manifested as unstable nuclear plasmids that can interact with centromeric plasmids resulting in a decrease in the number of observed events.

ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.