Compartmentalization of metabolic activities is a fundamental property of eucaryotic cells. The goal of my research is to identify the genetic and biochemical elements responsible for maintaining the integrity and structure of one subcellular compartment, the mitochondrion, and for coordinating its metabolic activities with intermediary metabolism. Novel genetic screens have allowed us to identify genes whose products are involved in these processes in the yeast Saccharomyces cerevisiae.
1) Isolating genes necessary for establishing and maintaining the compartmental integrity of mitochondria. Hybridization and sequence analysis over the past decade led to the identification of pseudogenes of mitochondrial origin in the nuclear genomes of many different organisms. Presumably, mitochondrial DNA (mtDNA) had escaped the mitochondrial compartment and been integrated into the host genome. In an attempt to observe this transfer of genetic information experimentally, a nuclear gene was placed in the mitochondrial compartment of the yeast Saccharomyces cerevisiae. This nuclear gene was not expressed in mitochondria, but DNA would spontaneously escape from mitochondria and migrate to the nucleus where it complemented a nuclear mutation of that same gene. The rate of DNA escape from mitochondria can be measured statistically and it is constant for a given yeast strain under given growth conditions (approximately 5 x 10-6 events/cell/cell division).
Although the mechanisms of DNA escape remain unknown, they could include transient breaches in mitochondrial membranes that occur during rearrangement of mitochondrial compartments, incomplete degradation of mtDNA during autophagy of mitochondria, or any number of processes important for maintaining the integrity of the mitochondrial compartment. In an attempt to learn more about these processes, a number of mutant yeast strains have been isolated that have an increased rate of DNA escape from mitochondria. The recessive nuclear mutations that lead to this increased rate of DNA escape have been designated yme, for yeast mitochondrial DNA escape. We have isolated recessive mutations in nine different nuclear genes and a dominant mutation in a tenth gene. We have also isolated multicopy plasmids bearing yeast genomic DNA that cause a dosage dependent increase in DNA escape from mitochondria.
2) Characterization of genes that affect the structure and integrity of the mitochondrial compartment. Several of the yme mutations have collateral growth phenotypes that are normally associated with defective mitochondrial activity. In three of these cases, the collateral phenotypes have allowed isolation of the wild type copy of the gene. One of these genes, YME1, encodes a protein localized to mitochondria that is involved in maintaining the proper super-structure of the mitochondrial compartment. The protein encoded by YME1 is homologous to a class of proteins that are reputed to be ATPases and are involved in biological processes ranging from cell division to secretion to organelle biogenesis. Yme1p also has sequence elements consistent with the protein having a zinc-dependent protease activity which has lead us to propose that Yme1p may act as an ATP-dependent protease. In support of this model for Yme1p function, we have demonstrated that conserved residues in the putative ATPase and zinc-binding motifs are required for Yme1p function. Furthermore, unassembled subunit II of cytochrome oxidase is a likely substrate for Yme1p. Our genetic analysis indicates that additional substrates for Yme1p must exist, and we are searching for these substrates by both genetic and biochemical methods. We are identifying gene products that interact with or function similarly to Yme1p by isolating genetic suppressors of yme1 mutations and cloning the wild type gene products of these suppressors. A bypass suppressor of yme1 mutations is encoded by a mutated version of a regulatory subunit of the ubiquitous 26S proteosome. We have also observed a genetic interaction between YME1 and ATP3, the gamma subunit of the mitochondrial ATP synthase. We are extending our genetic analysis of these suppressors by measuring enzymatic activities and determining the half-life of these and associated proteins.
We have also used both fluorescence and electron microscopy to examine yme1 strains. We have found altered mitochondrial morphology in yme1 strains consistent with our model of Yme1p being important for maintaining the correct compartment superstructure. These studies have also led us to begin investigations into the role of the vacuole in the turnover of the mitochondrial compartment, as electron micrographs suggest an increase in autophagy of mitochondria in yme1 strains. Our initial genetic studies indicate that the high rate of DNA escape from mitochondria can be suppressed by mutations that compromise the function of the vacuole. We tentatively conclude that the lack of Yme1p damages mitochondria such that they are marked for degradation. During autophagy, mtDNA can escape and the increased rate of DNA escape in yme1 cells is a reflection of an increased rate of autophagy.
We have recently cloned the YME2 and YME6 loci. In each case, these DNAs identified known genes. In the case of YME2, the loci had been identified as RNA12, a gene previously thought to be involved in RNA metabolism. However, our genetic characterization of this locus and the biochemical characterization of the encoded protein product lead us to believe that any effect this locus has upon cytoplasmic rRNA maturation is indirect. We have determined that Yme2p is localized to the inner mitochondrial membrane and that in combination with other mutations, yme2 strains are incapable of respiration. Furthermore, a dominant mutation exists in YME2 that interferes with cell growth at the restrictive temperature, independent of carbon source. We are pursuing the biochemical analysis of mitochondrial function in wild-type and mutant yeast in order to determine what essential process is interfered with in the mutant cells. We are actively pursuing the analysis of genes and gene products that interact with Yme2p by employing classic suppressor analysis and the two-hybrid system for identification of interacting proteins.
The YME6 locus is identical to that of a gene named MMM1. Mutations of MMM1 were found to severely affect mitochondrial morphology, preventing the formation of the normal mitochondrial reticulum and replacing it with abnormally large, rounded mitochondrial compartments. YME6/MMM1 encodes a protein localized to the outer membrane of mitochondria. We have determined that our mutant allele of YME6 functionally interacts with a mutant allele of YME4 resulting in a synthetic respiratory defect. Our efforts are directed at isolating the YME4 locus in an effort to determine the function of the gene products encoded by both YME6 and YME4.
3) Autophagy of the mitochondrial compartment. Mitochondria are dynamic structures that alter their organization and metabolic capacity in response to changing environmental conditions. Yeast that are grown on a fermentable carbon source such as glucose have reticulated mitochondria that comprise about 3% of the cell volume. Growth on nonfermentable carbon sources increases the proportion of mitochondrial volume in the cell to 12%. In contrast to the highly reticulated chondriome of actively growing yeast, cells that have entered stationary phase have smaller, more numerous mitochondrial compartments. In response to starvation or a switch from a nonfermentable to a fermentable carbon source, mitochondria and cytoplasmic constituents are autophagized by the vacuole. The mechanisms and regulation of these important metabolic events are largely unknown.
As noted above, we have identified several mutations that affect the structure of the mitochondrial compartment and consequently the ability of the organelle to support respiration and oxidative phosphorylation. At least one of these mutations apparently increases the rate of autophagy as evidenced by the suppression of the high rate of DNA escape by mutations in genes encoding vacuolar proteases. Therefore, the escape of DNA from mitochondria can serve as an indirect measure of autophagy of mitochondria by vacuoles. We have recently developed a direct means to measure autophagy of mitochondria. A phosphatase normally localized to the vacuole has been re-directed to the mitochondrial compartment. The phosphatase exists in an inactive form in mitochondria, but subsequent autophagy of mitochondria by the vacuole results in activation of the phosphatase due to proteolytic processing by a vacuolar protease. In response to starvation for nitrogen, yeast will autophagize bulk cytoplasm. We can demonstrate that mitochondria are also autophagized in response to this stress. We are using these two assays, the escape of DNA from mitochondria and the activation of a mitochondrially localized phosphatase, to identify environmental conditions and mutations that result in the autophagy of mitochondrial compartments.
Together, these genetic and biochemical experiments will help define the critical gene products and processes involved in maintaining the integrity of the mitochondrial compartment and integrating mitochondrial functions into the overall metabolic activities of the cell.
Corey L. Campbell and Peter E. Thorsness (1997) "Biochemical and Genetic Analysis of Vacuole-mediated Turnover of Mitochondria in Saccharomyces cerevisiae" Submitted.
Theodor Hanekamp and Peter E. Thorsness (1997) "Mitochondrial Endoproteases Afg3p, Rca1p, and Yme1p" in the Handbook of Proteolytic Enzymes, Academic Press (in press)
Theodor Hanekamp and Peter E. Thorsness (1996) "Inactivation of YME2, Which Encodes an Integral Inner Mitochondrial Membrane Protein, Causes Increased Escape of DNA from Mitochondria to the Nucleus in Saccharomyces cerevisiae". Molec. Cell. Biol. 16: 2764-2771
Eric R. Weber, Theodor Hanekamp, and Peter E. Thorsness (1996) "Biochemical and Functional Analysis of the YME1 gene product, an ATP and Zinc-Dependent Protease from S. cerevisiae." Molec. Biol. Cell 7: 307-317
Peter E. Thorsness and Eric R. Weber (1996) "Escape and Migration of Nucleic Acids Between Chloroplasts, Mitochondria and the Nucleus". International Review of Cytology 165: 207-234
Eric R. Weber, Robert S. Rooks, Karen S. Shafer, John W. Chase and Peter E. Thorsness (1995) "Mutations in the Mitochondrial ATP Synthase Gamma Subunit Suppress a Slow-Growth Phenotype of yme1 Yeast Lacking Mitochondrial DNA". Genetics 140: 435-442
Corey Campbell, Noriko Tanaka, Karen White and Peter E. Thorsness (1994) "Mitochondrial Morphological and Functional Defects in Yeast Caused by yme1 are Suppressed by Mutation of a 26S Protease Subunit Homologue". Molec. Biol. Cell 5: 899-905
Peter E. Thorsness, Karen H. White, and Thomas D. Fox (1993) "Inactivation of YME1, a Member of the ftsH-SEC18-PAS1-CDC48 Family of Putative ATPase-Encoding Genes, Causes Increased Escape of DNA from Mitochondria in Saccharomyces cerevisiae ". Molec. Cell. Biol. 13: 5418-5426
Peter E. Thorsness, Karen H. White, and Weoi-Choo Ong (1993) "AFG2, an Essential Gene in Yeast, Encodes a New Member of the Sec18p, Pas1p, Cdc48p, TBP-1 Family of Putative ATPases". Yeast 9: 1267-1271
Peter E. Thorsness and Thomas D. Fox (1993) "Nuclear Mutations in Saccharomyces cerevisiae that Affect the Escape of DNA from Mitochondria to the Nucleus". Genetics 134: 21-28
Peter E. Thorsness and Thomas D. Fox (1990) "Escape of DNA from Mitochondria to the Nucleus in Saccharomyces cerevisiae". Nature 346: 376-379
"Remember, none of this may be true." - Lots of people.
Return to the Department of Molecular Biology Web Page
25 March, 1997
thorsnes@uwyo.edu
voice: (307) 766-2038
fax: (307) 766-5098