Peter E. Thorsness

Escape and migration of nucleic acids between chloroplasts, mitochondria, and the nucleus.

The escape and migration of genetic information between mitochondria, chloroplasts, and nuclei have been an integral part of evolution and has a continuing impact on the biology of cells. The evolutionary transfer of functional genes and fragments of genes from chloroplasts to mitochondria, from chloroplasts to nuclei, and from mitochondria to nuclei has been documented for numerous organisms. Most documented instances of genetic material transfer have involved the transfer of information from mitochondria and chloroplasts to the nucleus. The pathways for the escape of DNA from organelles may include transient breaches in organellar membranes during fusion and/or budding processes, terminal degradation of organelles by autophagy coupled with the subsequent release of nucleic acids to the cytoplasm, illicit use of nucleic acid or protein import machinery, or fusion between heterotypic membranes. Some or all of these pathways may lead to the escape of DNA or RNA from organellar compartments with subsequent uptake of nucleic acids from the cytoplasm into the nucleus. Investigations into the escape of DNA from mitochondria in yeast have shown the rate of escape for gene-sized fragments of DNA from mitochondria and its subsequent migration to the nucleus to be roughly equivalent to the rate of spontaneous mutation of nuclear genes. Smaller fragments of mitochondrial DNA may appear in the nucleus even more frequently. Mutations of nuclear genes that define gene products important in controlling the rate of DNA escape from mitochondria in yeast also have been described. The escape of genetic material from mitochondria and chloroplasts has clearly had an impact on nuclear genetic organization throughout evolution and may also affect cellular metabolic processes.

Mechanisms of mitochondrial DNA escape to the nucleus in the yeast Saccharomyces cerevisiae.

Abstract 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.

YNT20, a bypass suppressor of yme1 yme2, encodes a putative 3′-5′ exonuclease localized in mitochondria of Saccharomyces cerevisiae

Abstract Mutation of YME genes in yeast results in a high rate of mitochondrial DNA escape to the nucleus. The synthetic respiratory growth defect of yme1 yme2 yeast strains is suppressed by recessive mutations in YNT20. Inactivation of YNT20 creates a cold-sensitive respiratory growth defect that is more pronounced in a yme1 background and which is suppressed by yme2. Inactivation of YNT20 causes a qualitative reduction in the rate of mitochondrial DNA escape in yme1, but not yme2, strains, suggesting that YNT20 plays a role in the yme1-mediated mitochondrial DNA escape pathway. YNT20p is a soluble mitochondrial protein that belongs to a subfamily of putative 3′-5′ exonucleases. Furthermore, conserved sequence elements in Yme2p suggest that this protein may also function as an exonuclease.

Yeast Vps13 promotes mitochondrial function and is localized at membrane contact sites

Loss of VPS13 produces multiple phenotypes. This study implicates VPS13 in the function of membrane contact sites and suggests that different phenotypes of the mutant result from defects in different contact sites. In yeast, mutations found in the VPS13A gene of ChAc patients have specific defects in the mitochondrial aspect of VPS13 function.

Hsp90 and mitochondrial proteases Yme1 and Yta10/12 participate in ATP synthase assembly in Saccharomyces cerevisiae

Hsc82 and Hsp82, the Hsp90 family proteins of yeast, are both required for fermentative growth at 37°C. Inactivation of either of the mitochondrial AAA proteases, Yme1 or Yta10/12, allows fermentative growth of hsc82∆ or hsp82∆ strains at 37°C. Genetic evidence indicates interaction of Hsc82/Hsp82 with the Yme1 and Yta10/Yta12 complexes in promoting F(1)F(o)-ATPase activity, with Hsc82 specifically required for F(1)-ATPase assembly. A previously reported mutation in Rpt3, one of the six ATPases of the proteasome, suppresses yme1∆ phenotypes and increases transcription of HSC82 but not HSP82. These genetic interactions describe a functional role for Hsp90 proteins in mitochondrial biogenesis.

Functional expression of human adenine nucleotide translocase 4 in Saccharomyces cerevisiae.

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells.

The Molecular Basis for Relative Physiological Functionality of the ADP/ATP Carrier Isoforms in Saccharomyces cerevisiae

AAC2 is one of three paralogs encoding mitochondrial ADP/ATP carriers in the yeast Saccharomyces cerevisiae, and because it is required for respiratory growth it has been the most extensively studied. To comparatively examine the relative functionality of Aac1, Aac2, and Aac3 in vivo, the gene encoding each isoform was expressed from the native AAC2 locus in aac1Δ aac3Δ yeast. Compared to Aac2, Aac1 exhibited reduced capacity to support growth of yeast lacking mitochondrial DNA or of yeast lacking the ATP/Mg-Pi carrier, both conditions requiring ATP import into the mitochondrial matrix through the ADP/ATP carrier. Sixteen AAC1/AAC2 chimeric genes were constructed and analyzed to determine the key differences between residues or sections of Aac1 and Aac2. On the basis of the growth rate differences of yeast expressing different chimeras, the C1 and M2 loops of the ADP/ATP carriers contain divergent residues that are responsible for the difference(s) between Aac1 and Aac2. One chimeric gene construct supported growth on nonfermentable carbon sources but failed to support growth of yeast lacking mitochondrial DNA. We identified nine independent intragenic mutations in this chimeric gene that suppressed the growth phenotype of yeast lacking mitochondrial DNA, identifying regions of the carrier important for nucleotide exchange activities.

Yme2p is a mediator of nucleoid structure and number in mitochondria of the yeast Saccharomyces cerevisiae

A large number of gene products have been identified that either directly or indirectly alter the inheritance of mitochondrial DNA. In yeast, we have used a unique genetic screen based on the transfer of DNA from mitochondria to nucleus to identify nuclear-encoded gene products that are targeted to mitochondria and impact the stable inheritance of mitochondrial DNA. A specific allele of one of these genes, yme2-4, prevents even the low wild-type rate of mitochondrial DNA transfer to the nucleus and imparts significant temperature-sensitive and respiratory-growth defects. Intra- and extragenic suppressors of the yme2-4 growth phenotypes were isolated and analysis of these interacting genes reveals that both YME2 and its suppressors influence the structure and number of mitochondrial nucleoids. The yme2-4 allele decreases the average number of mtDNA nucleoids found in cells and the sensitivity of DNA in toluene-treated mitochondria to digestion by DNA exonuclease, effects reversed by intra- and extragenic suppressors. The extragenic suppressor, a missense allele of ILV5, encodes an enzyme of the branched-chain amino acid biosynthetic pathway that is also a component of mitochondrial nucleoids. A null allele of ILV5 suppresses transfer of mitochondrial DNA to the nucleus and displays synthetic interactions with yme2-4.

Structural dynamics of the mitochondrial compartment

The metabolic activities of mitochondria have been extensively characterized. However, there is much less known about the morphogenic changes of the mitochondrial compartment during growth, development and aging of the cell and the consequences of those structural changes on cellular metabolism. There is a growing body of evidence for interactions of mitochondria with cytoskeletal components and changes of mitochondrial structure during development and in response to changing environmental conditions. Segregation and recombination of mitochondrial genomes are also processes dependent upon the dynamic nature of the mitochondrial compartment. These regulatory and structural aspects of mitochondrial compartment dynamics will play an important role in the analysis of mitochondrial function and pathology.

Formation of an Energized Inner Membrane in Mitochondria with a γ-Deficient F1-ATPase

ABSTRACT Eukaryotic cells require mitochondrial compartments for viability. However, the budding yeast Saccharomyces cerevisiae is able to survive when mitochondrial DNA suffers substantial deletions or is completely absent, so long as a sufficient mitochondrial inner membrane potential is generated. In the absence of functional mitochondrial DNA, and consequently a functional electron transport chain and F1Fo-ATPase, the essential electrical potential is maintained by the electrogenic exchange of ATP4− for ADP3− through the adenine nucleotide translocator. An essential aspect of this electrogenic process is the conversion of ATP4− to ADP3− in the mitochondrial matrix, and the nuclear-encoded subunits of F1-ATPase are hypothesized to be required for this process in vivo. Deletion of ATP3, the structural gene for the γ subunit of the F1-ATPase, causes yeast to quantitatively lose mitochondrial DNA and grow extremely slowly, presumably by interfering with the generation of an energized inner membrane. A spontaneous suppressor of this slow-growth phenotype was found to convert a conserved glycine to serine in the β subunit of F1-ATPase (atp2-227). This mutation allowed substantial ATP hydrolysis by the F1-ATPase even in the absence of the γ subunit, enabling yeast to generate a twofold greater inner membrane potential in response to ATP compared to mitochondria isolated from yeast lacking the γ subunit and containing wild-type β subunits. Analysis of the suppressing mutation by blue native polyacrylamide gel electrophoresis also revealed that the α3β3 heterohexamer can form in the absence of the γ subunit.