Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast

The MDM31 and MDM32 genes are required for normal distribution and morphology of mitochondria in the yeast Saccharomyces cerevisiae. They encode two related proteins located in distinct protein complexes in the mitochondrial inner membrane. Cells lacking Mdm31 and Mdm32 harbor giant spherical mitochondria with highly aberrant internal structure. Mitochondrial DNA (mtDNA) is instable in the mutants, mtDNA nucleoids are disorganized, and their association with Mmm1-containing complexes in the outer membrane is abolished. Mutant mitochondria are largely immotile, resulting in a mitochondrial inheritance defect. Deletion of either one of the MDM31 and MDM32 genes is synthetically lethal with deletion of either one of the MMM1, MMM2, MDM10, and MDM12 genes, which encode outer membrane proteins involved in mitochondrial morphogenesis and mtDNA inheritance. We propose that Mdm31 and Mdm32 cooperate with Mmm1, Mmm2, Mdm10, and Mdm12 in maintenance of mitochondrial morphology and mtDNA.     

Inactivation of YME2/RNA12, which encodes an integral inner mitochondrial membrane protein, causes increased escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae.

Inactivation of the yeast nuclear gene YMe2 causes an increased rate of DNA escape from mitochondria to the nucleus. Mutations in yme2 also show genetic interactions with yme1, a second gene that affects DNA escape from mitochondria to the nucleus. The yme1 cold-sensitive growth phenotype is suppressed by yme2 mutations. In addition, yme1 yme2 double mutants exhibit a synthetic growth defect on ethanol-glycerol medium at 30 degrees C. YME2 was isolated by complementation of the synthetic growth defect of yme1 yme2 strains and was found to be identical with the previously cloned RNA12 gene. The dominant temperature-sensitive mutation RNA12-1 prevents growth of yeast cells at 37 degrees C. YME2 encodes a protein with a predicted molecular weight of 96,681 and is an integral inner mitochondrial membrane protein. The larger carboxyl-terminal domain of the YME2 gene product faces the intermembrane space. Null alleles of yme2 display the same genetic interactions with yme1 and high rate of DNA escape from mitochondria as do the originally isolated yme2 mutant strains. Disruption of yme2 causes a strain-dependent growth defect on nonfermentable carbon sources.     

Nuclear Mutations in Saccharomyces Cerevisiae That Affect the Escape of DNA from Mitochondria to the Nucleus

We have inserted a yeast nuclear DNA fragment bearing the TRP1 gene and its associated origin of DNA replication, ARS1, into the functional mitochondrial chromosome of a strain carrying a chromosomal trp1 deletion. TRP1 was not phenotypically expressed within the organelle. However, this Trp(-) strain readily gave rise to respiratory competent Trp(+) clones that contained the TRP1/ARS1 fragment, associated with portions of mitochondrial DNA (mtDNA), replicating in their nuclei. Thus the Trp(+) clones arose as a result of DNA escaping from mitochondria and migrating to the nucleus. We have isolated 21 nuclear mutants in which the rate of mtDNA escape is increased by screening for increased rates of papillation to Trp(+). All 21 mutations were recessive and fell into six complementation groups, termed YME1-YME6. In addition to increasing the rate of mtDNA escape, yme1 mutations also caused a heat-sensitive respiratory deficient phenotype at 37° and a cold-sensitive growth defect on complete glucose medium at 14°. While the other yme mutations had no detectable growth phenotypes, synergistic interactions were observed in two double mutant combinations: a yme1, yme2 double mutant failed to respire at 30° and a yme4, yme6 double mutant failed to respire at all temperatures tested. None of the respiratory defects were caused by loss of functional mtDNA. These findings suggest that yme1, yme2, yme4 and yme6 mutations alter mitochondrial functions and thereby lead to an increased rate of DNA escape from the organelle.     

Mutations in the Mitochondrial Atp Synthase Gamma Subunit Suppress a Slow-Growth Phenotype of Yme1 Yeast Lacking Mitochondrial DNA

In Saccharomyces cerevisiae, inactivation of the nuclear gene YME1 causes several phenotypes associated with impairment of mitochondrial function. In addition to deficiencies in mitochondrial compartment integrity and respiratory growth, yme1 mutants grow extremely slowly in the absence of mitochondrial DNA. We have identified two genetic loci that, when mutated, act as dominant suppressors of the slow-growth phenotype of yme1 strains lacking mitochondrial DNA. These mutations only suppressed the slow-growth phenotype of yme1 strains lacking mitochondrial DNA and had no effect on other phenotypes associated with yme1 mutations. One allele of one linkage group had a collateral respiratory deficient phenotype that allowed the isolation of the wild-type gene. This suppressing mutation was in ATP3, a gene that encodes the gamma subunit of the mitochondrial ATP synthase. Recovery of two of the suppressing ATP3 alleles and subsequent sequence analysis placed the suppressing mutations at strictly conserved residues near the C terminus of Atp3p. Deletion of the ATP3 genomic locus resulted in an inability to utilize nonfermentable carbon sources. atp3 deletion strains lacking mitochondrial DNA grew slowly on glucose media but were not as compromised for growth as yme1 yeast lacking mitochondrial DNA.     

Biochemical and functional analysis of the YME1 gene product, an ATP and zinc-dependent mitochondrial protease from S. cerevisiae.

Inactivation of YME1 in yeast causes several distinct phenotypes: an increased rate of DNA escape from mitochondria, temperature-sensitive growth on nonfermentable carbon sources, extremely slow growth when mitochondrial DNA is completely absent from the cell, and altered morphology of the mitochondrial compartment. The protein encoded by YME1, Yme1p, contains two highly conserved sequence elements, one implicated in the binding and hydrolysis of ATP, and the second characteristic of active site residues found in neutral, zinc-dependent proteases. Both the putative ATPase and zinc-dependent protease elements are necessary for the function of Yme1p as genes having mutations in critical residues of either of these motifs are unable to suppress any of the phenotypes exhibited by yme1 deletion strains. Yme1p co-fractionates with proteins associated with the mitochondrial inner membrane, is tightly associated with this membrane, and is oriented with the bulk of the protein facing the matrix. Unassembled subunit II of cytochrome oxidase is stabilized in yme1 yeast strains. The data support a model in which Yme1p is an ATP and zinc-dependent protease associated with the matrix side of the inner mitochondrial membrane. Subunit II of cytochrome oxidase, when not assembled into a higher order complex, is a likely substrate of Yme1p.     

Apoptosis and aging in mitochondrial morphology mutants of<Emphasis Type="Italic">S. cerevisiae</Emphasis>

Cell viability during chronological aging and after apoptotic stimuli in some yeast mutants with altered mitochondrial morphology was followed; a function for the corresponding genes in the apoptotic process was assessed. MDM30 and DNM1, the genes encoding an F-box protein and the dynamin-related GTPase, respectively, are involved in triggering aging and apoptosis. In contrast, YME1, encoding a subunit of the mitochondrial inner membrane i-AAA proteinase complex, has a protective role in these processes. FIS1, the mitochondrial fission gene, might play a protective role after an apoptotic insult while it seems to promote cell death in aging cells.     

A screen for nigericin-resistant yeast mutants revealed genes controlling mitochondrial volume and mitochondrial cation homeostasis

Little is known about the regulation of ion transport across the inner mitochondrial membrane in Saccharomyces cerevisiae. To approach this problem, we devised a screening procedure for facilitating the identification of proteins involved in mitochondrial ion homeostasis. Taking advantage of the growth inhibition of yeast cells by electroneutral K(+)/H(+) ionophore nigericin, we screened for genetic mutations that would render cells tolerant to this drug when grown on a nonfermentable carbon source and identified several candidate genes including MDM31, MDM32, NDI1, YMR088C (VBA1), CSR2, RSA1, YLR024C, and YNL136W (EAF7). Direct examination of intact cells by electron microscopy indicated that mutants lacking MDM31 and/or MDM32 genes contain dramatically enlarged, spherical mitochondria and that these morphological abnormalities can be alleviated by nigericin. Mitochondria isolated from the Deltamdm31 and Deltamdm32 mutants exhibited limited swelling in an isotonic solution of potassium acetate even in the presence of an exogenous K(+)/H(+) antiport. In addition, growth of the mutants was inhibited on ethanol-containing media in the presence of high concentrations of salts (KCl, NaCl, or MgSO(4)) and their mitochondria exhibited two- (Deltamdm31 and Deltamdm32) to threefold (Deltamdm31Deltamdm32) elevation in magnesium content. Taken together, these data indicate that Mdm31p and Mdm32p control mitochondrial morphology through regulation of mitochondrial cation homeostasis and the maintenance of proper matrix osmolarity.     

Role of MMM1 in maintaining mitochondrial morphology in Neurospora crassa

Mmm1p is a protein required for maintenance of mitochondrial morphology in budding yeast. It was proposed that it is required to mediate the interaction of the mitochondrial outer membrane with the actin cytoskeleton. We report the cloning and characterization of MMM1 of the filamentous fungus Neurospora crassa, an organism that uses microtubules for mitochondrial transport. Mutation of the mmm-1 gene leads to a temperature-sensitive slow growth phenotype and female sterility. Mutant cells harbor abnormal giant mitochondria at all stages of the asexual life cycle, whereas actin filament-depolymerizing drugs have no effect on mitochondrial morphology. The MMM1 protein has a single transmembrane domain near the N terminus and exposes a large C-terminal domain to the cytosol. The protein can be imported into the outer membrane in a receptor-dependent manner. Our findings suggest that MMM1 is a factor of general importance for mitochondrial morphology independent of the cytoskeletal system used for mitochondrial transport.     

The human homologue of the yeast mitochondrial AAA metalloprotease Yme1p complements a yeast yme1 disruptant

In yeast, three AAA superfamily metalloproteases (Yme1p, Afg3p and Rca1p) are localized to the mitochondrial inner membrane where they perform roles in the assembly and turnover of the respiratory chain complexes. We have investigated the function of the proposed human orthologue of yeast Yme1p, encoded by the YME1L gene on chromosome 10p. Transfection of both HEK-293EBNA and yeast cells with a green fluorescent protein-tagged YME1L cDNA confirmed mitochondrial targeting. When expressed in a yme1 disruptant yeast strain, YME1L restored growth on glycerol at 37 degrees C. We propose that YME1L plays a phylogenetically conserved role in mitochondrial protein metabolism and could be involved in mitochondrial pathologies.     

Mutational spectrometry without phenotypic selection: human mitochondrial DNA.

By first separating mutant from nonmutant DNA sequences on the basis of their melting temperatures and then increasing the number of copies by high-fidelity DNA amplification, we have developed a method that allows observation of point mutations in biological samples at fractions at or above 10-6. Using this method, we have observed the hotspot point mutations that lie in 100 base pairs of the mitochondrial genome in samples of cultured cells and human tissues. To date, 19 mutants have been isolated, their fractions ranging from 4x10-4 down to the limit of detection. We performed specific tests to determine if the observed signals were artefacts arising from contamination, polymerase errors during PCR or DNA adducts created during the procedure. We also tested the possibilities that DNA replication mismatch intermediates, or endogenous DNA adducts that were originally present in the cells, were included with true mutants in our separation steps and converted to mutants during PCR. We show that while most of the mutants behave as double-stranded point mutants in the cells, some appear to arise at least in part from mismatch intermediates or cellular DNA adducts. This technology is therefore sufficient for the observation of the spectrum of point mutations in human mitochondrial DNA and is a tool for discovering the primary causes of these mutations.     

Nuclear mutations in Saccharomyces cerevisiae which increase the spontaneous mutation frequency in mitochondrial DNA.

Summary Fourteen mutants have been identified in which the frequency of spontaneous mutations in mitochondrial DNA is increased. As well as increasing the frequency of mutations to resistance to erythromycin, oligomycin and spiramycin, all the mutants also show changes in the frequency of spontaneous petite induction. None of the mutants has any effect on the frequency of spontaneous nuclear mutations. Nine of the mutants are in one complementation group and five are in another. The phenotype of both groups is caused by a single nuclear mutation.     

Expression of the Saccharomyces cerevisiae gene YME1 in the petite-negative yeast Schizosaccharomyces pombe converts it to petite-positive

Organisms that can grow without mitochondrial DNA are referred to as "petite-positive" and those that are inviable in the absence of mitochondrial DNA are termed "petite-negative." The petite-positive yeast Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1 phenotype can occur by virtue of dominant mutations in the alpha- and gamma-subunits of mitochondrial ATP synthase. These mutations are similar or identical to those occurring in the same subunits of the same enzyme that converts the petite-negative yeast Kluyveromyces lactis to petite-positive. Expression of YME1 in the petite-negative yeast Schizosaccharomyces pombe converts this yeast to petite-positive. No sequence closely related to YME1 was found by DNA-blot hybridization to S. pombe or K. lactis genomic DNA, and no antigenically related proteins were found in mitochondrial extracts of S. pombe probed with antisera directed against Yme1p. Mutations that block the formation of the F(1) component of mitochondrial ATP synthase are also petite-negative. Thus, the F(1) complex has an essential activity in cells lacking mitochondrial DNA and Yme1p can mediate that activity, even in heterologous systems.     

YME1L controls the accumulation of respiratory chain subunits and is required for apoptotic resistance, cristae morphogenesis, and cell proliferation

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.     

Isolation of mutants with aberrant mitochondrial morphology from Arabidopsis thaliana

To identify genes related to plant mitochondrial morphology and dynamics, novel mutants with respect to mitochondrial morphology were isolated from an ethyl methane sulphonate (EMS)-mutated population of Arabidopsis thaliana. Mitochondria were visualized by transforming Arabidopsis with a gene for a fusion protein consisting of GFP and a mitochondria-targeting pre-sequence. From 19,000 M2 populations, 17 mutants were isolated by fluorescent microscopic observations. All mitochondria in these mutants were longer and/or larger than wild-type mitochondria. The approximate chromosomal loci of the mutations of seven mutants that grew well were determined. The mitochondrial phenotypes of six of the mutants were recessive but the mitochondrial phenotype of the seventh mutant was dominant. Chromosomal rough mapping of the seven mutants showed that the mutations occurred at four different loci. At least one of these loci was novel, i.e., it was different from loci of other known mitochondrial morphology mutants of Arabidopsis and different from loci of Arabidopsis homologues of yeast genes related to mitochondrial morphology.     

Localization on mitochondrial DNA of mutations leading to a loss of rutamycin-sensitive adenosine triphosphatase.

Four cytoplasmic mutants of Saccharomyces cerevisiae showing loss of mitochondrial rutamycin-sensitive ATPase activity but having significant cytochrome oxidase and NADH-cytochrome c reductase have been isolated. Genetic studies indicate the mutations to be closely linked to each other and have been assigned to a new locus, PHO1. The mutations show a low frequency of recombination with the OL12 locus, suggesting a linkage to this marker. They are not, however, linked to the OLI1 locus. Linkage of the ATPase mutations to the OLI2 locus is also indicated by restoration of wild-type diploids by sigma- clones that retain the segment of mitochondrial DNA carrying OLI2. Based on the recombinants issued from crosses of the mutants with a triple drug-resistant strain and an analysis of the resistance markers present in sigma- clones that are effective in restoring a wild-type phenotype, the PHO1 locus has been placed in the segment of DNA located between PAR1 and OLI2.     

The mitochondrial inner membrane AAA metalloprotease family in metazoans

Three metalloproteases belonging to the AAA superfamily (Yme1p, Afg3p and Rca1p) are involved in protein turnover and respiratory chain complex assembly in the yeast inner mitochondrial membrane. Analysis of the completed genome sequences of Caenorhabditis elegans and Drosophila melanogaster indicates that this gene family typically comprises 3-4 members in metazoans. Phylogenetic analysis reveals three main branches represented, respectively, by Saccharomyces cerevisiae YME1, human SPG7 (paraplegin) and S. cerevisiae AFG3 and RCA1. mt-AAA metalloproteases are weak candidates for several previously studied Drosophila mutants. A full elucidation of the cellular and physiological roles of mt-AAA metalloproteases in metazoans will require the creation of targeted mutations.     

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.

The yeast nuclear gene YME1 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. yme1 mutations, including a deletion, cause four known recessive phenotypes: an elevation in the rate at which copies of TRP1 and ARS1, 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. The cloned YME1 gene complements all of these phenotypes. The gene product, Yme1p, is immunologically detectable as an 82-kDa protein present in mitochondria. Yme1p is a member of a family of homologous putative ATPases, including Sec18p, Pas1p, Cdc48p, TBP-1, and the FtsH protein. Yme1p is most similar to the Escherichia coli FtsH protein, an essential protein involved in septum formation during cell division. This observation suggests the hypothesis that Yme1p may play a role in mitochondrial fusion and/or division.     

Antimutators of mitochondrial and nuclear DNA in Saccharomyces cerevisiae. Relationship with gamma-ray sensitivity

Summary In Saccharomyces cerevisiae ten antimutator mutants have been isolated. The spontaneous occurrence of mitochondrial mutants resistant to erythromycin, oligomycin, and diuron is decreased 2-60-fold in these strains. The rate of forward and reverse spontaneous mutations of the nuclear genome is also reduced. The meiotic progenies arising from the crosses of seven mutants (LB₁, LB₂, LB₄, LB₅, LB₆, LB₇, LB₁₀) with an isogenic parental strain exhibit 2:2 segregations and therefore are the result of mutations in a single nuclear gene. The six mutants LB₁, LB₂, LB₄, LB₆, LB₇, LB₁₀ are semidominant and determine six complementation groups. The mutant LB₅ is dominant and therefore cannot be assigned to any complementation group. The mutants. LB₁, LB₄ and LB₁₀ are gamma-ray sensitive and, by tetrad analysis, it has been shown that gamma-ray sensitivity and spontaneous antimutability are the result of a single nuclear gene mutation. The other three mutants LB₃, LB₈ and LB₉ exhibit complex tetrad segregations typical of cytoplasmic inheritance and do not complement each other. However, although the mutations are semidominant, it has not been possible to detect any antimutator cytoductant among some 500 cytoductants carrying the kar1-1 nucleus. These results suggest that either several nuclear genes are involved in the expression of the antimutator phenotype or that the antimutator gene is located on nonchromosomal elements of the nucleus. The present study leads to the conclusion that a large number of nuclear genes are able to control simultaneously the spontaneous mutation rate of nuclear and mitochondrial genes. Since out of the ten antimutator mutants, three are also deficient in the repair of gamma-ray damage, it is also concluded that spontaneous and gamma-ray-induced lesions of DNA can be repaired by the same error-free process.     

The inner membrane protein Mdm33 controls mitochondrial morphology in yeast

Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Delta mdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein-protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.     

A protein complex containing Mdm10p,Mdm12p,and Mmm1p links mitochondrial membranes and DNA to the cytoskeleton-based segregation machinery

Previous studies indicate that two proteins, Mmm1p and Mdm10p, are required to link mitochondria to the actin cytoskeleton of yeast and for actin-based control of mitochondrial movement, inheritance and morphology. Both proteins are integral mitochondrial outer membrane proteins. Mmm1p localizes to punctate structures in close proximity to mitochondrial DNA (mtDNA) nucleoids. We found that Mmm1p and Mdm10p exist in a complex with Mdm12p, another integral mitochondrial outer membrane protein required for mitochondrial morphology and inheritance. This interpretation is based on observations that 1) Mdm10p and Mdm12p showed the same localization as Mmm1p; 2) Mdm12p, like Mdm10p and Mmm1p, was required for mitochondrial motility; and 3) all three proteins coimmunoprecipitated with each other. Moreover, Mdm10p localized to mitochondria in the absence of the other subunits. In contrast, deletion of MMM1 resulted in mislocalization of Mdm12p, and deletion of MDM12 caused mislocalization of Mmm1p. Finally, we observed a reciprocal relationship between the Mdm10p/Mdm12p/Mmm1p complex and mtDNA. Deletion of any one of the subunits resulted in loss of mtDNA or defects in mtDNA nucleoid maintenance. Conversely, deletion of mtDNA affected mitochondrial motility: mitochondria in cells without mtDNA move 2-3 times faster than mitochondria in cells with mtDNA. These observations support a model in which the Mdm10p/Mdm12p/Mmm1p complex links the minimum heritable unit of mitochondria (mtDNA and mitochondrial outer and inner membranes) to the cytoskeletal system that drives transfer of that unit from mother to daughter cells.