The N-terminal intrinsically disordered domain of mgm101p is localized to the mitochondrial nucleoid.

The mitochondrial genome maintenance gene, MGM101, is essential for yeasts that depend on mitochondrial DNA replication. Previously, in Saccharomyces cerevisiae, it has been found that the carboxy-terminal two-thirds of Mgm101p has a functional core. Furthermore, there is a high level of amino acid sequence conservation in this region from widely diverse species. By contrast, the amino-terminal region, that is also essential for function, does not have recognizable conservation. Using a bioinformatic approach we find that the functional core from yeast and a corresponding region of Mgm101p from the coral Acropora millepora have an ordered structure, while the N-terminal domains of sequences from yeast and coral are predicted to be disordered. To examine whether ordered and disordered domains of Mgm101p have specific or general functions we made chimeric proteins from yeast and coral by swapping the two regions. We find, by an in vivo assay in S.cerevisiae, that the ordered domain of A.millepora can functionally replace the yeast core region but the disordered domain of the coral protein cannot substitute for its yeast counterpart. Mgm101p is found in the mitochondrial nucleoid along with enzymes and proteins involved in mtDNA replication. By attaching green fluorescent protein to the N-terminal disordered domain of yeast Mgm101p we find that GFP is still directed to the mitochondrial nucleoid where full-length Mgm101p-GFP is targeted

In vivo methylation of mtDNA reveals the dynamics of protein–mtDNA interactions

To characterize the organization of mtDNA–protein complexes (known as nucleoids) in vivo, we have probed the mtDNA surface exposure using site-specific DNA methyltransferases targeted to the mitochondria. We have observed that DNA methyltransferases have different accessibility to different sites on the mtDNA based on the levels of protein occupancy. We focused our studies on selected regions of mtDNA that are believed to be major regulatory regions involved in transcription and replication. The transcription termination region (TERM) within the tRNALeu(UUR) gene was consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mitochondrial transcription termination factor 1 (mTERF1) to the site. Protection from methylation was also observed in other regions of the mtDNA, including the light and heavy strand promoters (LSP, HSP) and the origin of replication of the light strand (OL). Manipulations aiming at increasing or decreasing the levels of the mitochondrial transcription factor A (TFAM) led to decreased in vivo methylation, whereas manipulations that stimulated mtDNA replication led to increased methylation. We also analyzed the effect of ATAD3 and oxidative stress in mtDNA exposure. Our data provide a map of human mtDNA accessibility and demonstrate that nucleoids are dynamically associated with proteins

ERAL1 is associated with mitochondrial ribosome and elimination of ERAL1 leads to mitochondrial dysfunction and growth retardation

ERAL1, a homologue of Era protein in Escherichia coli, is a member of conserved GTP-binding proteins with RNA-binding activity. Depletion of prokaryotic Era inhibits cell division without affecting chromosome segregation. Previously, we isolated ERAL1 protein as one of proteins which were associated with mitochondrial transcription factor A by using immunoprecipitation. In this study, we analysed the localization and function of ERAL1 in mammalian cells. ERAL1 was localized in mitochondrial matrix and associated with mitoribosomal proteins including the 12S rRNA. siRNA knockdown of ERAL1 decreased mitochondrial translation, caused redistribution of ribosomal small subunits and reduced 12S rRNA. The knockdown of ERAL1 in human HeLa cells elevated mitochondrial superoxide production and slightly decreased mitochondrial membrane potential. The knockdown inhibited the growth of HeLa cells with an accumulation of apoptotic cells. These results suggest that ERAL1 is localized in a small subunit of the mitochondrial ribosome, plays an important role in the small ribosomal constitution, and is also involved in cell viability

Counseling with Kindergarten Children: an Autobiographical Experience

Frustration is a familiar emotion to this writer. For seventeen years, she has either taught or counseled in Milwaukee County. As a teacher she was plagued with problems of class size; curriculum content; parental, principal, or fellow teacher expectations; record completion; and deadlines. The most perplexing of all frustrations for her, as teacher, was being unable to connect emotionally with each child

Iron toxicity protection by truncated Ras2 GTPase in yeast strain lacking frataxin

Yeast strain deleted for the YFH1 gene, which encodes the orthologue of human frataxin, accumulates iron in mitochondria, constitutively activates the high-affinity iron import system in the plasma membrane, and is sensitive to high iron media. We have performed a genetic screen for mutants of a yfh1 deleted strain with increased resistance to high levels of iron. One of the identified mutations caused the deletion of the hypervariable C-terminal region of Ras2p GTPase. The effect of ras2 mutation on the growth of yfh1 null strain was masked by the addition of caffeine. We found that the ras2 mutation does not alter the expression of the iron regulon nor prevent mitochondrial iron accumulation in a yfh1 mutant context. The double yfh1 ras2 mutant has increased mRNA levels of CIT2 gene and augmented catalase activity. (C) 2003 Elsevier Inc. All rights reserved

Yeast mitochondrial biogenesis: a model system for humans?

Recently, our knowledge of yeast mitochondrial biogenesis has considerably progressed. This concerns the import machinery that guides preproteins synthesized on the cytoplasmic ribosomes through the mitochondrial outer and inner membranes, as well as the inner membrane insertion machinery of mitochondrially encoded polypeptides, or the proteins participating in the assembly and quality control of the respiratory complexes and ATP synthase. More recently, two new fields have emerged, biosynthesis of the iron-sulfur clusters and dynamics of the mitochondrion. Many of the newly discovered yeast proteins have homologues in human mitochondria. Thus, Saccharomyces cerevisiae has proven a particularly suitable simple organism for approaching the molecular bases of a growing number of human mitochondrial diseases caused by mutations in nuclear genes identified by positional cloning

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 Δmdm31 and Δmdm32 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- (Δmdm31 and Δmdm32) to threefold (Δmdm31Δmdm32) 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