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Seed germination provides an excellent model to study the process of mitochondrial biogenesis. It is a complex and strictly regulated process which requires a proper biogenesis of fully active organelles from existing promitochondrial structures. We have previously reported that the lack of the inner mitochondrial membrane protease FTSH4 delayed Arabidopsis seed germination. Here, we implemented a targeted mass spectrometry-based approach, Multiple Reaction Monitoring (MRM), with stable-isotope-labeled standard peptides for increased sensitivity, to quantify mitochondrial proteins in dry and germinating wild-type and ftsh4 mutant seeds, lacking the FTSH4 protease. Using total seed protein extracts we measured the abundance of the peptide targets belonging to the OXPHOS complexes, AOX1A, transport, and inner membrane scaffold as well as mitochondrial proteins that are highly specific to dry and germinating seeds. The MRM assay showed that the abundance of these proteins in ftsh4 did not differ substantially from that observed in wild-type at the level of dry seed and after stratification, but we observed a reduction in protein abundance in most of the examined OXPHOS subunits in the later stages of germination. These changes in OXPHOS protein levels in ftsh4 mutants were accompanied by a lower cytochrome pathway activity as well as an increased AOX1A amount at the transcript and protein level and alternative pathway activity. The analyses of the steady-state transcript levels of mitochondrial and nuclear genes encoding OXPHOS subunits did not show significant difference in their amount, indicating that the observed changes in the OXPHOS occurred at the post-transcriptional level. At the time when ftsh4 seeds were fully germinated, the abundance of the OXPHOS proteins in the mutant was either slightly lowered or comparable to these amounts in wild-type seeds at the similar developmental stage. By the implementation of an integrative approach combining targeted proteomics, quantitative transcriptomics, and physiological studies we have shown that the FTSH4 protease has an important role in the biogenesis of OXPHOS and thus biogenesis of mitochondria during germination of Arabidopsis seeds. [ABSTRACT FROM AUTHOR]

Oxygen fluctuations represent a common stressor in estuarine and intertidal environments and can compromise the mitochondrial integrity and function in marine organisms. We assessed the role of mitochondrial protection mechanisms (ATP-dependent and -independent mitochondrial proteases, and antioxidants) in tolerance to intermittent hypoxia or anoxia in three species of marine bivalves: hypoxia-tolerant hard clams (Mercenaria mercenaria) and oysters (Crassostrea virginica), and a hypoxia-sensitive subtidal scallop (Argopecten irradians). In clams and oysters, mitochondrial tolerance to hypoxia (18 h at 5% O2), anoxia (18 h at 0.1% O2) and subsequent reoxygenation was associated with the ability to maintain the steadystate activity of ATP-dependent and -independent mitochondrial proteases and an anticipatory upregulation of the total antioxidant capacity under the low oxygen conditions. No accumulation of endproducts of lipid or protein peroxidation was found during intermittent hypoxia oranoxiain clams and oysters (except foran increase in protein carbonyl concentration after hypoxia-reoxygenation in oysters). In contrast, hypoxia/anoxia and reoxygenation strongly suppressed activity of the ATP-dependent mitochondrial proteases in hypoxiasensitive scallops. This suppressionwas associated with accumulation of oxidatively damaged mitochondrial proteins (including carbonylated proteins and proteins conjugated with a lipid peroxidation product malondialdehyde) despite high total antioxidant capacity levels in scallop mitochondria. These findings highlight a key role of mitochondrial proteases in protection against hypoxia-reoxygenation stress and adaptations to frequent oxygen fluctuations in intertidal mollusks. [ABSTRACT FROM AUTHOR]

Background: The growth of Escherichia coli at elevated temperatures is limited due to the inherent instability of homoserine o-succinyltransferase, MetA, which is the first enzyme in the methionine biosynthesis pathway. MetA is also unstable under other stressful conditions, such as weak organic acids and oxidative stress. The MetA protein unfolds, even at 25°C, forms considerable aggregates at 37°C and completely aggregates at 44°C. Results: We extended the MetA mutation studies using a consensus concept based on statistics and sequence database analysis to predict the point mutations resulting in increased MetA stability. In this study, four single amino acid substitutions (Q96K, I124L, I229Y and F247Y) in MetA designed according to the consensus concept and using the I-mutant2.0 modeling tool conferred accelerated growth on the E. coli strain WE at 44°C. MetA mutants that enabled E. coli growth at higher temperatures did not display increased melting temperatures (Tm) or enhanced catalytic activity but did show improved in vivo stability at mild (37°C) and elevated (44°C) temperatures. Notably, we observed that the stabilized MetA mutants partially recovered the growth defects of E. coli mutants in which ATP-dependent proteases or the DnaK chaperone was deleted. These results suggest that the impaired growth of these E. coli mutants primarily reflect the inherent instability of MetA and, thus, the methionine supply. As further evidence, the addition of methionine recovered most of the growth defects in mutants lacking either ATP-dependent proteases or the DnaK chaperone. Conclusions: A collection of stable single-residue mutated MetA enzymes were constructed and investigated as background for engineering the stabilized mutants. In summary, the mutations in a single gene, metA, reframe the window of growth temperature in both normal and mutant E. coli strains. [ABSTRACT FROM AUTHOR]

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Mitochondria are crucial for both life and death of eukaryotic cells. Compromised mitochondrial integrity has severe cellular consequences and is linked to senescence and neurodegenerative disorders in humans. To maintain the functionality of proteins in mitochondria, quality-control mechanisms including signal transduction pathways counteracting mitochondrial stress have evolved. A network of molecular chaperones and proteases monitors protein integrity and prevents accumulation of damaged proteins. In this review, the current knowledge of elaborate defence strategies within mitochondria is summarized. [ABSTRACT FROM PUBLISHER]

The review characterizes the main enzymatic systems of selective proteolysis responsible for maintenance of intracellular proteome in prokaryotes, eukaryotes and archaea. The features of proteolytic components of the ATP-dependent proteases as well as similarity and diversity of their regulatory components belonging to AAA+ ATPases are discussed. [ABSTRACT FROM AUTHOR]

We report here the first crystal structure of the N-terminal domain of an A-type Lon protease. Lon proteases are ubiquitous, multidomain, ATP-dependent enzymes with both highly specific and non-specific protein binding, unfolding, and degrading activities. We expressed and purified a stable, monomeric 119-amino acid N-terminal subdomain of the Escherichia coli A-type Lon protease and determined its crystal structure at 2.03 Å (Protein Data Bank [PDB] code 2ANE). The structure was solved in two crystal forms, yielding 14 independent views. The domain exhibits a unique fold consisting primarily of three twisted β-sheets and a single long α-helix. Analysis of recent PDB depositions identified a similar fold in BPP1347 (PDB code 1ZBO), a 203-amino acid protein of unknown function from Bordetella parapertussis, crystallized as part of a structural genomics effort. BPP1347 shares sequence homology with Lon N-domains and with a family of other independently expressed proteins of unknown functions. We postulate that, as is the case in Lon proteases, this structural domain represents a general protein and polypeptide interaction domain. [ABSTRACT FROM AUTHOR]

Selective proteolysis is one of the mechanisms for the maintenance of cell homeostasis via rapid degradation of defective polypeptides and certain short-lived regulatory proteins. In prokaryotic cells, high-molecular-mass oligomeric ATP-dependent proteases are responsible for selective protein degradation. In eukaryotes, most polypeptides are attacked by the multicatalytic 26S proteasome, and the degradation of the majority of substrates involves their preliminary modification with the protein ubiquitin. The proteins undergoing the selective proteolysis often contain specific degradation signals necessary for their recognition by the corresponding proteases. [ABSTRACT FROM AUTHOR]

Lon is an oligomeric ATP-dependent protease that degrades defective or denatured proteins as well as some folded proteins for the control of cellular protein quality and metabolism. Lon from Thermococcus onnurineus NA1 was purified and crystallized at 295 K. A 2.0 Å resolution data set was collected using synchrotron radiation. The crystals belonged to space group P63, with unit-cell parameters a = 121.45, b = 121.45, c = 195.24 Å. Assuming the presence of two monomers in the asymmetric unit, the solvent content was estimated to be about 60.7%. [ABSTRACT FROM AUTHOR]