scholarly journals Supramolecular Organization of the Respiratory Chain in Neurospora crassa Mitochondria

2007 ◽  
Vol 6 (12) ◽  
pp. 2391-2405 ◽  
Author(s):  
Isabel Marques ◽  
Norbert A. Dencher ◽  
Arnaldo Videira ◽  
Frank Krause
Keyword(s):  
Neurospora Crassa ◽  
Respiratory Chain ◽  
Complex I ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Deletion Mutants ◽  
Supramolecular Organization ◽  
Wild Type ◽  
Respiratory Supercomplexes ◽  
Native Polyacrylamide Gel Electrophoresis

ABSTRACT The existence of specific respiratory supercomplexes in mitochondria of most organisms has gained much momentum. However, its functional significance is still poorly understood. The availability of many deletion mutants in complex I (NADH:ubiquinone oxidoreductase) of Neurospora crassa, distinctly affected in the assembly process, offers unique opportunities to analyze the biogenesis of respiratory supercomplexes. Herein, we describe the role of complex I in assembly of respiratory complexes and supercomplexes as suggested by blue and colorless native polyacrylamide gel electrophoresis and mass spectrometry analyses of mildly solubilized mitochondria from the wild type and eight deletion mutants. As an important refinement of the fungal respirasome model, we found that the standard respiratory chain of N. crassa comprises putative complex I dimers in addition to I-III-IV and III-IV supercomplexes. Three Neurospora mutants able to assemble a complete complex I, lacking only the disrupted subunit, have respiratory supercomplexes, in particular I-III-IV supercomplexes and complex I dimers, like the wild-type strain. Furthermore, we were able to detect the I-III-IV supercomplexes in the nuo51 mutant with no overall enzymatic activity, representing the first example of inactive respirasomes. In addition, III-IV supercomplexes were also present in strains lacking an assembled complex I, namely, in four membrane arm subunit mutants as well as in the peripheral arm nuo30.4 mutant. In membrane arm mutants, high-molecular-mass species of the 30.4-kDa peripheral arm subunit comigrating with III-IV supercomplexes and/or the prohibitin complex were detected. The data presented herein suggest that the biogenesis of complex I is linked with its assembly into supercomplexes.

scholarly journals The mitochondrial AAA protease FTSH3 regulates Complex I abundance by promoting its disassembly

2021 ◽  
Author(s):  
Aneta Ivanova ◽  
Abi S Ghifari ◽  
Oliver Berkowitz ◽  
James Whelan ◽  
Monika W Murcha
Keyword(s):  
Complex I ◽  
Nadh Dehydrogenase ◽  
Slow Growth ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Forward Genetic Screen ◽  
Slow Growth Rate ◽  
Partial Restoration ◽  
Screen Approach

Abstract ATP is generated in mitochondria by oxidative phosphorylation. Complex I (NADH:ubiquinone oxidoreductase or NADH dehydrogenase) is the first multisubunit protein complex of this pathway, oxidising NADH and transferring electrons to the ubiquinone pool. Typically Complex I mutants display a slow growth rate compared to wild-type plants. Here, using a forward genetic screen approach for restored growth of a Complex I mutant, we have identified the mitochondrial ATP dependent metalloprotease, Filamentous Temperature Sensitive H 3 (FTSH3), as a factor that is required for the disassembly of Complex I. An ethyl methanesulfonate-induced mutation in FTSH3, named rmb1 (restoration of mitochondrial biogenesis 1), restored Complex I abundance and plant growth. Complementation could be achieved with FTSH3 lacking proteolytic activity, suggesting the unfoldase function of FTSH3 has a role in Complex I disassembly. The introduction of the rmb1 to an additional, independent, and extensively characterised Complex I mutant, ndufs4, resulted in similar increases to Complex I abundance and a partial restoration of growth. These results show that disassembly or degradation of Complex I plays a role in determining its steady-state abundance and thus turnover may vary under different conditions.

scholarly journals Membrane Sequestration of PII Proteins and Nitrogenase Regulation in the Photosynthetic Bacterium Rhodobacter capsulatus

2007 ◽  
Vol 189 (16) ◽  
pp. 5850-5859 ◽  
Author(s):  
Pier-Luc Tremblay ◽  
Thomas Drepper ◽  
Bernd Masepohl ◽  
Patrick C. Hallenbeck
Keyword(s):  
Gel Electrophoresis ◽  
Membrane Fraction ◽  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Photosynthetic Bacterium ◽  
Wild Type ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Pii Proteins ◽  
High Level

ABSTRACT Both Rhodobacter capsulatus PII homologs GlnB and GlnK were found to be necessary for the proper regulation of nitrogenase activity and modification in response to an ammonium shock. As previously reported for several other bacteria, ammonium addition triggered the AmtB-dependent association of GlnK with the R. capsulatus membrane. Native polyacrylamide gel electrophoresis analysis indicates that the modification/demodification of one PII homolog is aberrant in the absence of the other. In a glnK mutant, more GlnB was found to be membrane associated under these conditions. In a glnB mutant, GlnK fails to be significantly sequestered by AmtB, even though it appears to be fully deuridylylated. Additionally, the ammonium-induced enhanced sequestration by AmtB of the unmodifiable GlnK variant GlnK-Y51F follows the wild-type GlnK pattern with a high level in the cytoplasm without the addition of ammonium and an increased level in the membrane fraction after ammonium treatment. These results suggest that factors other than PII modification are driving its association with AmtB in the membrane in R. capsulatus.

scholarly journals The Campylobacter jejuni NADH:Ubiquinone Oxidoreductase (Complex I) Utilizes Flavodoxin Rather than NADH

2007 ◽  
Vol 190 (3) ◽  
pp. 915-925 ◽  
Author(s):  
Dilan R. Weerakoon ◽  
Jonathan W. Olson
Keyword(s):  
Campylobacter Jejuni ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Respiratory Activity ◽  
Essential Gene ◽  
Electron Flow ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
The Novel ◽  
Wild Type ◽  
Transport Chain

ABSTRACT Campylobacter jejuni encodes 12 of the 14 subunits that make up the respiratory enzyme NADH:ubiquinone oxidoreductase (also called complex I). The two nuo genes not present in C. jejuni encode the NADH dehydrogenase, and in their place in the operon are the novel genes designated Cj1575c and Cj1574c. A series of mutants was generated in which each of the 12 nuo genes (homologues to known complex I subunits) was disrupted or deleted. Each of the nuo mutants will not grow in amino acid-based medium unless supplemented with an alternative respiratory substrate such as formate. Unlike the nuo genes, Cj1574c is an essential gene and could not be disrupted unless an intact copy of the gene was provided at an unrelated site on the chromosome. A nuo deletion mutant can efficiently respire formate but is deficient in α-ketoglutarate respiratory activity compared to the wild type. In C. jejuni, α-ketoglutarate respiration is mediated by the enzyme 2-oxoglutarate:acceptor oxidoreductase; mutagenesis of this enzyme abolishes α-ketoglutarate-dependent O2 uptake and fails to reduce the electron transport chain. The electron acceptor for 2-oxoglutarate:acceptor oxidoreductase was determined to be flavodoxin, which was also determined to be an essential protein in C. jejuni. A model is presented in which CJ1574 mediates electron flow into the respiratory transport chain from reduced flavodoxin and through complex I.

scholarly journals Transcriptional analysis of the response of Neurospora crassa to phytosphingosine reveals links to mitochondrial function

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 3134-3141 ◽  
Author(s):  
Arnaldo Videira ◽  
Takao Kasuga ◽  
Chaoguang Tian ◽  
Catarina Lemos ◽  
Ana Castro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Neurospora Crassa ◽  
Complex I ◽  
Time Course ◽  
Expression Profiles ◽  
Transcriptional Analysis ◽  
Mitochondrial Proteins ◽  
Wild Type ◽  
Expression Levels ◽  
Genes Encoding

Treatment of Neurospora crassa cells with phytosphingosine (PHS) induces programmed cell death (PCD) by an unknown mechanism. To determine the relationship between PHS treatment and PCD, we determined changes in global gene expression levels in N. crassa during a time-course of PHS treatment. Most genes having differential expression levels compared to untreated samples showed an increase in relative expression level upon PHS exposure. However, genes encoding mitochondrial proteins were highly enriched among ∼100 genes that showed a relative decrease in expression levels after PHS treatment, suggesting that repression of these genes might be related to the death-inducing effects of PHS. Since mutants in respiratory chain complex I are more resistant to both PHS and hydrogen peroxide (H2O2) than the wild-type strain, possibly related to the production of reactive oxygen species, we also compared gene expression profiles of a complex I mutant (nuo14) and wild-type in response to H2O2. Genes with higher expression levels in the mutant, in the presence of H2O2, are also significantly enriched in genes encoding mitochondrial proteins. These data suggest that complex I mutants cope better with drug-induced decrease in expression of genes encoding mitochondrial proteins and may explain their increased resistance to both PHS and H2O2. As a way of identifying new components required for PHS-induced death, we analysed the PHS sensitivity of 24 strains carrying deletions in genes that showed a significant alteration in expression pattern when the wild-type was exposed to the sphingolipid. Two additional mutants showing increased resistance to PHS were identified and both encode predicted mitochondrial proteins, further supporting the role of the mitochondria in PHS-induced PCD.

Defective valyl-tRNA synthetase hampers the mitochondrial respiratory chain in Neurospora crassa

2012 ◽  
Vol 448 (3) ◽  
pp. 297-306 ◽  
Author(s):  
Margarida Duarte ◽  
Arnaldo Videira
Keyword(s):  
Protein Synthesis ◽  
Neurospora Crassa ◽  
Mutant Strain ◽  
Respiratory Chain ◽  
Mitochondrial Respiratory Chain ◽  
Trna Synthetase ◽  
Wild Type ◽  
Respiratory Chain Deficiency ◽  
Cytosolic Protein ◽  
Mitochondrial Respiratory

Respiratory chain deficiency can result from alterations in mitochondrial and/or cytosolic protein synthesis due to the dual genetic origin of mitochondrial oxidative phosphorylation. In the present paper we report a point mutation (D750G) in the bifunctional VARS (valyl-tRNA synthetase) of the fungus Neurospora crassa, associated with a temperature-sensitive phenotype. Analysis of the mutant strain revealed decreased steady-state levels of VARS and a clear reduction in the rate of mitochondrial protein synthesis. We observed a robust induction of the mitochondrial alternative oxidase with a concomitant decrease in the canonical respiratory pathway, namely in cytochrome b and aa3 content. Furthermore, the mutant strain accumulates the peripheral arm of complex I and depicts decreased levels of complexes III and IV, consistent with severe impairment of the mitochondrial respiratory chain. The phenotypic alterations of the mutant strain are observed at the permissive growth temperature and exacerbated upon increase of the temperature. Surprisingly, glucose-6-phosphate dehydrogenase activities were similar in the wild-type and mutant strains, whereas mitochondrial activities for succinate dehydrogenase and alternative NADH dehydrogenases were increased in the mutant strain, suggesting that the VARSD−G mutation does not affect overall cytosolic protein synthesis. Expression of the wild-type vars gene rescues all of the mutant phenotypes, indicating that the VARSD−G mutation is a loss-of-function mutation that results in a combined respiratory chain deficiency.

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