scholarly journals Effects of disrupting the 21 kDa subunit of complex I from Neurospora crassa

1999 ◽  
Vol 342 (3) ◽  
pp. 551-554 ◽  
Author(s):  
Fátima FERREIRINHA ◽  
Margarida DUARTE ◽  
Ana M. P. MELO ◽  
Arnaldo VIDEIRA
Keyword(s):  
Neurospora Crassa ◽  
Conformational Changes ◽  
Complex I ◽  
Reductase Activity ◽  
Point Mutations ◽  
Nuclear Gene ◽  
Mitochondrial Diseases ◽  
Specific Protein ◽  
Gene Encoding

We have cloned and inactivated in vivo, by repeat-induced point mutations, the nuclear gene encoding a 21 kDa subunit of complex I from Neurospora crassa. Mitochondria from the nuo21 mutant lack this specific protein but retain other subunits of complex I in approximately normal amounts. In addition, this mutant is able to assemble an almost intact enzyme. The electron transfer activities from NADH to artificial acceptors of mitochondrial membranes from nuo21 differ from those of the wild-type strain, suggesting that the absence of the 21 kDa polypeptide results in conformational changes in complex I. Nevertheless, complex I of nuo21 is able to perform NADH:ubiquinone reductase activity, as judged by the observation that the respiration of mutant mitochondria is sensitive to inhibition by rotenone. We discuss these findings in relation to the involvement of complex I in mitochondrial diseases.

Respiratory Chain Complex I Is Essential for Sexual Development in Neurospora and Binding of Iron Sulfur Clusters Are Required for Enzyme Assembly

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 607-615
Author(s):  
Margarida Duarte ◽  
Arnaldo Videira
Keyword(s):  
Sexual Development ◽  
Complex I ◽  
Point Mutations ◽  
Nuclear Gene ◽  
Chain Complex ◽  
Early Developmental Stage ◽  
Iron Sulfur ◽  
Gene Coding ◽  
Intact Gene

Abstract We have cloned and disrupted in vivo, by repeat-induced point mutations, the nuclear gene coding for an iron sulfur subunit of complex I from Neurospora crassa, homologue of the mammalian TYKY protein. Analysis of the obtained mutant nuo21.3c revealed that complex I fails to assemble. The peripheral arm of the enzyme is disrupted while its membrane arm accumulates. Furthermore, mutated 21.3c-kD proteins, in which selected cysteine residues were substituted with alanines or serines, were expressed in mutant nuo21.3c. The phenotypes of these strains regarding the formation of complex I are similar to that of the original mutant, indicating that binding of iron sulfur centers to protein subunits is a prerequisite for complex I assembly. Homozygous crosses of nuo21.3c strain, and of other complex I mutants, are unable to complete sexual development. The crosses are blocked at an early developmental stage, before fusion of the nuclei of opposite mating types. This phenotype can be rescued only by transformation with the intact gene. Our results suggest that this might be due to the compromised capacity of complex I-defective strains in energy production.

scholarly journals Disruption of iron-sulphur cluster N2 from NADH:ubiquinone oxidoreductase by site-directed mutagenesis

2002 ◽  
Vol 364 (3) ◽  
pp. 833-839 ◽  
Author(s):  
Margarida DUARTE ◽  
Helena PÓPULO ◽  
Arnaldo VIDEIRA ◽  
Thorsten FRIEDRICH ◽  
Ulrich SCHULTE
Keyword(s):  
Complex I ◽  
Point Mutations ◽  
Nuclear Gene ◽  
Site Directed Mutagenesis ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Gene Encoding ◽  
Visible Spectra ◽  
Uv Visible

We have cloned and inactivated, by repeat-induced point mutations, the nuclear gene encoding the 19.3kDa subunit of complex I (EC 1.6.5.3) from Neurospora crassa, the homologue of the bovine PSST polypeptide. Mitochondria from mutant nuo19.3 lack the peripheral arm of complex I while its membrane arm accumulates. Transformation with wild-type cDNA rescues this phenotype and assembly of complex I is restored. To interfere with assembly of a proposed bound iron-sulphur cluster, site-directed mutants were constructed by introducing cDNA with altered codons for two adjacent cysteines, Cys-101 and Cys-102. The mutant complexes were purified and their enzymic activities and EPR and UV/visible spectra were analysed. Either of the mutations abolishes assembly of iron-sulphur cluster N2, showing that this redox group is bound to the 19.3kDa protein. We also observed an interference with the reduction of redox group X, suggesting that cluster N2 is the electron donor to this high-potential redox group.

scholarly journals Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane.

1986 ◽  
Vol 6 (2) ◽  
pp. 626-634 ◽  
Author(s):  
G S Adrian ◽  
M T McCammon ◽  
D L Montgomery ◽  
M G Douglas
Keyword(s):  
Transmembrane Protein ◽  
Nuclear Gene ◽  
Translocator Protein ◽  
Gene Encoding ◽  
Inner Membrane ◽  
Amino Terminal ◽  
Mitochondrial Inner Membrane ◽  
Membrane Anchoring ◽  
High Degree

The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization.

scholarly journals Specific protein binding to the simian virus 40 enhancer in vitro.

1986 ◽  
Vol 6 (6) ◽  
pp. 2098-2105 ◽  
Author(s):  
A G Wildeman ◽  
M Zenke ◽  
C Schatz ◽  
M Wintzerith ◽  
T Grundström ◽  
...  
Keyword(s):  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Specific Protein ◽  
Dnase I ◽  
Wild Type ◽  
Dnase I Footprinting ◽  
Nuclear Extracts

HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains.

scholarly journals Improvement of α-Amylase Production by Modulation of Ribosomal Component Protein S12 in Bacillus subtilis 168

2006 ◽  
Vol 72 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Kazuhiko Kurosawa ◽  
Takeshi Hosaka ◽  
Norimasa Tamehiro ◽  
Takashi Inaoka ◽  
Kozo Ochi
Keyword(s):  
Bacillus Subtilis ◽  
Antibiotic Production ◽  
Point Mutations ◽  
Protease Production ◽  
Gene Encoding ◽  
Translational Activity ◽  
Amylase Production ◽  
Ribosomal Protein S12

ABSTRACT The capacity of ribosomal modification to improve antibiotic production by Streptomyces spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (str) also enhances α-amylase (and protease) production by a strain of Bacillus subtilis as estimated by measuring the enzyme activity. The str mutations are point mutations within rpsL, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various rpsL mutations K56R (which corresponds to position 42 in E. coli) was particularly effective at enhancing α-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg2+ concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in α-amylase production seen in the affected strain.

scholarly journals Redox-induced activation of the proton pump in the respiratory complex I

2015 ◽  
Vol 112 (37) ◽  
pp. 11571-11576 ◽  
Author(s):  
Vivek Sharma ◽  
Galina Belevich ◽  
Ana P. Gamiz-Hernandez ◽  
Tomasz Róg ◽  
Ilpo Vattulainen ◽  
...  
Keyword(s):  
Proton Pump ◽  
Conformational Changes ◽  
Complex I ◽  
Large Scale ◽  
Reductase Activity ◽  
Proton Pumping ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Respiratory Chains ◽  
Dynamics Simulations

Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH2) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions.

scholarly journals Demonstration of a New Pathogenic Mutation in Human Complex I Deficiency: A 5-bp Duplication in the Nuclear Gene Encoding the 18-kD (AQDQ) Subunit

1998 ◽  
Vol 62 (2) ◽  
pp. 262-268 ◽  
Author(s):  
Lambert van den Heuvel ◽  
Wim Ruitenbeek ◽  
Roel Smeets ◽  
Zully Gelman-Kohan ◽  
Orly Elpeleg ◽  
...  
Keyword(s):  
Complex I ◽  
Nuclear Gene ◽  
Pathogenic Mutation ◽  
Gene Encoding ◽  
Complex I Deficiency ◽  
Human Complex

Early role of Fsp1 in epithelial-mesenchymal transformation

1997 ◽  
Vol 273 (4) ◽  
pp. F563-F574 ◽  
Author(s):  
Hirokazu Okada ◽  
Theodore M. Danoff ◽  
Raghuram Kalluri ◽  
Eric G. Neilson
Keyword(s):  
Growth Factor ◽  
Wound Repair ◽  
Transforming Growth Factor ◽  
Mesenchymal Cell ◽  
Transforming Growth Factor Β1 ◽  
Specific Protein ◽  
Early Event ◽  
Gene Encoding ◽  
Mesenchymal Transformation

A seamless plasticity exists among cells shifting between epithelial and mesenchymal phenotypes during early development and again later, in adult tissues, following wound repair or organ remodeling in response to injury. Fsp1, a gene encoding a fibroblast-specific protein associated with mesenchymal cell morphology and motility, is expressed during epithelial-mesenchymal transformations (EMT) in vivo. In the current study, we identified several cytokines that induce Fsp1 in cultured epithelial cells. A combination of these factors, however, was most efficacious at completing the process of EMT. The optimal combination identified were two of the cytokines classically associated with fibrosis, i.e., transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). To confirm that it was the induction of Fsp1 by these cytokines mediating EMT, we used antisense oligomers to block Fsp1 production and subsequently measured cell motility and markers of EMT phenotype. The antisense oligomers suppressed Fsp1 expression and epithelial transformation; therefore, we conclude that the appearance of Fsp1 is an important early event in the pathway toward EMT.

Disruption of a Nuclear Gene Encoding a Mitochondrial Gamma Carbonic Anhydrase Reduces Complex I and Supercomplex I+III2 Levels and Alters Mitochondrial Physiology in Arabidopsis

2005 ◽  
Vol 350 (2) ◽  
pp. 263-277 ◽  
Author(s):  
Mariano Perales ◽  
Holger Eubel ◽  
Jesco Heinemeyer ◽  
Alejandro Colaneri ◽  
Eduardo Zabaleta ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Complex I ◽  
Nuclear Gene ◽  
Gene Encoding ◽  
Gamma Carbonic Anhydrase
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