Composition of complex I from Neurospora crassa and disruption of two “accessory” subunits (original) (raw)
Related papers
Effects of disrupting the 21 kDa subunit of complex I from Neurospora crassa
Biochemical Journal, 1999
We have cloned and inactivated in i o, 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 wildtype strain, suggesting that the absence of the 21 kDa polypeptide Abbreviation used : SMP, submitochondrial particles. 1 To whom correspondence should be addressed, at Instituto de Biologia Molecular e Celular (e-mail asvideir!icbas.up.pt). 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.
Biochemical Journal, 1993
We have cloned and sequenced a cDNA encoding a 17.8 kDa subunit of the hydrophobic fragment of complex I from Neurospora crassa. The deduced primary structure of this subunit was partially confirmed by automated Edman degradation of the isolated polypeptide. The sequence data obtained indicate that the 17.8 kDa subunit is made as an extended precursor of 20.8 kDa. Resistance of the polypeptide to alkaline extraction from mitochondrial membranes and the existence of a putative membrane-spanning domain suggests that the 17.8 kDa subunit is an intrinsic (bitopic) membrane protein. The in vitro synthesized precursor of the 17.8 kDa subunit can be efficiently imported into isolated mitochondria, where it is cleaved to the mature species by the metal-dependent matrix-processing peptidase. The in vitro imported mature subunit is found mainly exposed to the mitochondrial intermembrane space. However, a significant fraction of the imported polypeptide acquires the same membrane topology as t...
Biochemical Journal, 1992
The 20.9 kDa subunit of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa is a nuclear-coded component of the hydrophobic arm of the enzyme. We have determined the primary structure of this subunit by sequencing a full-length cDNA and a cleavage product of the isolated polypeptide. The deduced protein sequence is 189 amino acid residues long and contains a putative membrane-spanning domain. Striking similarity over a 60 amino-acid-residue domain with the M (matrix) protein of para-influenza virus was found. No other relationship with already known sequences could be detected, leaving the function of this subunit in complex I still undefined. The biogenetic pathway of this polypeptide was studied using a mitochondrial import system in vitro. The 20.9 kDa subunit synthesized in vitro is efficiently imported into isolated mitochondria, where it obtains distinct features of the endogenous subunit. Our results suggest that the 20.9 kDa polypeptide is made on cytosolic rib...
Supramolecular Organization of the Respiratory Chain in Neurospora crassa Mitochondria
Eukaryotic Cell, 2007
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.
Journal of Biological Chemistry, 1998
In Neurospora crassa, the mitochondrial arginine biosynthetic enzymes, N-acetylglutamate kinase (AGK) and N-acetyl-␥-glutamyl-phosphate reductase (AGPR), are generated by processing of a 96-kDa cytosolic polyprotein precursor (pAGK-AGPR). The proximal kinase and distal reductase domains are separated by a short connector region. Substitutions of arginines at positions ؊2 and ؊3 upstream of the N terminus of the AGPR domain or replacement of threonine at position ؉3 in the mature AGPR domain revealed a second processing site at position ؊20. Substitution of arginine at position ؊22, in combination with changes at ؊2 and ؊3, prevented cleavage of the precursor and identified two proteolytic cleavage sites, Arg-Gly2Tyr-Leu-Thr at the N terminus of the AGPR domain and Arg-Gly-Tyr2Ser-Thr located 20 residues upstream. Inhibitors of metal-dependent peptidases blocked proteolytic cleavage at both sites. Amino acid residues required for proteolytic cleavage in the connector were identified, and processing was abolished by mutations changing these residues. The unprocessed AGK-AGPR fusion had both catalytic activities, including feedback inhibition of AGK, and complemented AGK ؊ AGPR ؊ mutants. These results indicate that cleavage of pAGK-AGPR is not required for functioning of these enzymes in the mitochondria.
Biochemical Journal, 1977
A new procedure for the purification of the arom multienzyme complex from Neurospora crassa is presented. Important factors are the inactivation of proteinases by phenylmethanesulphonyl fluoride and the use of cellulose phosphate as an affinity adsorbent. A homogeneous enzyme, with a specific shikimate dehydrogenase activity of 70units/mg of protein, is obtained in 25% yield. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, combined with cross-linking studies using dimethyl suberimidate, suggest that the complex is composed of two subunits of molecular weight 165000. Glycerol-density-gradient centrifugation indicates a molecular weight for the intact complex ofabout 270000. Evidence for the effects ofproteolysis, both during the preparation and on storage of the purified complex, is presented, and previous reports in the literature of the occurrence of multiple subunits are discussed in this light.
[26] Biosynthesis and assembly of nuclear-coded mitochondrial membrane proteins in Neurospora crassa
Biomembranes Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts), 1983
Section I. Prokaryotic Membranes A. General Methods 1. Genetic Analysis of Protein Export in Escherichia JONATHAN BECKWITH AND coli THOMAS J. SILHAVY 3 2. Isolation and Characterization of Mutants of Esche-THOMAS J. SILHAVY AND richia coli Kl2 Affected in Protein Localization JONATHAN BECKWITH 3. Purification and Characterization of Leader Pepti-P. B. WOLFE, C. ZWIZINSKI, dase from Escherichia coli AND WILLIAM WICKNER 40 4. Molecular Genetics of Escherichia coli Leader Pep-TAKAYASU DATE, tidase PAMELA SILVER, AND WILLIAM WICKNER 5. Pulse-Labeling Studies of Membrane Assembly and WILLIAM WICKNER, Protein Secretion in Intact Cells: M13 Coat Pro-TAKAYASU DATE, tein RICHARD ZIMMERMANN, AND KOREAKI ITO 57 6. Synthesis of Proteins by Membrane-Associated PNANG C. TAI, Polysomes and Free Polysomes MICHAEL P. CAULFIELD, AND BERNARD D. DAVIS 62 7. Preparation of Free and Membrane-Bound Poly-LINDA L. RANDALL AND somes from Escherichia coli SIMON J. S. HARDY 70 8. Analysis of Cotranslational Proteolytic Processing of LARS-GÖRAN JOSEFSSON Nascent Chains Using Two-Dimensional Gel AND LINDA L. RANDALL Electrophoresis B. Outer Membrane 9. Proteins Forming Large Channels from Bacterial HIROSHI NIKAIDO and Mitochondrial Outer Membranes: Porins and Phage Lambda Receptor Protein 10. Phage λ Receptor (LamB Protein) in Escherichia coli MAXIME SCHWARTZ 11. Synthesis and Assembly of the Outer Membrane IAN CROWLESMITH AND Proteins OmpA and OmpF of Escherichia coli KONRAD GAMON 12. Isolation of Mutants of the Major Outer Membrane JACK COLEMAN, Lipoprotein of Escherichia coli for the Study of Its SUMIKO INOUYE, Assembly AND ΜASAYORI INOUYE ν VI TABLE OF CONTENTS C. Inner Membrane 13. Analysis of Μ13 Procoat Assembly into Membranes COLIN WATTS, in Vitro JOEL M. GOODMAN, PAMELA SILVER, AND WILLIAM WICKNER 14. Insertion of Proteins into Bacterial Membranes PETER MODEL AND MARJORIE RUSSEL 15. Influence of Membrane Potential on the Insertion ROBERT C. LANDICK, and Transport of Proteins in Bacterial Membranes CHARLES J. DANIELS, AND DALE L. OXENDER 16. Penicillinase Secretion in Vivo and in Vitro JENNIFER Β. K. NIELSEN 17. Lactose Permease of Escherichia coli J. K. WRIGHT, R. M. TEATHER, AND P.OVERATH 18. Cloning of the Structural Genes of the Escherichia DAVID A. JANS AND coli Adenosinetriphosphatase Complex FRANK GIBSON 19. Biogenesis of an Oligomeric Membrane Protein WILLIAM S. A. BRUSILOW, Complex: The Proton Translocating ATPase of ROBERT P. GUNSALUS, AND Escherichia coli ROBERT D. SIMONI 20. Analysis of Escherichia coli ATP Synthase Subunits JOHN E. WALKER AND by DNA and Protein Sequencing NICHOLAS J. GAY 21. Biogenesis of Purple Membrane in Halobacteria DOROTHEA-CH. NEUGEBAUER, HORST-PETER ZINGSHEIM, AND DIETER OESTERHELT 22. Isolation of the Bacterioopsin Gene by Colony Hy-HEIKE VOGELSANG, bridization WOLFGANG OERTEL, AND DIETER OESTERHELT Section II. Mitochondria 23. Assessing Import of Proteins into Mitochondria: An SUSAN M. GASSER AND Overview RICK HAY 245 24. Molecular Cloning of Middle-Abundant mRNAs ADELHEID VIEBROCK, from Neurospora crassa ANGELA PERZ, AND WALTER SEBALD 254 25. Biogenesis of Cytochrome c in Neurospora crassa BERND HENNIG AND WALTER NEUPERT 261 26. Biosynthesis and Assembly of Nuclear-Coded RICHARD ZIMMERMANN Mitochondrial Membrane Proteins in Neurospora AND WALTER NEUPERT 275 crassa TABLE OF CONTENTS VÜ 27. Isolation and Properties of the Porin of the Outer Mitochondrial Membrane from Neurospora crassa 28. Synthesis and Assembly of Subunit 6 of the Mitochondrial ATPase in Yeast 29. Preparation and Use of Antibodies against Insoluble Membrane Proteins 30. Processing of Mitochondrial Polypeptide Precursors in Yeast 31. Pulse Labeling of Yeast Cells and Spheroplasts 32. Import of Polypeptides into Isolated Yeast Mitochondria 33. A Yeast Mitochondrial Chelator-Sensitive Protease That Processes Cytoplasmically Synthesized Protein Precursors: Isolation from Yeast and Assay 34. Selection and Characterization of Nuclear Genes Coding Mitochondrial Proteins: Genetic Complementation of Yeast pet Mutants 35. Transformation of Nuclear Respiratory Deficient Mutants of Yeast 36. Analysis of Yeast Mitochondrial Genes 37. Genetics and Biogenesis of Cytochrome b 38. Synthesis and Intracellular Transport of Mitochondrial Matrix Proteins in Rat Liver: Studies in Vivo and in Vitro 39. Biosynthesis of Cytochrome c and Its Posttranslational Transfer into Mitochondria 40. Isolation of Mammalian Mitochondrial DNA and RNA and Cloning of the Mitochondrial Genome 41. Analysis of Human Mitochondrial RNA
Isolation of a bifunctional domain from the pentafunctional arom enzyme complex of Neurospora crassa
Biochemical Journal, 1983
Limited proteolysis of the arom enzyme complex of Neurospora crassa by trypsin or subtilisin yielded a stable fragment of Mr 68 000. This fragment, which was purified by two-dimensional polyacrylamide-gel electrophoresis, was shown by activity staining to contain the shikimate dehydrogenase active site, and by substrate labelling with 3-dehydroquinate and NaB3H4 to contain the 3-dehydroquinase active site. The fragment thus constitutes a bifunctional domain containing the two enzymic activities that are known, from genetic evidence, to be located adjacently at the C-terminal end of the pentafunctional arom polypeptide. The arom enzyme complex of the mould Neurospora crassa catalyses five reactions on the early common pathway of aromatic-amino-acid biosynthesis (see Scheme 1). When purified rapidly in the presence of proteinase inhibitors the arom complex was found to be composed of two subunits, each of Mr 165000 (Lumsden & Coggins, 1977; Gaertner & Cole, 1977). Peptide mapping demonstrated that these subunits are identical (Lumsden & Coggins, 1978), establishing the pentafunctional character of the arom polypeptide chain. Limited proteolysis has proved a useful tool in the study of the structure of multifunctional enzymes as it offers a method of isolating functional domains