Transport of Cytoplasmically Synthesized Proteins into the Mitochondria in a Cell Free System from Neurospora crassa (original) (raw)

Different Transport Pathways of Individual Precursor Proteins in Mitochondria

European Journal of Biochemistry, 1981

Transport of mitochondrial precursor proteins into mitochondria of Neurospora crassa was studied in a cellfree reconstituted system. Precursors were synthesized in a reticulocyte lysate programmed with Neurospora mRNA and transported into isolated mitochondria in the absence of protein synthesis. Uptake of the following precursors was investigated: apocytochrome c, ADP/ATP carrier and subunit 9 of the oligomycin‐sensitive ATPase.Addition of high concentrations of unlabelled chemically prepared apocytochrome c (1–10 μM) inhibited the appearance in the mitochondrial of labelled cytochrome c synthesized in vitro because the unlabelled protein dilutes the labelled one and because the translocation system has a limited capacity [apparent V is 1–3 pmol × min−1× (mg mitochondrial protein)−1]. Concentrations of added apocytochrome c exceeding the concentrations of precursor proteins synthesized in vitro by a factor of about 104 did not inhibit the transfer of ADP/ATP carrier or ATPase subun...

[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

Protein import into mitochondria of Neurospora crassa

Fungal Genetics and Biology, 2002

Biogenesis of mitochondria requires import of several hundreds of different nuclear-encoded preproteins needed for mitochondrial structure and function. Import and sorting of these preproteins is a multistep process facilitated by complex proteinaceous machineries located in the mitochondrial outer and inner membranes. The translocase of the mitochondrial outer membrane, the TOM complex, comprises receptors which specifically recognize mitochondrial preproteins and a protein conducting channel formed by TOM40. The TOM complex is able to insert resident proteins into the outer membrane and to translocate proteins into the intermembrane space. For import of inner membrane or matrix proteins, the TOM complex cooperates with translocases of the inner membrane, the TIM complexes. During the past 30 years, intense research on fungi enabled the identification and mechanistic characterization of a number of different proteins involved in protein translocation. This review focuses on the contributions of the filamentous fungus Neurospora crassa to our current understanding of mitochondrial protein import, with special emphasis on the structure and function of the TOM complex. Ó

Assembly of mitochondria: Synthesis and intracellular transfer of mitochondrial proteins

Biosystems, 1980

The majority of mitochondrial proteins are synthesized on cytoplasmic ribosomes and transferred to the mitochondria where they are assembled to supramolecular structures. The intracellular transfer of these proteins appears to occur by a post-translational mechanism, i.e., it involves extramitochondrial precursor forms which are translocated in a step independent from translation. The synthesis and transfer of individual proteins was investigated in vivo, or in vitro employing homologous and heterologous cell free systems for protein synthesis. Cytochrome c was initially made as the apoprotein. This precursor protein was converted to the holoprotein on uptake by mitochondria in reconstituted systems. Integrity of mitechondria was essential for the ape to holo conversion. In the case of the ADP/ATP carrier protein, an integral transmembrane protein of the inner mitochondrial membrane, the initial translation product had the same apparent molecular weight as the mature protein. It was found in soluble form in the post-ribosomal supernatant. Citrate synthase, a matrix protein, was synthesized as a precursor with an apparent molecular weight of 47 000. Transfer to the mitochondria was accompanied by cleavage to yield a molecular weight of 45000. The significance of these results in relation to the mechanisms of intracellular transfer and of assembly of the individual proteins is discussed.

[27] Isolation and properties of the porin of the outer mitochondrial membrane from 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 100 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 153 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 188 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

The Intracellular Site of Synthesis of Mitochondrial Ribosomal Proteins in Neurospora Crassa

The Journal of Cell Biology, 1972

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to 14C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-3H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c)...