Home LiteratureArticle Details
PMID: 8268801 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Import of cytochrome b2 to the mitochondrial intermembrane space: the tightly folded heme-binding domain makes import dependent upon matrix ATP.

Protein science : a publication of the Protein Society ·Vol. 2 ·No. 11 ·1993-11-00 ·Pages 1901-17

Glick BS, Wachter C, Reid GA, Schatz G

Abstract

Cytochrome b2 is synthesized as a precursor in the cytoplasm and imported to the intermembrane space of yeast mitochondria. We show here that the precursor contains a tightly folded heme-binding domain and that translocation of this domain across the outer membrane requires ATP. Surprisingly, it is ATP in the mitochondrial matrix rather than external ATP that drives import of the heme-binding domain. When the folded structure of the heme-binding domain is disrupted by mutation or by urea denaturation, import and correct processing take place in ATP-depleted mitochondria. These results indicate that (1) cytochrome b2 reaches the intermembrane space without completely crossing the inner membrane, and (2) some precursors fold outside the mitochondria but remain translocation-competent, and import of these precursors in vitro does not require ATP-dependent cytosolic chaperone proteins.

MeSH Terms
Adenosine Triphosphate/metabolism Binding Sites Biological Transport, Active Cell Compartmentation Heme/metabolism Intracellular Membranes/metabolism L-Lactate Dehydrogenase/drug effects,genetics,metabolism L-Lactate Dehydrogenase (Cytochrome) Mitochondria/metabolism Models, Biological Peptide Fragments/metabolism Protein Denaturation Protein Folding Protein Precursors/drug effects,genetics,metabolism Recombinant Fusion Proteins/metabolism Saccharomyces cerevisiae/metabolism Tetrahydrofolate Dehydrogenase/genetics,metabolism Urea/pharmacology
Chemicals
Peptide Fragments Protein Precursors Recombinant Fusion Proteins Heme Adenosine Triphosphate Urea L-Lactate Dehydrogenase L-Lactate Dehydrogenase (Cytochrome) Tetrahydrofolate Dehydrogenase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Glick B S
Biocenter, University of Basel, Switzerland.
Wachter C
Reid G A
Schatz G
References (75)
75 references, click to expand
  1. Translocation can drive the unfolding of a preprotein domain.
    EMBO J. 1993 Jan;12(1):243-53 PMID: 8428582
  2. Protein folding in a cell-free translation system. The fate of the precursor to mitochondrial aspartate aminotransferase.
    J Biol Chem. 1993 Feb 25;268(6):3925-37 PMID: 8440686
  3. A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins.
    EMBO J. 1993 Apr;12(4):1579-86 PMID: 8096814
  4. Loss of mitochondrial hsp60 function: nonequivalent effects on matrix-targeted and intermembrane-targeted proteins.
    Mol Cell Biol. 1993 May;13(5):3050-7 PMID: 8097278
  5. Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal.
    EMBO J. 1993 Jun;12(6):2295-302 PMID: 8508764
  6. The signal that sorts yeast cytochrome b2 to the mitochondrial intermembrane space contains three distinct functional regions.
    EMBO J. 1993 Jun;12(6):2303-11 PMID: 8389693
  7. Protein import into mitochondria: a paradigm for the translocation of polypeptides across membranes.
    Curr Opin Cell Biol. 1993 Aug;5(4):694-700 PMID: 8257609
  8. Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.
    J Cell Biol. 1989 Oct;109(4 Pt 1):1421-8 PMID: 2529262
  9. Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells.
    Cell. 1989 Nov 17;59(4):591-601 PMID: 2573430
  10. Protein sorting to mitochondria: evolutionary conservations of folding and assembly.
    Science. 1990 Feb 23;247(4945):930-8 PMID: 2406905
  11. Molecular structure of flavocytochrome b2 at 2.4 A resolution.
    J Mol Biol. 1990 Apr 20;212(4):837-63 PMID: 2329585
  12. Complete amino acid sequence of the heme-binding core in bakers' yeast cytochrome b2 (L-(+)-lactate dehydrogenase).
    Biochimie. 1976;58(3):305-16 PMID: 776230
  13. Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly.
    Science. 1990 May 18;248(4957):850-4 PMID: 2188360
  14. Import of precursor proteins into mitochondria: site of polypeptide unfolding.
    J Biol Chem. 1990 Jun 5;265(16):9444-51 PMID: 2345178
  15. Purification and identification of a cytosolic factor required for import of precursors of mitochondrial proteins into mitochondria.
    Arch Biochem Biophys. 1990 Aug 1;280(2):299-304 PMID: 2369121
  16. Energy requirements for unfolding and membrane translocation of precursor proteins during import into mitochondria.
    J Biol Chem. 1990 Sep 25;265(27):16324-9 PMID: 2144529
  17. How do polypeptides cross the mitochondrial membranes?
    Cell. 1990 Nov 2;63(3):447-50 PMID: 2225059
  18. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins.
    Nature. 1990 Nov 8;348(6297):137-43 PMID: 2234077
  19. Polypeptides traverse the mitochondrial envelope in an extended state.
    FEBS Lett. 1990 Nov 26;275(1-2):190-4 PMID: 2148157
  20. A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein.
    EMBO J. 1990 Dec;9(13):4315-22 PMID: 2265609
  21. Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast.
    EMBO J. 1991 Feb;10(2):247-54 PMID: 1991446
  22. Molecular chaperones.
    Annu Rev Biochem. 1991;60:321-47 PMID: 1679318
  23. Sequential action of mitochondrial chaperones in protein import into the matrix.
    EMBO J. 1991 Nov;10(11):3273-80 PMID: 1915294
  24. More similarity between bakers'yeast L-(+)-lactate dehydrogenase and liver microsomal cytochrome b5.
    Nature. 1975 May 29;255(5507):422-3 PMID: 165435
  25. Spontaneous dissociation of a cytochrome core and a biglobular flavoprotein after mild trypsinolysis of the bifunctional Saccharomyces cerevisiae flavocytochrome b2.
    Eur J Biochem. 1980 Oct;111(2):357-67 PMID: 7007037
  26. Study of a zone highly sensitive to proteases in flavocytochrome b2 from Saccharomyces cerevisiae.
    Eur J Biochem. 1981 Nov;120(2):279-87 PMID: 7032912
  27. Imported mitochondrial proteins cytochrome b2 and cytochrome c1 are processed in two steps.
    Proc Natl Acad Sci U S A. 1982 Jan;79(2):267-71 PMID: 7043457
  28. Biogenesis of mitochondrial ubiquinol:cytochrome c reductase (cytochrome bc1 complex). Precursor proteins and their transfer into mitochondria.
    J Biol Chem. 1982 Sep 10;257(17):10364-71 PMID: 6286652
  29. Import of proteins into mitochondria. Import and maturation of the mitochondrial intermembrane space enzymes cytochrome b2 and cytochrome c peroxidase in intact yeast cells.
    J Biol Chem. 1982 Nov 10;257(21):13068-74 PMID: 6290492
  30. Sequencing of the nuclear gene for the yeast cytochrome c1 precursor reveals an unusually complex amino-terminal presequence.
    EMBO J. 1984 Sep;3(9):2137-43 PMID: 6092058
  31. The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix.
    FEBS Lett. 1984 Dec 10;178(2):306-10 PMID: 6096170
  32. A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences.
    EMBO J. 1984 Dec 20;3(13):3143-8 PMID: 6526014
  33. The amino-terminal region of an imported mitochondrial precursor polypeptide can direct cytoplasmic dihydrofolate reductase into the mitochondrial matrix.
    EMBO J. 1984 Dec 20;3(13):3149-56 PMID: 6098467
  34. Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes.
    Cell. 1985 Nov;43(1):339-50 PMID: 2866845
  35. The presequences of two imported mitochondrial proteins contain information for intracellular and intramitochondrial sorting.
    Cell. 1986 Mar 14;44(5):801-12 PMID: 3004746
  36. Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome b2).
    EMBO J. 1985 Dec 1;4(12):3265-72 PMID: 3004948
  37. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria.
    Nature. 1986 Jul 17-23;322(6076):228-32 PMID: 3016548
  38. Commercially available firefly luciferase reagents.
    Methods Enzymol. 1986;133:51-70 PMID: 3821551
  39. 17th Sir Hans Krebs lecture. Signals guiding proteins to their correct locations in mitochondria.
    Eur J Biochem. 1987 May 15;165(1):1-6 PMID: 3552675
  40. Mitochondrial protein import: nucleoside triphosphates are involved in conferring import-competence to precursors.
    Cell. 1987 Jun 19;49(6):815-23 PMID: 2884042
  41. Transport of proteins to the mitochondrial intermembrane space: the 'matrix-targeting' and the 'sorting' domains in the cytochrome c1 presequence.
    EMBO J. 1987 Aug;6(8):2433-9 PMID: 2822392
  42. Transport of proteins to the mitochondrial intermembrane space: the 'sorting' domain of the cytochrome c1 presequence is a stop-transfer sequence specific for the mitochondrial inner membrane.
    EMBO J. 1987 Aug;6(8):2441-8 PMID: 2822393
  43. The role of protein structure in the mitochondrial import pathway. Unfolding of mitochondrially bound precursors is required for membrane translocation.
    J Biol Chem. 1987 Nov 15;262(32):15605-9 PMID: 3680214
  44. Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide.
    Cell. 1987 Dec 24;51(6):1027-37 PMID: 2826012
  45. Mitochondrial precursor proteins are imported through a hydrophilic membrane environment.
    Eur J Biochem. 1987 Dec 1;169(2):289-93 PMID: 2891506
  46. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides.
    Nature. 1988 Apr 28;332(6167):800-5 PMID: 3282178
  47. 70K heat shock related proteins stimulate protein translocation into microsomes.
    Nature. 1988 Apr 28;332(6167):805-10 PMID: 3282179
  48. Seventy-kilodalton heat shock proteins and an additional component from reticulocyte lysate stimulate import of M13 procoat protein into microsomes.
    EMBO J. 1988 Sep;7(9):2875-80 PMID: 3181144
  49. 70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria.
    J Cell Biol. 1988 Dec;107(6 Pt 1):2051-7 PMID: 3058716
  50. Binding of a tightly folded artificial mitochondrial precursor protein to the mitochondrial outer membrane involves a lipid-mediated conformational change.
    J Biol Chem. 1989 Feb 15;264(5):2951-6 PMID: 2536727
  51. The role of stress proteins in membrane biogenesis.
    Trends Biochem Sci. 1988 Oct;13(10):384-8 PMID: 3072700
  52. Protein import into the yeast mitochondrial matrix. A new translocation intermediate between the two mitochondrial membranes.
    J Biol Chem. 1991 Nov 5;266(31):21083-9 PMID: 1657948
  53. Protein import into isolated yeast mitochondria.
    Methods Cell Biol. 1991;34:389-99 PMID: 1943812
  54. Mitochondrial preproteins en route from the outer membrane to the inner membrane are exposed to the intermembrane space.
    FEBS Lett. 1991 Nov 18;293(1-2):85-8 PMID: 1835702
  55. Protein folding causes an arrest of preprotein translocation into mitochondria in vivo.
    J Cell Biol. 1991 Dec;115(6):1601-9 PMID: 1757464
  56. Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.
    EMBO J. 1992 Feb;11(2):449-56 PMID: 1371459
  57. Protein translocation across mitochondrial membranes.
    Bioessays. 1992 Jan;14(1):17-23 PMID: 1532121
  58. Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space.
    Cell. 1992 Mar 20;68(6):1163-75 PMID: 1347713
  59. Isolation and characterization of the cytochrome domain of flavocytochrome b2 expressed independently in Escherichia coli.
    Biochem J. 1992 Apr 1;283 ( Pt 1):87-90 PMID: 1567382
  60. What drives the translocation of proteins?
    Proc Natl Acad Sci U S A. 1992 May 1;89(9):3770-4 PMID: 1349170
  61. Import of proteins into mitochondria.
    Annu Rev Genet. 1991;25:21-44 PMID: 1812807
  62. Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism.
    Cell. 1992 May 29;69(5):809-22 PMID: 1350514
  63. Presequence binding factor-dependent and -independent import of proteins into mitochondria.
    J Biol Chem. 1992 Jul 5;267(19):13119-22 PMID: 1618812
  64. Targeting proteins to mitochondria: a current overview.
    Biochem J. 1992 Jun 15;284 ( Pt 3):609-20 PMID: 1622383
  65. Bacterial protein translocation: kinetic and thermodynamic role of ATP and the protonmotive force.
    Trends Biochem Sci. 1992 Jun;17(6):219-23 PMID: 1502724
  66. Differential scanning calorimetry of thermal unfolding of the methionine repressor protein (MetJ) from Escherichia coli.
    Biochemistry. 1992 Oct 13;31(40):9717-24 PMID: 1390748
  67. The translation machinery and 70 kd heat shock protein cooperate in protein synthesis.
    Cell. 1992 Oct 2;71(1):97-105 PMID: 1394434
  68. Secondary structure and folding of a functional chloroplast precursor protein.
    J Biol Chem. 1992 Oct 5;267(28):19907-13 PMID: 1400307
  69. Mutations in a 19-amino-acid hydrophobic region of the yeast cytochrome c1 presequence prevent sorting to the mitochondrial intermembrane space.
    Mol Cell Biol. 1992 Oct;12(10):4677-86 PMID: 1328858
  70. Protein targeting to and translocation across the membrane of the endoplasmic reticulum.
    Curr Opin Cell Biol. 1992 Aug;4(4):573-80 PMID: 1419037
  71. Putting energy into mitochondrial protein import.
    Curr Opin Cell Biol. 1992 Aug;4(4):646-51 PMID: 1419046
  72. Transport of proteins into the various subcompartments of mitochondria.
    FEBS Lett. 1992 Nov 16;313(1):2-7 PMID: 1426263
  73. Protein sorting in mitochondria.
    Trends Biochem Sci. 1992 Nov;17(11):453-9 PMID: 1455515
  74. Role of ATP in the intramitochondrial sorting of cytochrome c1 and the adenine nucleotide translocator.
    EMBO J. 1992 Dec;11(13):4787-94 PMID: 1334457
  75. YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism.
    Cell. 1992 Dec 24;71(7):1143-55 PMID: 1473150
Article Info
Journal
Protein science : a publication of the Protein Society
Abbr.
Protein Sci
ISSN
0961-8368
DOI
10.1002/pro.5560021112
Published
1993-11-00
Pages
1901-17
Language
English
Region
United States
NLM ID
9211750
PMCID
PMC2142280
Subset
IM
Grants
NIGMS NIH HHS · 2 R01 GM37803 · United States
External Links
PubMed source DOI: 10.1002/pro.5560021112 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2