Home LiteratureArticle Details
PMID: 3023841 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

The nine amino-terminal residues of delta-aminolevulinate synthase direct beta-galactosidase into the mitochondrial matrix.

Molecular and cellular biology ·Vol. 6 ·No. 2 ·1986-02-00 ·Pages 355-64

Keng T, Alani E, Guarente L

Abstract

delta-Aminolevulinate synthase, the first enzyme in the heme biosynthetic pathway, is encoded by the nuclear gene HEM1. The enzyme is synthesized as a precursor in the cytoplasm and imported into the matrix of the mitochondria, where it is processed to its mature form. Fusions of beta-galactosidase to various lengths of amino-terminal fragments of delta-aminolevulinate synthase were constructed and transformed into yeast cells. The subcellular location of the fusion proteins was determined by organelle fractionation. Fusion proteins were found to be associated with the mitochondria. Protease protection experiments involving the use of intact mitochondria or mitoplasts localized the fusion proteins to the mitochondrial matrix. This observation was confirmed by fractionation of the mitochondrial compartments and specific activity measurements of beta-galactosidase activity. The shortest fusion protein contains nine amino acid residues of delta-aminolevulinate synthase, indicating that nine amino-terminal residues are sufficient to localize beta-galactosidase to the mitochondrial matrix. The amino acid sequence deduced from the DNA sequence of HEM1 showed that the amino-terminal region of delta-aminolevulinate synthase was largely hydrophobic, with a few basic residues interspersed.

MeSH Terms
5-Aminolevulinate Synthetase/genetics Amino Acid Sequence Base Sequence DNA Restriction Enzymes Galactosidases/genetics Genetic Complementation Test Heme/biosynthesis Mitochondria/enzymology Plasmids Saccharomyces cerevisiae/enzymology,genetics,growth & development beta-Galactosidase/genetics
Chemicals
Heme 5-Aminolevulinate Synthetase DNA Restriction Enzymes Galactosidases beta-Galactosidase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Keng T
Alani E
Guarente L
References (44)
44 references, click to expand
  1. Plasmid-mediated complementation of a delta-aminolevulinic-acid-requiring Saccharomyces cerevisiae mutant.
    Gene. 1984 May;28(2):195-9 PMID: 6376285
  2. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  3. How mitochondria import proteins.
    Biochim Biophys Acta. 1984 Jan 27;779(1):65-87 PMID: 6318829
  4. Use of gene fusions to study outer membrane protein localization in Escherichia coli.
    Proc Natl Acad Sci U S A. 1977 Dec;74(12):5411-5 PMID: 414221
  5. Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria.
    Proc Natl Acad Sci U S A. 1984 Jul;81(13):3983-7 PMID: 6330727
  6. Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria.
    Eur J Biochem. 1982 Jun 15;125(1):109-16 PMID: 6213410
  7. 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
  8. Import of proteins into mitochondria. Partial purification of a matrix-located protease involved in cleavage of mitochondrial precursor polypeptides.
    J Biol Chem. 1983 Apr 25;258(8):4937-43 PMID: 6300104
  9. Import of proteins into mitochondria. Energy-dependent uptake of precursors by isolated mitochondria.
    J Biol Chem. 1982 Nov 10;257(21):13034-41 PMID: 6215405
  10. Two nuclear mutations that block mitochondrial protein import in yeast.
    Proc Natl Acad Sci U S A. 1984 Aug;81(15):4819-23 PMID: 6235522
  11. Isolation and sequence of the structural gene for cytochrome c oxidase subunit VI from Saccharomyces cerevisiae.
    J Biol Chem. 1984 Dec 25;259(24):15401-7 PMID: 6210289
  12. Transport of ADP/ATP carrier into mitochondria. Precursor imported in vitro acquires functional properties of the mature protein.
    J Biol Chem. 1984 Mar 25;259(6):3487-91 PMID: 6323427
  13. Compilation of published signal sequences.
    Nucleic Acids Res. 1984 Jul 11;12(13):5145-64 PMID: 6379599
  14. Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.
    J Bacteriol. 1979 Jul;139(1):19-31 PMID: 110778
  15. A simple method of preparing large amounts of phiX174 RF 1 supercoiled DNA.
    Biochim Biophys Acta. 1973 Apr 11;299(4):516-20 PMID: 4575181
  16. Lactic dehydrogenase and cytochrome b2 of baker's yeast; purification and crystallization.
    Biochem J. 1959 Mar;71(3):492-9 PMID: 13638255
  17. A rapid boiling method for the preparation of bacterial plasmids.
    Anal Biochem. 1981 Jun;114(1):193-7 PMID: 6269464
  18. 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
  19. Regulation of genes controlling synthesis of the galactose pathway enzymes in yeast.
    Genetics. 1966 Sep;54(3):911-6 PMID: 5970626
  20. A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus.
    EMBO J. 1984 Dec 20;3(13):3157-64 PMID: 6396085
  21. Different transport pathways of individual precursor proteins in mitochondria.
    Eur J Biochem. 1981 Jun 1;116(3):455-60 PMID: 6167439
  22. The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix.
    EMBO J. 1985 Aug;4(8):2061-8 PMID: 2998781
  23. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria.
    J Biol Chem. 1982 Nov 10;257(21):13028-33 PMID: 6290489
  24. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase.
    Cell. 1982 Jan;28(1):145-54 PMID: 7039847
  25. Nucleotide sequence of the yeast nuclear gene for cytochrome c peroxidase precursor. Functional implications of the pre sequence for protein transport into mitochondria.
    J Biol Chem. 1982 Dec 25;257(24):15054-8 PMID: 6294090
  26. Assembly of cytochrome c. Apocytochrome c is bound to specific sites on mitochondria before its conversion to holocytochrome c.
    Eur J Biochem. 1981 Dec;121(1):203-12 PMID: 6276170
  27. Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae.
    Proc Natl Acad Sci U S A. 1981 Apr;78(4):2199-203 PMID: 6264467
  28. Transformation of yeast.
    Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929-33 PMID: 347451
  29. 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
  30. A leader peptide is sufficient to direct mitochondrial import of a chimeric protein.
    EMBO J. 1985 May;4(5):1129-35 PMID: 3891325
  31. Spectrophotometric measurements of the enzymatic formation of fumaric and cis-aconitic acids.
    Biochim Biophys Acta. 1950 Jan;4(1-3):211-4 PMID: 15403927
  32. Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.
    J Biol Chem. 1977 May 10;252(9):2846-54 PMID: 323256
  33. The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles.
    EMBO J. 1983;2(7):1105-11 PMID: 16453466
  34. A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site.
    Proc Natl Acad Sci U S A. 1982 Dec;79(23):7410-4 PMID: 6760197
  35. Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein.
    EMBO J. 1984 Dec 1;3(12):2831-7 PMID: 6098449
  36. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
    Anal Biochem. 1976 May 7;72:248-54 PMID: 942051
  37. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination.
    Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963-5 PMID: 6575390
  38. Import of proteins into mitochondria. Extramitochondrial pools and post-translational import of mitochondrial protein precursors in vivo.
    J Biol Chem. 1982 Nov 10;257(21):13062-7 PMID: 6215406
  39. Cytochrome c oxidase from bakers' yeast. I. Isolation and properties.
    J Biol Chem. 1973 Feb 25;248(4):1346-54 PMID: 4346952
  40. Calcium-dependent bacteriophage DNA infection.
    J Mol Biol. 1970 Oct 14;53(1):159-62 PMID: 4922220
  41. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast.
    Methods Enzymol. 1983;101:181-91 PMID: 6310321
  42. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.
    Mol Cell Biol. 1984 Oct;4(10):1985-98 PMID: 6390181
  43. 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
  44. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.
    Anal Biochem. 1981 Apr;112(2):195-203 PMID: 6266278
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.6.2.355-364.1986
Published
1986-02-00
Pages
355-64
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC367524
Subset
IM
External Links
PubMed source DOI: 10.1128/mcb.6.2.355-364.1986 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: [email protected]

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