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Abstract

  1. Top of page
  2. Abstract
  3. REFERENCES

The membrane topology of two alkane-inducible cytochromes P450 from the yeast Candida tropicalis, alk1 and alk2, was tested by construction of fusion proteins with part of invertase and histidinol dehydrogenase (invHIS4C) and expression in a Saccharomyces cerevisiae his4 mutant. Depending on the localization of invHIS4C on the endoplasmic reticulum (ER) cytoplasmic or luminal side, the enzyme converts histidinol to histidine and allows the his4 yeast strain to grow on histidinol-supplemented medium. The N-terminal segments of alk1 and alk2 were fused to invHIS4C at three different locations that follow the first alk1 and alk2 transmembrane domains or a second putative transmembrane domain of alk1. The combination of this in vivo assay with subcellular immunoprecipitations of the expressed fusion proteins allowed us to establish that both P450s contain only one transmembrane domain with their N-terminus located in the ER lumen. Deletions performed in these fusion proteins removing the first transmembrane domain of alk1 (TM) resulted in a less efficient targeting to the ER membrane but did not prevent their insertion in these membranes. Furthermore deletion of a negatively charged peptide preceding the first alk1 transmembrane domain (L) in an invHIS4C protein fused after this domain caused the N-terminal to have a positive net charge and to be oriented in the cytoplasm thus translocating the remaining protein into the ER lumen. The presence of the second hydrophobic segment, however, prevented the complete translocation of this fusion protein into the ER lumen. This study describes the first assessment of P450 membrane topology using an in vivo technique.

Abbreviations
aa

amino acid, ADHI, gene coding for yeast alcohol dehydrogenase I (ADHI)

alk1

alkane-inducible cytochrome P450alk1

alk2

alkane-inducible cytochrome P450alk2

CYP52A1

gene coding for alk1

CYP52A2

gene coding for alk2

EndoH

endoglycosidase H

ER

endoplasmic reticulum

HIS4C

histidinol dehydrogenase

HMG-CoA reductase

3-hydroxy-3-methylglutaryl coenzyme A reductase

HMG1

gene coding for HMG-CoA reductase

invHIS4C

fusion protein between an N-terminal invertase and C-terminal histidinol dehydrogenase segment according to [12]

invHIS4C

gene coding for invHIS4C

P450

cytochrome P450

REFERENCES

  1. Top of page
  2. Abstract
  3. REFERENCES
  • 1
    Omura, T. & Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem. 239, 23702378.
  • 2
    Omura, T. & Sato, R. (1964) The carbon monooxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties, J. Biol. Chem. 239, 23792385.
  • 3
    Ozols, J., Heinemann, F. S. & Johnson, E. F. (1985) The complete amino acid sequence of constitutive form of liver microsomal cytochrome P450, J. Biol. Chem. 260, 54275434.
  • 4
    Szczesna-Skorupa, E., Browne, N., Mead, D. & Kemper, B. (1988) Positive charges at the NH2-terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory signal peptide, Proc. Natl Acad. Sci. USA 85, 738742.
  • 5
    Sakaguchi, M., Mihara, K. & Sato, R. (1987) A short aminoterminal segment of microsomal cytochrome P-450 functions both as an insertion and as a stop-transfer sequence, EMBO J. 6, 24252431.
  • 6
    Monier, S., Van Luc, P., Kreiblich, G., Sabatini, D. D. & Adesnik, M. (1988) Signals for the incorporation and orientation of cytochrome P450 the endoplasmic reticulum membrane, J. Cell Biol. 107, 457470.
  • 7
    DeLemos-Chiarandini, C., Frey, A. B., Sabatini, D. D. & Kreibich, G. (1987) Determination of membrane topology of the phenobarbital-inducible rat liver cytochrome P450 isoenzyme PB-4 using site-specific antibodies, J. Cell Biol. 104, 209219.
  • 8
    Vergères, G., Winterhalter, K. H. & Richter, C. (1989) Identification of the membrane anchor of microsomal rat liver cytochrome P450, Biochemistry 28, 36503655.
  • 9
    Nelson, D. R. & Strobel, H. W. (1988) On the membrane topology of vertebrate cytochrome P450 proteins, J. Biol. Chem. 263, 60386050.
  • 10
    Bernhardt, R., Kraft, R., Otto, A. & Rückpaul, K. (1988) A simple determination of the sideness of the NH2-terminus in the membrane-bound P-450 LM2, Biochem. Intern. 17, 11431150.
  • 11
    Chen, S. & Zhou, D. (1992) Functional domains of aromatase cytochrome P450 inferred from comparative analyses of amino acid sequence and substantiated by site-ditected experiments, J. Biol. Chem. 267, 2258722594.
  • 12
    Sengstag, C., Stirling, C., Schekman, R. & Rine, J. (1990) Genetic and biochemical evaluation of eukaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxyl-3-methyl-glutaryl coenzyme A reductase, Mol. Cell. Biol. 10, 672680.
  • 13
    Deshaies, R. J. & Schekman, R. (1987) A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum, J. Cell Biol. 105, 633645.
  • 14
    Green, N. & Walter, P. (1992) C-terminal sequences can inhibit the on membrane proteins into the endoplasmic reticulum of Saccharomyces cerevisiae, Mol. Cell. Biol. 12, 276282.
  • 15
    Sanglard, D. & Loper, J. C. (1989) Characterization of the alkane-cytochrome P450 (P450alk) gene from the yeast Candida tropicalis: identification of a new P450 gene family, Gene 76, 121136.
  • 16
    Seghezzi, W., Sanglard, D. & Fiechter A. (1991) Characterization of a second alkane-inducible cytochrome P450-encoding gene, CYP52A2, from Candida tropicalis, Gene 106, 5160.
  • 17
    Rothstein, R. (1985) Cloning in yeast, in DNA cloning (Glover, D. M., ed.) vol. 4, pp. 4566, IRL Press, Oxford .
  • 18
    Kyte, L. & Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein, J. Mol. Biol. 157, 105132.
  • 19
    Hartmann, E., Rapoport, T. A. & Lodisch, H. F. (1989) Predicting the orientation of eukaryotic membrane-spanning domains, Proc. Natl Acad. Sci. USA 86, 57865790.
  • 20
    Szczesna-Skorupa, E. & Kemper, B. (1989) NH2-terminal substitutof basic amino acids induce translocation accross the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2, J. Cell Biol. 108, 12371243.
  • 21
    Sakaguchi, M., Tomiyoshi, R., Kuroiwa, T., Mihara, K. & Omura, T. (1992) Functions of signal and signal-anchor sequences are determined by the balance between the hydrophobic segment and the N-terminal charge, Proc. Natl Acad. Sci. USA 89, 1619.
  • 22
    d'Enfert, C., Barlowe, C., Nishikawa, S. I., Nakano, A. & Schekman, R. (1991) Structural and functional dissection of a membrane glycoprotein required for vesicle budding from the endoplasmic reticulum, Mol. Cell Biol. 11, 57275734.
  • 23
    Deshaies, R. J. & Schekman, R. (1990) Structural and functional dissection of Sec62p, a membrane-bound component of the yeast endoplasmic reticulum protein import machinery, Mol. Cell Biol. 10, 60246035.
  • 24
    Feldheim, D., Rothblatt, J. & Schekman, R. (1992) Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation, Mol. Cell. Biol. 12, 32883296.
  • 25
    Hann, B. C. & Walter, P. (1991) The signal recognition particle in S. cerevisiae, Cell 67, 131143.
  • 26
    Stirling, C. J. & Hewett, E. W. (1992) The Saccharomyces cerevisiae SEC65 gene encodes a component of the yeast signal recognition particle with homology to human SRP19, Nature 356, 534537.
  • 27
    Rothblatt, J. A., Deshaies, R. J., Sanders, S. L., Daum, G. & Schekman, R. (1989) Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum, J. Cell Biol. 109, 26412652.
  • 28
    Stirling, C. J., Rothblatt, J., Hosobushi, M., Deshaies, R. & Schekman, R. (1992) Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum, Mol. Biol. Cell 3, 129142.
  • 29
    Needleman, S. B. & Wunsch, C. D. (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins, J. Mol. Biol. 48, 443453.
  • 30
    Ammerer, G. (1983) Expression of genes in yeast using the ADCI promoter, Methods Enzymol. 101, 192201.