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. 1989 Apr 1;108(4):1237–1243. doi: 10.1083/jcb.108.4.1237

NH2-terminal substitutions of basic amino acids induce translocation across the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2

PMCID: PMC2115505  PMID: 2494191

Abstract

Insertion of rabbit cytochrome P450IIC2 and its modified form, [2-lys,3- arg]P450IIC2, into microsomal membranes was studied in an in vitro transcription/translation/translocation system. Cytochrome P450IIC2, synthesized in the presence of chicken oviduct microsomal membranes, was resistant to extraction by alkaline solutions, but was sensitive to proteolytic digestion. In contrast, when [2-lys,3-arg]-P450IIC2 was synthesized in the presence of membranes, two new species migrating more slowly during gel electrophoresis were observed. After treatment with endoglycosidase H, the more slowly migrating species comigrated with [2-lys,3-arg]P450IIC2 synthesized in the absence of membranes, indicating that the proteins had been glycosylated. Both the glycosylated and nonglycosylated forms of [2-lys,3-arg]P450IIC2 were resistant to proteolytic digestion and to extraction from the membranes by alkaline solutions. Similar results were obtained for a truncated species, [2-lys,3-arg]P450IIC2(1-55), except that only a single glycosylated species was observed, consistent with the single remaining glycosylation site. In contrast to the proteolytic processing observed previously in a hybrid [2-lys,3-arg]P450IIC2/parathyroid hormone protein, little or no cleavage of the NH2-terminal peptide of [2-lys,3- arg]P450IIC2 was observed in the presence of membranes. Since cleavage in the hybrid protein occurred after glycine 25, which is derived from [2-lys,3-arg]P450IIC2, cytochrome P450 sequences COOH terminal to the cleavage site must decrease cleavage efficiency. These results demonstrate that cytochrome P450, which is normally localized on the cytoplasmic side of the membrane, can be entirely translocated to the luminal side when two basic amino acids precede the hydrophobic core of its NH2-terminal insertion/stop-transfer signal. None of the several internal hydrophobic regions of cytochrome P450, previously proposed as membrane spanning, function as a stop-transfer signal.

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Selected References

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  1. Audigier Y., Friedlander M., Blobel G. Multiple topogenic sequences in bovine opsin. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5783–5787. doi: 10.1073/pnas.84.16.5783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bar-Nun S., Kreibich G., Adesnik M., Alterman L., Negishi M., Sabatini D. D. Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes. Proc Natl Acad Sci U S A. 1980 Feb;77(2):965–969. doi: 10.1073/pnas.77.2.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyd D., Manoil C., Beckwith J. Determinants of membrane protein topology. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8525–8529. doi: 10.1073/pnas.84.23.8525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Lemos-Chiarandini C., Frey A. B., Sabatini D. D., Kreibich G. Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies. J Cell Biol. 1987 Feb;104(2):209–219. doi: 10.1083/jcb.104.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eisenberg D. Three-dimensional structure of membrane and surface proteins. Annu Rev Biochem. 1984;53:595–623. doi: 10.1146/annurev.bi.53.070184.003115. [DOI] [PubMed] [Google Scholar]
  7. Garoff H. Using recombinant DNA techniques to study protein targeting in the eucaryotic cell. Annu Rev Cell Biol. 1985;1:403–445. doi: 10.1146/annurev.cb.01.110185.002155. [DOI] [PubMed] [Google Scholar]
  8. Habener J. F., Rosenblatt M., Kemper B., Kronenberg H. M., Rich A., Potts J. T., Jr Pre-proparathyroid hormone; amino acid sequence, chemical synthesis, and some biological studies of the precursor region. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2616–2620. doi: 10.1073/pnas.75.6.2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  11. Leighton J. K., DeBrunner-Vossbrinck B. A., Kemper B. Isolation and sequence analysis of three cloned cDNAs for rabbit liver proteins that are related to rabbit cytochrome P-450 (form 2), the major phenobarbital-inducible form. Biochemistry. 1984 Jan 17;23(2):204–210. doi: 10.1021/bi00297a005. [DOI] [PubMed] [Google Scholar]
  12. Lennarz W. J. Protein glycosylation in the endoplasmic reticulum: current topological issues. Biochemistry. 1987 Nov 17;26(23):7205–7210. doi: 10.1021/bi00397a001. [DOI] [PubMed] [Google Scholar]
  13. Mead D. A., Skorupa E. S., Kemper B. Single stranded DNA SP6 promoter plasmids for engineering mutant RNAs and proteins: synthesis of a 'stretched' preproparathyroid hormone. Nucleic Acids Res. 1985 Feb 25;13(4):1103–1118. doi: 10.1093/nar/13.4.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  15. Mohana Rao J. K., Argos P. A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta. 1986 Jan 30;869(2):197–214. doi: 10.1016/0167-4838(86)90295-5. [DOI] [PubMed] [Google Scholar]
  16. Monier S., Van Luc P., Kreibich G., Sabatini D. D., Adesnik M. Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane. J Cell Biol. 1988 Aug;107(2):457–470. doi: 10.1083/jcb.107.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mostov K. E., DeFoor P., Fleischer S., Blobel G. Co-translational membrane integration of calcium pump protein without signal sequence cleavage. Nature. 1981 Jul 2;292(5818):87–88. doi: 10.1038/292087a0. [DOI] [PubMed] [Google Scholar]
  18. Nelson D. R., Strobel H. W. On the membrane topology of vertebrate cytochrome P-450 proteins. J Biol Chem. 1988 May 5;263(13):6038–6050. [PubMed] [Google Scholar]
  19. Ozols J., Heinemann F. S., Johnson E. F. The complete amino acid sequence of a constitutive form of liver microsomal cytochrome P-450. J Biol Chem. 1985 May 10;260(9):5427–5434. [PubMed] [Google Scholar]
  20. Paterson R. G., Lamb R. A. Ability of the hydrophobic fusion-related external domain of a paramyxovirus F protein to act as a membrane anchor. Cell. 1987 Feb 13;48(3):441–452. doi: 10.1016/0092-8674(87)90195-4. [DOI] [PubMed] [Google Scholar]
  21. Sakaguchi M., Mihara K., Sato R. A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence. EMBO J. 1987 Aug;6(8):2425–2431. doi: 10.1002/j.1460-2075.1987.tb02521.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sakaguchi M., Mihara K., Sato R. Signal recognition particle is required for co-translational insertion of cytochrome P-450 into microsomal membranes. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3361–3364. doi: 10.1073/pnas.81.11.3361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Szczesna-Skorupa E., Browne N., Mead D., Kemper B. 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 U S A. 1988 Feb;85(3):738–742. doi: 10.1073/pnas.85.3.738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Szczesna-Skorupa E., Mead D. A., Kemper B. Mutations in the NH2-terminal domain of the signal peptide of preproparathyroid hormone inhibit translocation without affecting interaction with signal recognition particle. J Biol Chem. 1987 Jun 25;262(18):8896–8900. [PubMed] [Google Scholar]
  25. Tarr G. E., Black S. D., Fujita V. S., Coon M. J. Complete amino acid sequence and predicted membrane topology of phenobarbital-induced cytochrome P-450 (isozyme 2) from rabbit liver microsomes. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6552–6556. doi: 10.1073/pnas.80.21.6552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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