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. 1987 Oct;6(10):3111–3116. doi: 10.1002/j.1460-2075.1987.tb02620.x

Import of alcohol oxidase into peroxisomes of Saccharomyces cerevisiae.

B Distel 1, M Veenhuis 1, H F Tabak 1
PMCID: PMC553751  PMID: 2826130

Abstract

Saccharomyces cerevisiae is unable to grow on methanol because it lacks the enzymes required for its metabolism. To study the possibility of whether or not the methanol oxidation pathway of Hansenula polymorpha can be transferred to S. cerevisiae, the gene coding for alcohol oxidase, a peroxisomal homo-octameric flavoprotein, was introduced into S. cerevisiae. Transformed cells contain varying amounts of alcohol oxidase depending on the plasmid used. Immunocytochemical experiments indicate that the protein is imported into peroxisomes, whether organelle proliferation is induced or not. Cells lack alcohol oxidase activity however, and only the monomeric, non-functional, form of the protein is found. These findings indicate that the H. polymorpha peroxisomal targeting signal of alcohol oxidase is recognized in S. cerevisiae and protein monomers are imported.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baldari C., Cesareni G. Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences. Gene. 1985;35(1-2):27–32. doi: 10.1016/0378-1119(85)90154-4. [DOI] [PubMed] [Google Scholar]
  2. Bilibin A. F., Gracheva N. M. Lekarstvennaia bolezn' v klinike infektsionnykh zabolevanii. Sov Med. 1974 Jul;0(7):3–10. [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borst P. How proteins get into microbodies (peroxisomes, glyoxysomes, glycosomes). Biochim Biophys Acta. 1986 May 5;866(4):179–203. doi: 10.1016/0167-4781(86)90044-8. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. 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;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Cannon S., Wang P., Roy H. Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein. J Cell Biol. 1986 Oct;103(4):1327–1335. doi: 10.1083/jcb.103.4.1327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Daum G., Böhni P. C., Schatz G. 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):13028–13033. [PubMed] [Google Scholar]
  8. Goodman J. M., Scott C. W., Donahue P. N., Atherton J. P. Alcohol oxidase assembles post-translationally into the peroxisome of Candida boidinii. J Biol Chem. 1984 Jul 10;259(13):8485–8493. [PubMed] [Google Scholar]
  9. Janowicz Z. A., Eckart M. R., Drewke C., Roggenkamp R. O., Hollenberg C. P., Maat J., Ledeboer A. M., Visser C., Verrips C. T. Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha. Nucleic Acids Res. 1985 May 10;13(9):3043–3062. doi: 10.1093/nar/13.9.3043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jones E. W. Proteinase mutants of Saccharomyces cerevisiae. Genetics. 1977 Jan;85(1):23–33. doi: 10.1093/genetics/85.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Klebe R. J., Harriss J. V., Sharp Z. D., Douglas M. G. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene. 1983 Nov;25(2-3):333–341. doi: 10.1016/0378-1119(83)90238-x. [DOI] [PubMed] [Google Scholar]
  12. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lazarow P. B., Fujiki Y. Biogenesis of peroxisomes. Annu Rev Cell Biol. 1985;1:489–530. doi: 10.1146/annurev.cb.01.110185.002421. [DOI] [PubMed] [Google Scholar]
  15. Ledeboer A. M., Edens L., Maat J., Visser C., Bos J. W., Verrips C. T., Janowicz Z., Eckart M., Roggenkamp R., Hollenberg C. P. Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res. 1985 May 10;13(9):3063–3082. doi: 10.1093/nar/13.9.3063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  17. Needleman R. B., Tzagoloff A. Breakage of yeast: a method for processing multiple samples. Anal Biochem. 1975 Apr;64(2):545–549. doi: 10.1016/0003-2697(75)90466-2. [DOI] [PubMed] [Google Scholar]
  18. Oudshoorn P., Van Steeg H., Swinkels B. W., Schoppink P., Grivell L. A. Subunit II of yeast QH2:cytochrome-c oxidoreductase. Nucleotide sequence of the gene and features of the protein. Eur J Biochem. 1987 Feb 16;163(1):97–103. doi: 10.1111/j.1432-1033.1987.tb10741.x. [DOI] [PubMed] [Google Scholar]
  19. Panzeri L., Philippsen P. Centromeric DNA from chromosome VI in Saccharomyces cerevisiae strains. EMBO J. 1982;1(12):1605–1611. doi: 10.1002/j.1460-2075.1982.tb01362.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  21. Vaessen R. T., Kreike J., Groot G. S. Protein transfer to nitrocellulose filters. A simple method for quantitation of single proteins in complex mixtures. FEBS Lett. 1981 Feb 23;124(2):193–196. doi: 10.1016/0014-5793(81)80134-2. [DOI] [PubMed] [Google Scholar]
  22. Van Loon A. P., Van Eijk E., Grivell L. A. Biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast. Discoordinate synthesis of the 11-kd subunit in response to increased gene copy number. EMBO J. 1983;2(10):1765–1770. doi: 10.1002/j.1460-2075.1983.tb01655.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Veenhuis M., Harder W., van Dijken J. P., Mayer F. Substructure of crystalline peroxisomes in methanol-grown Hansenula polymorpha: evidence for an in vivo crystal of alcohol oxidase. Mol Cell Biol. 1981 Oct;1(10):949–957. doi: 10.1128/mcb.1.10.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Veenhuis M., van Dijken J. P., Harder W. Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha. Arch Microbiol. 1976 Dec 1;111(1-2):123–135. doi: 10.1007/BF00446559. [DOI] [PubMed] [Google Scholar]
  25. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  26. Zaret K. S., Sherman F. DNA sequence required for efficient transcription termination in yeast. Cell. 1982 Mar;28(3):563–573. doi: 10.1016/0092-8674(82)90211-2. [DOI] [PubMed] [Google Scholar]

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