Abstract
Native isocitrate lyase from castor bean and a C-terminally truncated variant were expressed in Saccharomyces cerevisiae under the control of a galactose-inducible promoter. Both forms of isocitrate lyase were targeted to the yeast peroxisomes. They co-fractionated with catalase on sucrose-density-gradient centrifugation of a post-nuclear supernatant prepared from cells grown on oleic acid plus galactose, but were found in the cytosolic fractions when the cells were grown under conditions that repress peroxisome formation. The endogenous S. cerevisiae isocitrate lyase was found solely in the cytoplasmic fractions, even under growth conditions that induce peroxisome proliferation. This result shows that the presence of isocitrate lyase in peroxisomes is not essential for a functional glyoxylate cycle. Although the heterologous enzyme was transported to peroxisomes it was not enzymically active. Immunocytochemical studies provide independent evidence that the plant enzyme is imported into the matrix of yeast peroxisomes.
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- Aitchison J. D., Murray W. W., Rachubinski R. A. The carboxyl-terminal tripeptide Ala-Lys-Ile is essential for targeting Candida tropicalis trifunctional enzyme to yeast peroxisomes. J Biol Chem. 1991 Dec 5;266(34):23197–23203. [PubMed] [Google Scholar]
- Barth G., Scheuber T. Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein. Mol Gen Genet. 1993 Nov;241(3-4):422–430. doi: 10.1007/BF00284696. [DOI] [PubMed] [Google Scholar]
- Behari R., Baker A. The carboxyl terminus of isocitrate lyase is not essential for import into glyoxysomes in an in vitro system. J Biol Chem. 1993 Apr 5;268(10):7315–7322. [PubMed] [Google Scholar]
- Cooper T. G., Beevers H. Mitochondria and glyoxysomes from castor bean endosperm. Enzyme constitutents and catalytic capacity. J Biol Chem. 1969 Jul 10;244(13):3507–3513. [PubMed] [Google Scholar]
- Courtois-Verniquet F., Douce R. Lack of aconitase in glyoxysomes and peroxisomes. Biochem J. 1993 Aug 15;294(Pt 1):103–107. doi: 10.1042/bj2940103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Distel B., Veenhuis M., Tabak H. F. Import of alcohol oxidase into peroxisomes of Saccharomyces cerevisiae. EMBO J. 1987 Oct;6(10):3111–3116. doi: 10.1002/j.1460-2075.1987.tb02620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Erdmann R., Veenhuis M., Mertens D., Kunau W. H. Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5419–5423. doi: 10.1073/pnas.86.14.5419. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Evers M. E., Höhfeld J., Kunau W. H., Harder W., Veenhuis M. Physiological studies on the utilization of oleic acid by Saccharomyces cerevisiae in relation to microbody development. FEMS Microbiol Lett. 1991 Dec 15;69(1):73–78. doi: 10.1016/0378-1097(91)90649-u. [DOI] [PubMed] [Google Scholar]
- Fernández E., Moreno F., Rodicio R. The ICL1 gene from Saccharomyces cerevisiae. Eur J Biochem. 1992 Mar 15;204(3):983–990. doi: 10.1111/j.1432-1033.1992.tb16720.x. [DOI] [PubMed] [Google Scholar]
- Gietl C., Faber K. N., van der Klei I. J., Veenhuis M. Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3151–3155. doi: 10.1073/pnas.91.8.3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Glover J. R., Andrews D. W., Subramani S., Rachubinski R. A. Mutagenesis of the amino targeting signal of Saccharomyces cerevisiae 3-ketoacyl-CoA thiolase reveals conserved amino acids required for import into peroxisomes in vivo. J Biol Chem. 1994 Mar 11;269(10):7558–7563. [PubMed] [Google Scholar]
- Gould S. J., Keller G. A., Hosken N., Wilkinson J., Subramani S. A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol. 1989 May;108(5):1657–1664. doi: 10.1083/jcb.108.5.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heupel R., Markgraf T., Robinson D. G., Heldt H. W. Compartmentation studies on spinach leaf peroxisomes : evidence for channeling of photorespiratory metabolites in peroxisomes devoid of intact boundary membrane. Plant Physiol. 1991 Jul;96(3):971–979. doi: 10.1104/pp.96.3.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- KORNBERG H. L., KREBS H. A. Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle. Nature. 1957 May 18;179(4568):988–991. doi: 10.1038/179988a0. [DOI] [PubMed] [Google Scholar]
- Kawamoto S., Tanaka A., Yamamura M., Teranishi Y., Fukui S. Microbody of n-alkane-grown yeast. Enzyme localization in the isolated microbody. Arch Microbiol. 1977 Feb 4;112(1):1–8. doi: 10.1007/BF00446647. [DOI] [PubMed] [Google Scholar]
- Kionka C., Kunau W. H. Inducible beta-oxidation pathway in Neurospora crassa. J Bacteriol. 1985 Jan;161(1):153–157. doi: 10.1128/jb.161.1.153-157.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kragler F., Langeder A., Raupachova J., Binder M., Hartig A. Two independent peroxisomal targeting signals in catalase A of Saccharomyces cerevisiae. J Cell Biol. 1993 Feb;120(3):665–673. doi: 10.1083/jcb.120.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Liu H., Tan X., Veenhuis M., McCollum D., Cregg J. M. An efficient screen for peroxisome-deficient mutants of Pichia pastoris. J Bacteriol. 1992 Aug;174(15):4943–4951. doi: 10.1128/jb.174.15.4943-4951.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- López-Boado Y. S., Herrero P., Fernández T., Fernández R., Moreno F. Glucose-stimulated phosphorylation of yeast isocitrate lyase in vivo. J Gen Microbiol. 1988 Sep;134(9):2499–2505. doi: 10.1099/00221287-134-9-2499. [DOI] [PubMed] [Google Scholar]
- Marzioch M., Erdmann R., Veenhuis M., Kunau W. H. PAS7 encodes a novel yeast member of the WD-40 protein family essential for import of 3-oxoacyl-CoA thiolase, a PTS2-containing protein, into peroxisomes. EMBO J. 1994 Oct 17;13(20):4908–4918. doi: 10.1002/j.1460-2075.1994.tb06818.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matsuoka M., McFadden B. A. Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in Escherichia coli. J Bacteriol. 1988 Oct;170(10):4528–4536. doi: 10.1128/jb.170.10.4528-4536.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McCammon M. T., Veenhuis M., Trapp S. B., Goodman J. M. Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae. J Bacteriol. 1990 Oct;172(10):5816–5827. doi: 10.1128/jb.172.10.5816-5827.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McCollum D., Monosov E., Subramani S. The pas8 mutant of Pichia pastoris exhibits the peroxisomal protein import deficiencies of Zellweger syndrome cells--the PAS8 protein binds to the COOH-terminal tripeptide peroxisomal targeting signal, and is a member of the TPR protein family. J Cell Biol. 1993 May;121(4):761–774. doi: 10.1083/jcb.121.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Motley A., Hettema E., Distel B., Tabak H. Differential protein import deficiencies in human peroxisome assembly disorders. J Cell Biol. 1994 May;125(4):755–767. doi: 10.1083/jcb.125.4.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Olsen L. J., Ettinger W. F., Damsz B., Matsudaira K., Webb M. A., Harada J. J. Targeting of glyoxysomal proteins to peroxisomes in leaves and roots of a higher plant. Plant Cell. 1993 Aug;5(8):941–952. doi: 10.1105/tpc.5.8.941. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Onyeocha I., Behari R., Hill D., Baker A. Targeting of castor bean glyoxysomal isocitrate lyase to tobacco leaf peroxisomes. Plant Mol Biol. 1993 Jun;22(3):385–396. doi: 10.1007/BF00015970. [DOI] [PubMed] [Google Scholar]
- Robertson E. F., Hoyt J. C., Reeves H. C. Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli. J Biol Chem. 1988 Feb 15;263(5):2477–2482. [PubMed] [Google Scholar]
- Small G. M., Szabo L. J., Lazarow P. B. Acyl-CoA oxidase contains two targeting sequences each of which can mediate protein import into peroxisomes. EMBO J. 1988 Apr;7(4):1167–1173. doi: 10.1002/j.1460-2075.1988.tb02927.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Subramani S. Protein import into peroxisomes and biogenesis of the organelle. Annu Rev Cell Biol. 1993;9:445–478. doi: 10.1146/annurev.cb.09.110193.002305. [DOI] [PubMed] [Google Scholar]
- Swinkels B. W., Gould S. J., Subramani S. Targeting efficiencies of various permutations of the consensus C-terminal tripeptide peroxisomal targeting signal. FEBS Lett. 1992 Jun 29;305(2):133–136. doi: 10.1016/0014-5793(92)80880-p. [DOI] [PubMed] [Google Scholar]
- Tsukamoto T., Hata S., Yokota S., Miura S., Fujiki Y., Hijikata M., Miyazawa S., Hashimoto T., Osumi T. Characterization of the signal peptide at the amino terminus of the rat peroxisomal 3-ketoacyl-CoA thiolase precursor. J Biol Chem. 1994 Feb 25;269(8):6001–6010. [PubMed] [Google Scholar]
- Van der Leij I., Van den Berg M., Boot R., Franse M., Distel B., Tabak H. F. Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure. J Cell Biol. 1992 Oct;119(1):153–162. doi: 10.1083/jcb.119.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Waterham H. R., Titorenko V. I., Haima P., Cregg J. M., Harder W., Veenhuis M. The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals. J Cell Biol. 1994 Nov;127(3):737–749. doi: 10.1083/jcb.127.3.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]