Abstract
Previous work has shown that the firefly (Photinus pyralis) luciferase contains a C-terminal peroxisomal targeting signal consisting of the tripeptide Ser-Lys-Leu. This report describes the microinjection of two proteins, (i) luciferase and (ii) albumin conjugated to a peptide ending in the sequence Ser-Lys-Leu, into mammalian cells grown in tissue culture. Following microinjection, incubation of the cells at 37 degrees C resulted in peroxisomal transport of these exogenous proteins into catalase-containing vesicles. The translocation was both time and temperature dependent. The transport could be inhibited by coinjection of synthetic peptides bearing various peroxisomal targeting signal motifs. These proteins could be transported into peroxisomes in normal human fibroblast cell lines but not in cell lines derived from patients with Zellweger syndrome. These results demonstrate that microinjection of peroxisomal proteins yields an authentic in vivo system with which to study peroxisomal transport. Furthermore, these results reveal that the process of peroxisomal transport does not involve irreversible modification of the protein, that artificial hybrid substrates can be transported and used as tools to study peroxisomal transport, and that the defect in Zellweger syndrome is indeed the inability to transport proteins containing the Ser-Lys-Leu targeting signal into the peroxisomal lumen.
Full text
PDF










Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Brul S., Westerveld A., Strijland A., Wanders R. J., Schram A. W., Heymans H. S., Schutgens R. B., van den Bosch H., Tager J. M. Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis. J Clin Invest. 1988 Jun;81(6):1710–1715. doi: 10.1172/JCI113510. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feramisco J. R., Clark R., Wong G., Arnheim N., Milley R., McCormick F. Transient reversion of ras oncogene-induced cell transformation by antibodies specific for amino acid 12 of ras protein. Nature. 1985 Apr 18;314(6012):639–642. doi: 10.1038/314639a0. [DOI] [PubMed] [Google Scholar]
- Feramisco J. R. Microinjection of fluorescently labeled alpha-actinin into living fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3967–3971. doi: 10.1073/pnas.76.8.3967. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fujiki Y., Lazarow P. B. Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro. J Biol Chem. 1985 May 10;260(9):5603–5609. [PubMed] [Google Scholar]
- Fujiki Y., Rachubinski R. A., Lazarow P. B. Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free polysomes. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7127–7131. doi: 10.1073/pnas.81.22.7127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fujiki Y., Rachubinski R. A., Mortensen R. M., Lazarow P. B. Synthesis of 3-ketoacyl-CoA thiolase of rat liver peroxisomes on free polyribosomes as a larger precursor. Induction of thiolase mRNA activity by clofibrate. Biochem J. 1985 Mar 15;226(3):697–704. doi: 10.1042/bj2260697. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldman B. M., Blobel G. Biogenesis of peroxisomes: intracellular site of synthesis of catalase and uricase. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5066–5070. doi: 10.1073/pnas.75.10.5066. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gould S. G., Keller G. A., Subramani S. Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J Cell Biol. 1987 Dec;105(6 Pt 2):2923–2931. doi: 10.1083/jcb.105.6.2923. [DOI] [PMC free article] [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]
- Gould S. J., Keller G. A., Schneider M., Howell S. H., Garrard L. J., Goodman J. M., Distel B., Tabak H., Subramani S. Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J. 1990 Jan;9(1):85–90. doi: 10.1002/j.1460-2075.1990.tb08083.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gould S. J., Keller G. A., Subramani S. Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. J Cell Biol. 1988 Sep;107(3):897–905. doi: 10.1083/jcb.107.3.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gould S. J., Krisans S., Keller G. A., Subramani S. Antibodies directed against the peroxisomal targeting signal of firefly luciferase recognize multiple mammalian peroxisomal proteins. J Cell Biol. 1990 Jan;110(1):27–34. doi: 10.1083/jcb.110.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Imanaka T., Small G. M., Lazarow P. B. Translocation of acyl-CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential. J Cell Biol. 1987 Dec;105(6 Pt 2):2915–2922. doi: 10.1083/jcb.105.6.2915. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kamijo K., Taketani S., Yokota S., Osumi T., Hashimoto T. The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein superfamily. J Biol Chem. 1990 Mar 15;265(8):4534–4540. [PubMed] [Google Scholar]
- Lamb N. J., Fernandez A., Conti M. A., Adelstein R., Glass D. B., Welch W. J., Feramisco J. R. Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase. J Cell Biol. 1988 Jun;106(6):1955–1971. doi: 10.1083/jcb.106.6.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lazarow P. B., Black V., Shio H., Fujiki Y., Hajra A. K., Datta N. S., Bangaru B. S., Dancis J. Zellweger syndrome: biochemical and morphological studies on two patients treated with clofibrate. Pediatr Res. 1985 Dec;19(12):1356–1364. doi: 10.1203/00006450-198512000-00030. [DOI] [PubMed] [Google Scholar]
- Lazarow P. B., Fujiki Y., Small G. M., Watkins P., Moser H. Presence of the peroxisomal 22-kDa integral membrane protein in the liver of a person lacking recognizable peroxisomes (Zellweger syndrome). Proc Natl Acad Sci U S A. 1986 Dec;83(23):9193–9196. doi: 10.1073/pnas.83.23.9193. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meinkoth J. L., Ji Y., Taylor S. S., Feramisco J. R. Dynamics of the distribution of cyclic AMP-dependent protein kinase in living cells. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9595–9599. doi: 10.1073/pnas.87.24.9595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miura S., Mori M., Takiguchi M., Tatibana M., Furuta S., Miyazawa S., Hashimoto T. Biosynthesis and intracellular transport of enzymes of peroxisomal beta-oxidation. J Biol Chem. 1984 May 25;259(10):6397–6402. [PubMed] [Google Scholar]
- Miyazawa S., Osumi T., Hashimoto T., Ohno K., Miura S., Fujiki Y. Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus. Mol Cell Biol. 1989 Jan;9(1):83–91. doi: 10.1128/mcb.9.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rachubinski R. A., Fujiki Y., Mortensen R. M., Lazarow P. B. Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver. J Cell Biol. 1984 Dec;99(6):2241–2246. doi: 10.1083/jcb.99.6.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Riabowol K. T., Vosatka R. J., Ziff E. B., Lamb N. J., Feramisco J. R. Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells. Mol Cell Biol. 1988 Apr;8(4):1670–1676. doi: 10.1128/mcb.8.4.1670. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robbi M., Lazarow P. B. Synthesis of catalase in two cell-free protein-synthesizing systems and in rat liver. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4344–4348. doi: 10.1073/pnas.75.9.4344. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roggenkamp R., Janowicz Z., Stanikowski B., Hollenberg C. P. Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet. 1984;194(3):489–493. doi: 10.1007/BF00425563. [DOI] [PubMed] [Google Scholar]
- Santos M. J., Imanaka T., Shio H., Lazarow P. B. Peroxisomal integral membrane proteins in control and Zellweger fibroblasts. J Biol Chem. 1988 Jul 25;263(21):10502–10509. [PubMed] [Google Scholar]
- Santos M. J., Imanaka T., Shio H., Small G. M., Lazarow P. B. Peroxisomal membrane ghosts in Zellweger syndrome--aberrant organelle assembly. Science. 1988 Mar 25;239(4847):1536–1538. doi: 10.1126/science.3281254. [DOI] [PubMed] [Google Scholar]
- Santos M. J., Ojeda J. M., Garrido J., Leighton F. Peroxisomal organization in normal and cerebrohepatorenal (Zellweger) syndrome fibroblasts. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6556–6560. doi: 10.1073/pnas.82.19.6556. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Small G. M., Santos M. J., Imanaka T., Poulos A., Danks D. M., Moser H. W., Lazarow P. B. Peroxisomal integral membrane proteins in livers of patients with Zellweger syndrome, infantile Refsum's disease and X-linked adrenoleukodystrophy. J Inherit Metab Dis. 1988;11(4):358–371. doi: 10.1007/BF01800425. [DOI] [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]
- Tolbert N. E. Metabolic pathways in peroxisomes and glyoxysomes. Annu Rev Biochem. 1981;50:133–157. doi: 10.1146/annurev.bi.50.070181.001025. [DOI] [PubMed] [Google Scholar]
- Wanders R. J., Kos M., Roest B., Meijer A. J., Schrakamp G., Heymans H. S., Tegelaers W. H., van den Bosch H., Schutgens R. B., Tager J. M. Activity of peroxisomal enzymes and intracellular distribution of catalase in Zellweger syndrome. Biochem Biophys Res Commun. 1984 Sep 28;123(3):1054–1061. doi: 10.1016/s0006-291x(84)80240-5. [DOI] [PubMed] [Google Scholar]
- Wolvetang E. J., Tager J. M., Wanders R. J. Latency of the peroxisomal enzyme acyl-CoA:dihydroxyacetonephosphate acyltransferase in digitonin-permeabilized fibroblasts: the effect of ATP and ATPase inhibitors. Biochem Biophys Res Commun. 1990 Aug 16;170(3):1135–1143. doi: 10.1016/0006-291x(90)90511-k. [DOI] [PubMed] [Google Scholar]
- Zellweger H. Peroxisomes and peroxisomal disorders. Ala J Med Sci. 1988 Jan;25(1):54–58. [PubMed] [Google Scholar]
- del Valle R., Soto U., Necochea C., Leighton F. Detection of an ATPase activity in rat liver peroxisomes. Biochem Biophys Res Commun. 1988 Nov 15;156(3):1353–1359. doi: 10.1016/s0006-291x(88)80781-2. [DOI] [PubMed] [Google Scholar]