Abstract
Oleosins of Brassica napus L. (oilseed rape) synthesized by in-vitro translation were found to be very efficiently targeted to microsomal membranes but only poorly translocated to oil bodies or emulsified oil. The use of other bilayer membranes as controls showed that this interaction was specific. The rate of oleosin synthesis in the presence of microsomes was enhanced about threefold, indicative of the involvement of the signal-recognition particle in the targeting process. There is no evidence for the cleavage of the protein during targeting and the protein sequence reveals no consensus cleavage site for the signal peptide. Protection experiments using Proteinase K revealed that about 6 kDa of the protein is exposed on the cytoplasmic side of the ER but the remainder is protected. Carbonate (pH 11) washing of microsomal membranes after in-vitro translation confirmed that oleosins have a domain which remains inserted in the ER rather than the protein being transported completely into the lumen of the ER. These results indicate that oleosins are transported via the ER prior to their accumulation on oil bodies.
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Abbreviations
- cDNA:
-
copy DNA
- SDS-PAGE:
-
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
References
Au, D.M.Y., Kang, A.S., Murphy, D.J. (1989) An immunologically related family of apolipoproteins associated with triacylglycerol storage in the Cruciferae. Arch. Biochem. Biophys 273, 516–526
Bergfield, R., Hong, Y.-N., Kühnl, T., Schopfer, P. (1978) Formation of oleosomes (storage lipid bodies) during embryogenesis and their breakdown during seedling development in cotyledons of Sinapis alba L. Planta 143, 297–307
Cummins, I., Murphy, D.J. (1990) Mechanism of oil body synthesis and maturation in developing seeds. In: Plant lipid biochemistry, structure and utilization, pp. 231–233, Quinn, P.J., Harwood, J.L., eds. Portland Press, London
Cummins, I., and Murphy, D.J. (1992) cDNA sequence of a sunflower oleosin and transcript tissue-specificity. Plant Mol. Biol. 19, 873–876
Ericson, M.L., Rödin, J., Lenman, M., Glimelius, K., Josefson, L.-G., Rask, L. (1986) Structure of the rapeseed 1.7S storage protein napin, and its precursor. J. Biol. Chem. 261, 14576–14581
Fernandez, D.E., Qu, R., Huang, A.H.C., Staehelin, L.A. (1988) Immunogold localization of the L3 protein of maize lipid bodies during germination and seedling growth. Plant Physiol. 86, 270–274
Fujiki, Y., Hubbard, S., Fowler, S., Lazarow, P.B. (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J. Cell Biol. 93, 97–102
Hatzopoulos, P., Franz, G., Choy, L., Sung, R.Z. (1990) Interaction of nuclear factors with upstream sequences of a lipid body membrane protein gene from carrot. Plant Cell. 2, 457–467
Hills, M.J., Beevers, H. (1987) Ca2+ stimulated neutral lipase activity in castor bean lipid bodies. Plant Physiol. 84, 272–276
Ichihara, K. (1982) Formation of oleosomes in maturing safflower seeds. Agric. Biochem. 46, 1767–1773
Kalinski, A., Loer, D.S., Weisemann, J.M., Matthews, B.F., Herman, E.M. (1991) Isoforms of soybean seed oil body membrane protein 24 kDa oleosin are encoded by closely related cDNAs. Plant Mol. Biol. 17, 1095–1098
Keddie, J.S., Edwards, E.-W., Gibbons, T., Shaw, C.H., Murphy, D.J. (1992) Sequence of an oleosin cDNA from Brassica napus. Plant Mol. Biol. 19, 1079–1083
Keddie, J.S., Hubner, G., Slocombe, S.P., Jarvis, R.P., Cummins, I., Edwards, E.W., Shaw, C.H., Murphy, D.J. (1992) Cloning and characterisation of an oleosin gene from Brassica napus. Plant Mol. Biol. 19, 443–453
Murphy, D.J. (1990) Storage lipid bodies in plants and other organisms. Progr. Lipid Res. 29, 299–324
Murphy, D.J., Cummins, I. (1989) Purification and immunogold localization of the major oil-body membrane protein of oilseed rape. Plant Science 60, 47–54
Murphy, D.J., Keen, J.N., O'Sullivan, J.N., Au, D.M.Y., Edwards, E.W., Jackson, P.J., Cummins, I., Gibbons, T., Shaw, C.H., Ryan, A.J. (1991) A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins. Biochim. Biophys. Acta 1088, 86–94
Lee, W.S., Tzen, J.T.C., Kridl, J.C., Rakde, S.E., Huang, A.H.C. (1991) Maize oleosin is correctly targeted to seed oil bodies in Brassica napus transformed with the maize oleosin gene. Proc. Natl. Acad. Sci. USA 88, 6181–6185
Nilsson, I., von Heijne, G. (1990) Fine-tuning the topology of a polytopic membrane protein: role of positively and negatively charged amino acids. Cell. 62, 1135–1141
Prescott, A., Martin, C. (1987) A rapid method for the quantitative assessment of levels of specific mRNAs in plants. Plant Mol. Biol. Reporter 4, 219–224
Pugsley, A.P. (1989) Protein targeting. Academic Press, San Diego
Qu, R., Huang, A.H.C. (1990) Oleosin kD18 on the surface of oil bodis in maize. Genomic and cDNA Sequences. J. Mol. Biol. 265, 2238–2243
Qu, R., Wang, S., Lin, Y., Vance, V.B., Huang, A.H.C. (1986) Characteristics and biosynthesis of membrane proteins of lipid bodies in the scutella of maize (Zea mays L.). Biochem. J. 235, 57–65
Schägger, H., von Jagow, G.V. (1987) Tricine sodium-dodecyl sulphate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1–100 kD. Anal. Biochem. 166, 368–379
Siegel, V., Walter, P. (1988) Functional dissection of the signal recognition particle. Trends Biochem. Sci. 13, 314–316
Vance, V.B., Huang, A.H.C. (1987) The major protein from lipid bodies of maize. J. Biol. Chem. 262, 11275–11279
Wanner, G., Formanek, H., Theimer, R.R. (1981) The ontogeny of lipid bodies (spherosomes) in plant cells. Planta 151, 109–123
Wanner, G., Theimer, R.R. (1978) Membranous appendices of spherosomes (oleosomes). Possible role in fat utilization in germinating oil seeds. Planta 140, 163–169
Ye, R.D., Wun, T.-C., Sadler, J.E. (1988) Mammalian protein secretion without signal peptide removal. J. Biol. Chem. 263, 4869–4875
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We gratefully acknowledge financial support from the Agriculture and Food Research Council and the John Innes Centre, Norwich for providing laboratory accommodation. We also thank James Keddie (J.I.C.) for his advice on the oleosin cDNA and Steve Rawsthorne (J.I.C.) and Martin Trick (J.I.C.) for their comments on the manuscript.
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Hills, M.J., Watson, M.D. & Murphy, D.J. Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.). Planta 189, 24–29 (1993). https://doi.org/10.1007/BF00201339
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DOI: https://doi.org/10.1007/BF00201339