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. 1997 Aug;9(8):1481–1493. doi: 10.1105/tpc.9.8.1481

Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting.

B M Abell 1, L A Holbrook 1, M Abenes 1, D J Murphy 1, M J Hills 1, M M Moloney 1
PMCID: PMC157013  PMID: 9286116

Abstract

An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N- and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-beta-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-beta-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.

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Selected References

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  1. Borel A. C., Simon S. M. Biogenesis of polytopic membrane proteins: membrane segments assemble within translocation channels prior to membrane integration. Cell. 1996 May 3;85(3):379–389. doi: 10.1016/s0092-8674(00)81116-2. [DOI] [PubMed] [Google Scholar]
  2. Campos N., Boronat A. Targeting and topology in the membrane of plant 3-hydroxy-3-methylglutaryl coenzyme A reductase. Plant Cell. 1995 Dec;7(12):2163–2174. doi: 10.1105/tpc.7.12.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chrispeels M. J., Raikhel N. V. Short peptide domains target proteins to plant vacuoles. Cell. 1992 Feb 21;68(4):613–616. doi: 10.1016/0092-8674(92)90134-x. [DOI] [PubMed] [Google Scholar]
  4. Chuck S. L., Lingappa V. R. Pause transfer: a topogenic sequence in apolipoprotein B mediates stopping and restarting of translocation. Cell. 1992 Jan 10;68(1):9–21. doi: 10.1016/0092-8674(92)90202-n. [DOI] [PubMed] [Google Scholar]
  5. Chuck S. L., Yao Z., Blackhart B. D., McCarthy B. J., Lingappa V. R. New variation on the translocation of proteins during early biogenesis of apolipoprotein B. Nature. 1990 Jul 26;346(6282):382–385. doi: 10.1038/346382a0. [DOI] [PubMed] [Google Scholar]
  6. Do H., Falcone D., Lin J., Andrews D. W., Johnson A. E. The cotranslational integration of membrane proteins into the phospholipid bilayer is a multistep process. Cell. 1996 May 3;85(3):369–378. doi: 10.1016/s0092-8674(00)81115-0. [DOI] [PubMed] [Google Scholar]
  7. Gordon D. A., Jamil H., Gregg R. E., Olofsson S. O., Borén J. Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step. J Biol Chem. 1996 Dec 20;271(51):33047–33053. doi: 10.1074/jbc.271.51.33047. [DOI] [PubMed] [Google Scholar]
  8. Hills M. J., Watson M. D., Murphy D. J. Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.). Planta. 1993 Jan;189(1):24–29. doi: 10.1007/BF00201339. [DOI] [PubMed] [Google Scholar]
  9. Huang A. H. Oleosins and oil bodies in seeds and other organs. Plant Physiol. 1996 Apr;110(4):1055–1061. doi: 10.1104/pp.110.4.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Höfte H., Chrispeels M. J. Protein sorting to the vacuolar membrane. Plant Cell. 1992 Aug;4(8):995–1004. doi: 10.1105/tpc.4.8.995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Innerarity T. L., Borén J., Yamanaka S., Olofsson S. O. Biosynthesis of apolipoprotein B48-containing lipoproteins. Regulation by novel post-transcriptional mechanisms. J Biol Chem. 1996 Feb 2;271(5):2353–2356. doi: 10.1074/jbc.271.5.2353. [DOI] [PubMed] [Google Scholar]
  12. Iturriaga G., Jefferson R. A., Bevan M. W. Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco. Plant Cell. 1989 Mar;1(3):381–390. doi: 10.1105/tpc.1.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klein T. M., Harper E. C., Svab Z., Sanford J. C., Fromm M. E., Maliga P. Stable genetic transformation of intact Nicotiana cells by the particle bombardment process. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8502–8505. doi: 10.1073/pnas.85.22.8502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Knott T. J., Pease R. J., Powell L. M., Wallis S. C., Rall S. C., Jr, Innerarity T. L., Blackhart B., Taylor W. H., Marcel Y., Milne R. Complete protein sequence and identification of structural domains of human apolipoprotein B. Nature. 1986 Oct 23;323(6090):734–738. doi: 10.1038/323734a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Lee K., Bih F. Y., Learn G. H., Ting J. T., Sellers C., Huang A. H. Oleosins in the gametophytes of Pinus and Brassica and their phylogenetic relationship with those in the sporophytes of various species. Planta. 1994;193(3):461–469. doi: 10.1007/BF00201827. [DOI] [PubMed] [Google Scholar]
  17. Lee K., Ratnayake C., Huang A. H. Genetic dissection of the co-expression of genes encoding the two isoforms of oleosins in the oil bodies of maize kernel. Plant J. 1995 Apr;7(4):603–611. doi: 10.1046/j.1365-313x.1995.7040603.x. [DOI] [PubMed] [Google Scholar]
  18. Loer D. S., Herman E. M. Cotranslational Integration of Soybean (Glycine max) Oil Body Membrane Protein Oleosin into Microsomal Membranes. Plant Physiol. 1993 Mar;101(3):993–998. doi: 10.1104/pp.101.3.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Martoglio B., Hofmann M. W., Brunner J., Dobberstein B. The protein-conducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer. Cell. 1995 Apr 21;81(2):207–214. doi: 10.1016/0092-8674(95)90330-5. [DOI] [PubMed] [Google Scholar]
  20. McLeod R. S., Wang Y., Wang S., Rusiñol A., Links P., Yao Z. Apolipoprotein B sequence requirements for hepatic very low density lipoprotein assembly. Evidence that hydrophobic sequences within apolipoprotein B48 mediate lipid recruitment. J Biol Chem. 1996 Aug 2;271(31):18445–18455. doi: 10.1074/jbc.271.31.18445. [DOI] [PubMed] [Google Scholar]
  21. Murphy D. J. Structure, function and biogenesis of storage lipid bodies and oleosins in plants. Prog Lipid Res. 1993;32(3):247–280. doi: 10.1016/0163-7827(93)90009-l. [DOI] [PubMed] [Google Scholar]
  22. Napier J. A., Stobart A. K., Shewry P. R. The structure and biogenesis of plant oil bodies: the role of the ER membrane and the oleosin class of proteins. Plant Mol Biol. 1996 Aug;31(5):945–956. doi: 10.1007/BF00040714. [DOI] [PubMed] [Google Scholar]
  23. Ng D. T., Brown J. D., Walter P. Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J Cell Biol. 1996 Jul;134(2):269–278. doi: 10.1083/jcb.134.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Patel S. B., Grundy S. M. Interactions between microsomal triglyceride transfer protein and apolipoprotein B within the endoplasmic reticulum in a heterologous expression system. J Biol Chem. 1996 Aug 2;271(31):18686–18694. doi: 10.1074/jbc.271.31.18686. [DOI] [PubMed] [Google Scholar]
  25. Plant A. L., van Rooijen G. J., Anderson C. P., Moloney M. M. Regulation of an Arabidopsis oleosin gene promoter in transgenic Brassica napus. Plant Mol Biol. 1994 May;25(2):193–205. doi: 10.1007/BF00023237. [DOI] [PubMed] [Google Scholar]
  26. Qu R., Wang S. M., Lin Y. H., Vance V. B., Huang A. H. Characteristics and biosynthesis of membrane proteins of lipid bodies in the scutella of maize (Zea mays L.). Biochem J. 1986 Apr 1;235(1):57–65. doi: 10.1042/bj2350057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reizer J., Reizer A., Saier M. H., Jr The MIP family of integral membrane channel proteins: sequence comparisons, evolutionary relationships, reconstructed pathway of evolution, and proposed functional differentiation of the two repeated halves of the proteins. Crit Rev Biochem Mol Biol. 1993;28(3):235–257. doi: 10.3109/10409239309086796. [DOI] [PubMed] [Google Scholar]
  28. Ross J. H., Murphy D. J. Characterization of anther-expressed genes encoding a major class of extracellular oleosin-like proteins in the pollen coat of Brassicaceae. Plant J. 1996 May;9(5):625–637. doi: 10.1046/j.1365-313x.1996.9050625.x. [DOI] [PubMed] [Google Scholar]
  29. Spiess M., Lodish H. F. An internal signal sequence: the asialoglycoprotein receptor membrane anchor. Cell. 1986 Jan 17;44(1):177–185. doi: 10.1016/0092-8674(86)90496-4. [DOI] [PubMed] [Google Scholar]
  30. Thoyts P. J., Millichip M. I., Stobart A. K., Griffiths W. T., Shewry P. R., Napier J. A. Expression and in vitro targeting of a sunflower oleosin. Plant Mol Biol. 1995 Oct;29(2):403–410. doi: 10.1007/BF00043664. [DOI] [PubMed] [Google Scholar]
  31. Verwoerd T. C., Dekker B. M., Hoekema A. A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res. 1989 Mar 25;17(6):2362–2362. doi: 10.1093/nar/17.6.2362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wickner W. T., Lodish H. F. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. doi: 10.1126/science.4048938. [DOI] [PubMed] [Google Scholar]
  33. Yao Z., McLeod R. S. Synthesis and secretion of hepatic apolipoprotein B-containing lipoproteins. Biochim Biophys Acta. 1994 May 13;1212(2):152–166. doi: 10.1016/0005-2760(94)90249-6. [DOI] [PubMed] [Google Scholar]
  34. Yeung S. J., Chen S. H., Chan L. Ubiquitin-proteasome pathway mediates intracellular degradation of apolipoprotein B. Biochemistry. 1996 Oct 29;35(43):13843–13848. doi: 10.1021/bi9618777. [DOI] [PubMed] [Google Scholar]
  35. van Rooijen G. J., Moloney M. M. Structural requirements of oleosin domains for subcellular targeting to the oil body. Plant Physiol. 1995 Dec;109(4):1353–1361. doi: 10.1104/pp.109.4.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. van Rooijen G. J., Terning L. I., Moloney M. M. Nucleotide sequence of an Arabidopsis thaliana oleosin gene. Plant Mol Biol. 1992 Apr;18(6):1177–1179. doi: 10.1007/BF00047721. [DOI] [PubMed] [Google Scholar]

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