Abstract
Outer envelope membranes were isolated from purified chloroplasts of pea leaves. The sidedness of the vesicles was analyzed by (i) aqueous polymer-two phase partitioning, (ii) the effect of limited proteolysis on the outer-envelope proteins (OEP) 86 and OEP 7 in intact organelles and isolated membranes, (iii) fluorescence-microscopy and finally (iv) binding of precursor polypeptides to isolated outer-membrane vesicles. The results demonstrate that purified outer envelope membranes occur largely (>90%) as right-side-out vesicles.
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Abbreviations
- FITC:
-
fluorescein isothiocyanate
- IEP:
-
Pinner-envelope protein
- OE:
-
outer-envelope protein
- pSSU:
-
precursor form of the small subunit of ribulose bisphosphate carboxylaseoxygenase
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
References
Albertsson, P.-Å. (1971) Partition of cell particles and makro molecules, Wiley, New York
Albertsson, P.-Å., Andersson, B., Larsson, C., Akerlund, H.-E. (1982) Phase-partition method for purification and analysis of cell organelles and membrane vesicles. Methods Biochem. Anal. 28, 115–150
Andersson, B. (1986) Characterization of the thylakoid membrane by subfractionation analysis. Methods Enzymol. 118, 325–338
Arnon, D.J. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15
Block, M.A., Dorne, A.-J., Joyard, J., Douce, R. (1983a) Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. J. Biol. Chem. 258, 13273–13280
Block, M.A., Dorne, A.-J., Joyard, J., Douce, R. (1983b) Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. J. Biol. Chem. 258, 13281–13286
Bonner, W.M., Laskey, R.A. (1974) A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83–88
Cline, K., Andrews, J., Mersey, B., Newcomb, B.H., Keegstra, K. (1981) Separation and characterization of inner and outer envelope membranes of pea chloroplasts. Proc. Natl. Acad. Sci. USA 78, 3595–3999
Cline, K., Werner-Washburne, M., Andrews, J., Keegstra, K. (1984) Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. Plant Physiol. 75, 675–678
Cline, K., Keegstra, K., Staehelin, L.A. (1985) Freeze fracture electron microscopic analysis of ultrarapidly frozen envelope membranes on intact chloroplasts and after purification. Protoplasma 125, 111–123
Douce, R., Block, M.A., Dorne, A.J., Joyard, J. (1984) The plastid envelope membranes: their structure, composition and role in chloroplast biogenesis. Subcell. Biochem. 10, 1–84
Flügge, U.-I., Benz, R. (1984) Pore forming activity in the outer membrane of the chloroplast envelope. FEBS Lett. 169, 85–89
Flügge, U.-I., Heldt, H.W. (1981) The phosphate translocator of the chloroplast envelope: isolation of the carrier protein and reconstitution of transport. Biochim. Biophys. Acta 638, 296–304
Harlow, E., Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory
Joyard, J., Billecocq, A., Bartlett, S. G., Block, M. A., Chua, N. H., Douce, R. (1983) Localization of polypeptides to the cytosolic side of the outer envelope membrane of spinach chloroplasts. J. Biol. Chem. 258, 10000–10006
Keegstra, K., Youssif, A.E. (1986) Isolation and characterization of chloroplast envelope membranes. Methods Enzymol. 118, 316–325
Keegstra, K., Olsen, L.J., Theg, S.M. (1989) Chloroplastic precursors and their transport across the envelope membranes. Annu. Rev. Plant Physiol. 40, 471–501
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680–685
Larsson, C., Kjellbohm, P., Widell, S., Lundberg, T. (1984) Sidedness of plant plasma membrane vesicles purified by partitioning in aqueous two phase systems. FEBS Lett. 171, 271–276
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275
Lubben, T.H., Keegstra, K. (1986) Efficient in vitro import of a cytosolic heat shock protein into pea chloroplasts. Proc. Natl. Acad. Sci. USA 83, 5502–5506
Nakatani, A.J., Barber, J. (1977) An improved method for isolating chloroplasts retaining their outer membranes. Biochim. Biophys. Acta 416, 510–512
Salomon, M., Fischer, K., Flügge, U.-I., Soll, J. (1990) Sequence analysis and protein import studies of an outer chloroplast envelope polypeptide. Proc. Natl. Acad. Sci. USA 87, 5778–5782
Soll, J. (1987) α-Tocopherol and plastoquinone synthesis in chloroplast membranes. Methods Enzymol. 148, 383–392
Soll, J., Berger, V., Bennett, J. (1989) Adenylate effects on protein phosphorylation in the interenvelope lumen of pea chloroplasts. Planta 177, 393–400
Towbin, A., Staehlin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354
Waegemann, K., Soll, J. (1991) Characterization of the protein import apparatus in isolated outer envelopes of chloroplasts. Plant J. 1, 149–159
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We thank P. Å. Albertsson, Lund, Sweden, for introducing one of us (S. E.) to the technique of phase partitioning. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 246) and Fonds der Chemischen Industrie.
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Waegemann, K., Eichacker, S. & Soll, J. Outer envelope membranes from chloroplasts are isolated as right-side-out vesicles. Planta 187, 89–94 (1992). https://doi.org/10.1007/BF00201628
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DOI: https://doi.org/10.1007/BF00201628