Abstract
We have developed a fast and reliable method for the separation of two membrane fractions respectively enriched in outer and inner envelope membranes from isolated, intact, purified spinach chloroplasts kept in a hypertonic medium (0.6 M mannitol). This separation was achieved by osmotically shrinking the inner envelope membrane, thus widening the intermembrane space, and then subsequently removing the "loosened" outer envelope membrane by applying low pressure to the shrunken chloroplasts and slowly extruding them through the small aperture of a Yeda press under controlled conditions. By centrifugation of the mixture obtained through a discontinuous sucrose gradient, we were able to separate two membrane fractions having different densities (fraction 2 or light fraction, d = 1.08 g/cm3, and fraction 3 or heavy fraction, d = 1.13 g/cm3). The recent characterization of polypeptides localized on the outer envelope membrane from spinach chloroplasts, E10 and E24 (Joyard, J., Billecocq, A., Bartlett, S. G., Block, M. A., Chua, N.-H., and Douce, R. J. Biol. Chem., 258, 10000-10006) enabled us to characterize our two membrane fractions. Analyses of the polypeptides by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and immunoblotting have shown that fraction 2 (light fraction) was completely devoid of polypeptide E30, which is involved in the transport of phosphate across the inner envelope membrane, but was enriched in polypeptides E10 and E24. The reverse was true for fraction 3 (heavy fraction). Under these conditions, it is clear that fraction 2 is strongly enriched in outer envelope membrane whereas fraction 3 consisted mostly of inner envelope membrane. Indeed, by immunoelectrophoresis, we were able to demonstrate that, on a protein basis, fraction 2 contained about 90% of outer membrane, whereas fraction 3 contained about 80% of inner membrane. Further characterization of the outer envelope membrane was achieved by using thermolysin, a nonpenetrant protease.
Highlights
We have developed a fast and reliablme ethod for the tion of envelope membranes in a reasonably pure state [1]
As shownin suspension of intact spinach chloroplasts kept in a hypertoniTc able I, most of the chlorophyll was present in fraction 4 medium through the apertureof a Yeda press
From the area peptides [6] whereas E30isaninner envelope membrane under therockets, we calculated that, ona protein polypeptide [23,24,25], our results demonstrate that fraction 2 basis, fraction 2 contained approximately 90% of outer memis mostly devoid of inner envelope membrane but is strongly brane proteins, and fraction 3 approximately 80% of inner enrichedinouter envelope membrane.Fraction 3 reacted membrane proteins (Fig. 5)
Summary
From the Equipe deRecherche Associee au Centre National de la Recherche Scientifique No 847, Physiologie Cellulaire Vegetale, Departement de Recherche Fondamentale, Laboratoire de Biologie Vegetale, Centre d'Etudes Nucleaires et Uniuersite Scientifiqw et Medicale de Grenoble, 85X, F-38041 Grenoble-Cedex, France. In the pa1s0t years, the biochemhypertonic medium (0.6 M mannitol) This separation ical properties of the plastid envelope membranes have been was achieved by osmotically shrinking the inner en- elucidated in detail [1,2,3]. Thechloroplast envelope consists of two distinct membranes which together provide a flexible boundary between thechloroplastandthesurrounding cytosol [1].A gentle osmotic shock of intact andpurified chloroplasts, followed by density gradient centrifugation allows the efficient prepara-. Centrifugation for 90 min a t 23,000 rpm (Beckman L2 65 B, SW 27 rotor) resulted in the separation of membranes into two bands which were at the interfaceof the sucroselayers (Fig. 1). CIOICUPeS the shrunken chloroplasts remained intact after Yeda press treatment
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