A comparison of chloroplast membrane surfaces visualized by freeze-etch and negative staining techniques; and ultrastructural characterization of membrane fractions obtained from digitonin-treated spinach chloroplasts

Abstract

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.