Lamellae-bound inclusions in isolated spinach chloroplasts I. ultrastructure and isolation

Abstract

Summary Intact spinach chloroplasts isolated from deribbed spinach leaves stored at 4°C in the dark contain intrathylakoidal inclusions in dilated parts of both grana lamellae and stroma lamellae. The substructure of these thylakoid-bound inclusions consists of amorphous or paracrystalline material. Alternating electron-dense and transparent lines arranged in a parallel array were observed when the inclusions were thin-sectioned perpendicular to the thylakoid plane. When the membrane-inclusions are cut parallel to the plane of the lamellae the contents of dilated membranes reveals a crystalline pattern of squares, arising from the intersection of two sets of parallel lines crossing at an angle of about 90°. Fibril-like linear structures with the same perodicity of the electron-dense lines were detected on the stroma-bound thylakoid surface. It is assumed that the structures observed inside and outside the thylakoids are identical and that they may permeate the thylakoid membrane from the stroma into the intralamellar lumen where crystal formation takes place. Moreover, an aggregation of membrane proteins facing the inner space of the thylakoid lumen to crystalline array is discussed as a reason causing crystal formation. Washing of the intact chloroplasts in a hypotonic medium resulted in a loss of stroma components. The structure of the lamellae-surrounded inclusions, however, remained unaltered after this step. Fractionation of the washed lamellae system in a discontinuous sucrose gradient yielded an inclusion-enriched membrane fraction that accounted for approximately 10% of the total chloroplast RuDPCase (E.C. 4.1.1.39) activity when lamellae were ruptured by French press treatment. After SDS-gel electrophoresis the polypeptide pattern of this fraction showed peptides identical in their electrophoretic mobility with those of RuDPCase. Moreover, ATPase was present in this fraction. The effects of washing with EDTA-solution, treatment with a French press or ultrasonication on the ultrastructure of the inclusion-containing membrane fraction are described.