Polypeptide composition of envelopes of spinach chloroplasts: two major proteins occupy 90% of outer envelope...

Outer and inner envelope membranes of spinach chloroplasts were isolated using floatation centrifugation followed by sedimentation sucrose density gradient centrifugation after disruption of intact chloroplasts by freezing and thawing. Two major fractions with buoyant densities of 1.11 and 1.08 g cm-3 and a minor fraction with a density of 1.15 g cm-3 were obtained. They were identified as inner and outer envelope and thylakoid fractions, respectively by analyzing their polypeptide composition by high-resolution SDS-PAGE and the N-terminal sequences of their protein components. Due to the refinement of the isolation procedure, most of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which had always been observed as a contaminant, was eliminated from the outer envelope fraction. Application of high-resolution SDS-PAGE revealed that this fraction was rich in the low-molecular-mass outer envelope protein, E6.7 [Salomon et al. (1990) Proc. Natl. Acad. Sci. USA 87: 5778] and a protein with a molecular mass of 15 kDa which is homologous to the 16 kDa outer envelope protein of pea [Pohlmeyer et al. (1997) Proc. Natl. Acad. Sci. USA 94: 9504]. The two proteins account for 90% of the total proteins present in outer envelope membranes. Proteins which are suggested to function in translocation of nuclear-encoded polypeptides were not identified in the envelopes from spinach in the present study. Differences in the protein composition of outer envelope membranes are discussed based on the developemental stages of chloroplasts.