POLYPEPTIDE COMPOSITION OF ENVELOPES OF SPINACH-CHLOROPLASTS - 2 MAJOR PROTEINS OCCUPY 90-PERCENT OF OUTER ENVELOPE MEMBRANES

Outer and inner envelope membranes of spinach chloroplasts were isolat ed using floatation centrifugation followed by sedimentation sucrose d ensity gradient centrifugation after disruption of intact chloroplasts by freezing and thawing. Two major fractions with buoyant densities o f 1.11 and 1.08 g cm(-3) and a minor fraction viith a density of 1.15 g cm(-3) mere obtained. They were identified as inner and outer envelo pe and thylakoid fractions, respectively, by analyzing their polypepti de composition by high-resolution SDS-PAGE and the N-terminal sequence s of their protein components. Due to the refinement of the isolation procedure, most of the ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO), which had always been observed as a contaminant, was elimi nated from the outer envelope fraction. Application of high-resolution SDS-PAGE revealed that this fraction was rich in the low-molecular-ma ss outer envelope protein, E6.7 [Salomon ct al. (1990) PI oc. Natl. Ac ad. Sci. USA 87: 5778] and a protein with a molecular mass of 15 kDa w hich is homologous to the 16 kDa enter envelope protein of pea [Pohlme yer ct al, (1997) Proc. Natl. Acad. Sci, USA 94: 9504]. The two protei ns account for 90% of the total proteins present in enter envelope mem branes. Proteins which are suggested to function in translocation of n uclear-encoded polypeptides were not identified in the envelopes from spinach in the present study. Differences in the protein composition o f outer envelope membranes are discussed based on the developemental s tages of chloroplasts.