Purification and characterization of a 22‐kDa protein in chloroplasts from green spores of the fern Osmunda japonica

Abstract

Changes in the polypeptide composition of chloroplasts were investigated during germination of green spores of the fern Osmunda japonica. The polypeptide composition of chloroplasts was appreciably changed during a germination time course of 48 h. Levels of five polypeptides with apparent molecular masses of 47, 44, 42, 22 and 18.5 kDa in the soluble fraction of chloroplasts and three polypeptides with molecular masses of 24, 22 and 15 kDa in the thylakoid membranes decreased during germination. In contrast, no decrease of chloroplast polypeptides was observed in the spores incubated with cycloheximide for 48 h. A new 22‐kDa protein was isolated from thylakoid membranes of spores and the amino‐terminal sequence of the purified protein was determined. High levels of alanine and glycine were found in the basic protein (pl > 10.3). This protein, with a native molecular mass of 80 kDa, was characterized by a subunit band observed at a molecular mass of 22 kDa on SDS‐PAGE and by the disappearance of the band during spore germination. Protease activity against the 22‐kDa protein was observed in an extract prepared from chloroplasts of quiescent spores. A hypothetical cytosolic proteinaceous factor is implicated in the regulation of protein degradation in chloroplasts.

Characterization of a protease that acts specifically on the 22‐kDa protein in thylakoid membranes from green spores of the fern Osmunda japonica

“…Chloroplasts and thylakoid membranes were prepared from the quiescent spores, as previously described ( Inoue et al 1995). To isolate thylakoid membranes, chloroplasts (0.5 mg Chl ml −1 ) were homogenized in 10 m M Tris‐HCl (pH 7.4), centrifuged at 25 000 g for 20 min, and the pellet was resuspended in 50 m M Tris‐HCl (pH 7.4), 10 m M NaCl and 5 m M MgCl 2 to a chlorophyll concentration of 0.5 mg Chl ml −1 .…”

“…Chlorophyll concentration was determined according to Arnon (1949). For spore germination, sterilized spores of 100 mg were incubated in 50 ml of 1/5 diluted Murashige and Skoog's mineral medium ( Murashige and Skoog 1962), as previously described ( Inoue et al 1995).…”

“…When a purified 22‐kDa protein was necessary in Fig. 7, the preparation was further purified by a combination of hydroxylapatite chromatography, G3000SW size‐fractionation HPLC, and reversed‐phase HPLC, as previously described ( Inoue et al 1995).…”

“…Although some studies have been published on protein synthesis in chloroplasts isolated from gametophytes ( Raghavan and DeMaggio 1971) and on DNA synthesis in chloroplasts during germination ( Raghavan 1993), very few studies have focused on protein degradation in the chloroplasts of green spores during germination. Recently, we reported that five polypeptides in the soluble fraction of chloroplasts and three polypeptides in the thylakoid membranes in chloroplasts from green spores of the fern Osmunda japonica decreased during spore germination ( Inoue et al 1995). We focused on the thylakoid proteins.…”

“…An important step in understanding the molecular basis for the degradation mechanism of the thylakoid protein during germination is to isolate both the substrate protein and its specific protease. The 22‐kDa protein was purified in our previous study ( Inoue et al 1995). The purpose of this study is to isolate a specific protease for the 22‐kDa protein from thylakoid membranes and determine the properties of the enzyme.…”