Light-independent degradation of stromal proteins in intact chloroplasts isolated from Pisum sativum L. leaves: requirement for divalent cations

Abstract

Intact chloroplasts were isolated from mature pea (Pisum sativum L.) leaves in order to study the degradation of several stromal proteins in organello. Changes in the abundances of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), glutamine synthetase (EC 6.3.1.2) and ferredoxin-dependent glutamine:a-ketoglutarate aminotransferase (glutamate synthase; EC 1.4.7.1) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie-staining of the gels or immunoblotting using speci®c antibodies for the dierent enzymes. Degradation of several stromal proteins was strongly stimulated when intact chloroplasts were incubated in the light in the presence of dithiothreitol. Since free radicals may arti®cially accumulate in the chloroplast under such conditions and interfere with the stability of stromal proteins, the general relevance of these processes remains questionable. In the absence of light, proteolysis proceeded slowly in isolated chloroplasts and was not stimulated by dithiothreitol. Inhibition by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or excess zinc ions as well as the requirement for divalent cations suggested that a zinccontaining metalloprotease participated in this process. Furthermore, light-independent degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase was enhanced in chloroplasts isolated from leaves in which senescence was accelerated by nitrogen starvation. Our results indicate that light-independent stromal protein degradation in intact chloroplasts may be analogous to proteolysis that occurs in intact leaves during senescence.

Proteolytic activity and nitrogen remobilisation in senescing leaves of phenological forms of Fagus sylvatica

“…Endopeptidases are essential for the first stages of peptide bond cleavage and are therefore important for the initiation of the catabolism of chloroplast proteins, primarily Rubisco (Hortensteiner and Feller 2002). Additionally, aminopeptidase activity in chloroplasts reportedly contributes to the complete degradation of stromal proteins by degrading the peptides generated by endopeptidase activity (Roulin and Feller 1998). Carboxypeptidases, which have not been detected in intact plastids (Feller and Fischer 1994), play a role in the degradation of plastid proteins in lytic vacuoles (Hortensteiner and Feller 2002;Yang et al 2004).…”

The Degradation of the Large Subunit of Ribulose-1,5-bisphosphate Carboxylase/oxygenase into the 44-kDa Fragment in the Lysates of Chloroplasts Incubated in Darkness

“…However, protein purification or gene identification of EP1 has not yet been carried out and its physiological role is still unclear. Roulin and Feller (1998) studied light-independent degradation of several stromal proteins including Rubisco in intact chloroplasts from pea leaves and suggested that EP1 might participate in the degradation process because the addition of metal-ion chelators suppressed the decrease in the amounts of stromal proteins. However, the degradation process of Rubisco has not been seriously studied and no degradation products of LSU have been reported in chloroplasts incubated in darkness.…”

Senescence in wheat leaves: is a cysteine endopeptidase involved in the degradation of the large subunit of Rubisco?

“…A plastidial zinc protease able to hydrolyze Rubisco has been reported by Bushnell et al (1993). Further evidence for the involvement of a zinc-containing metalloprotease in the degradation of stromal proteins inside the plastids was obtained from experiments with intact chloroplasts isolated from pea leaves (Roulin and Feller 1998a). A role for activated oxygen species in the degradation of Rubisco in intact chloroplasts seems likely.…”