Stromal protein phosphorylation in spinach (Spinacia oleracea) chloroplasts

Abstract

When intact spinach chloroplasts were supplied with [32P]Pi, stromal protein phosphorylation was found to occur in the dark. On illumination the thylakoid protein kinase was activated and the amount of label found in thylakoid proteins quickly exceeded that incorporated into stromal protein, such that the latter was found to account for only 10-15% of the total radioactivity bound to chloroplast proteins after 5 min illumination. The rate of phosphorylation of stromal polypeptides was unchanged by light. After SDS/polyacrylamide-gel electrophoresis, more than 15 labelled polypeptides of stromal origin were observed. A polypeptide with an Mr of approx. 70 000 had the highest specific activity of labelling. Both the large and small subunits of the ribulose-1,5-bisphosphate carboxylase were phosphorylated. The level of phosphorylation of stromal protein was increased by CO2 fixation in intact chloroplasts. This increase was not observed in the absence of NaHCO3 or in the presence of the phosphoribulokinase inhibitor DL-glyceraldehyde. These effects appeared to be largely due to changes in the phosphorylation state of the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Studies with the reconstituted chloroplast system showed that the thylakoid protein kinase(s) played no part in the phosphorylation of stromal protein. The rate and level of phosphorylation of stromal protein was unaffected by the activation state of the thylakoid protein kinase and was unchanged when thylakoids were omitted from the reaction medium. The phosphorylation of stromal proteins is therefore catalysed by a discrete soluble protein kinase.

Purification of a Protein Phosphatase from Chloroplast Stroma Capable of Dephosphorylating the Light-Harvesting Complex-II

“…Many stromal proteins undergo reversible phosphorylation (Foyer, 1985), and the stromal protein phosphatase, along with other unidentified protein phosphatases, could catalyze the dephosphorylation of some or a11 of these proteins. The results of our experiment in which we examined the dephosphorylation of the major stromal phosphoprotein may not be conclusive.…”

“…dephosphorylation in the chloroplast has focused mainly on the LHC-I1 (Bennett, 1977), although many thylakoid proteins (Markwell et al, 1984;Silverstein et al, 1993) and stromal proteins (Foyer, 1985) become phosphorylated. The reversible phosphorylation of LHC-I1 is coincident with the state 1 to state 2 transition governing photosynthetic quanta1 energy distribution (Allen, 1995).…”

Protein Tyrosine Kinases and Protein Tyrosine Phosphatases Are Involved in Abscisic Acid-Dependent Processes in Arabidopsis Seeds and Suspension Cells

“…Mann and Turner (1988) have shown that the large subunit of Rubisco is phosphorylated on Tyr residues in Rhodomicrobium vannielii. The phosphorylation of this subunit may be involved in the assembly of Rubisco (Foyer, 1985). When ABA inhibits germination, the dephosphorylation on Tyr residues of the large subunit of Rubisco could impair the assembly of the enzyme.…”

In Vitro Phosphorylation of Maize Leaf Phosphoenolpyruvate Carboxylase

“…Phosphorylation/inactivation of PDC and PPDK has also been established in situ (6,24). In contrast, phosphorylation of Rubisco has unknown effects on the enzyme (12). Data on hydroxymethyl glutaryl CoA reductase have indicated that it may also be phosphorylated (25 Immunoblots.…”

“…To our knowledge, only three specific soluble leaf proteins have been identified as being phosphorylated: pyruvate dehydrogenase complex (PDC3; [24]), PPDK (1,9), and the large and small subunits of Rubisco (12). Phosphorylation of the mitochondrial 2 Present address: Department of Biochemistry, 21 1 Schweitzer Hall, University of Missouri, Columbia, MO 6521 1. I Abbreviations: PDC, pyruvate dehydrogenase complex; PPDK, pyruvate, Pi dikinase; PEP, phosphoenolpyruvate; PEPCase, PEP carboxylase; AP5A, P'P5-di(adenosine-5')pentaphosphate; IEF, isoelectric focusing; Rubisco, ribulosebisphosphate carboxylase/oxygenase.…”