Purification of a small phosphoprotein from chloroplasts and characterisation of its phosphoryl group

Summary

Introduction

The phosphorylation pattern of chloroplast proteins changes dramatically upon a switch in external conditions, e.g. a light–dark transition in vivo (Bennett, 1991) or a change in nucleoside triphosphate concentration in vitro (Ranjeva & Boudet, 1987). In the presence of very low concentrations of ATP or GTP (5–30 nm) and short labelling kinetics, only one protein of low molecular mass (about 19 kDa) is detected in soluble chloroplast proteins (Soll & Bennett, 1988; Soll, Berger & Bennett, 1989). This chapter describes the purification and analysis of this novel phosphoprotein.

Characterisation of the 19 kDa phosphoprotein

The 19 kDa protein was first detected as a phosphoprotein in highly purified intact chloroplasts from pea and spinach (Soll & Bennett, 1988). Upon subfractionation of chloroplasts on sucrose gradients, the phosphorylated protein could be detected in the total soluble extract of chloroplasts and the envelope membrane fraction. The total soluble extract contains stromal proteins as well as proteins from the intermembrane space, which are mostly liberated during lysis of the plastids. The hydrophilic nature of the 19 kDa protein was verified by Triton X-114/water phase partioning: the protein was exclusively found in the water phase. Mild sonication of mixed envelope membranes from chloroplasts also resulted in an almost complete release of the protein from the membrane. Differential labelling of the 19 kDa protein in intact and lysed chloroplasts of this soluble or peripheral membrane protein suggests its localisation outside the stromal space, perhaps in the inter-envelope lumen or the outer envelope membrane (Soll, Steidl & Schröder, 1991).