The Rubisco large subunit binding protein

Abstract

Newly synthesized Rubisco large subunits made by isolated intact chloroplasts fromPisum sativumare bound non-covalently to another protein, termed the Rubisco large subunit binding protein. This protein is implicated in the assembly of Rubisco in higher plant chloroplasts. The binding protein has been purified fromPisum sativumin the form of an oligomer of relative molecular mass (Mr) about 720000. Analysis on polyacrylamide gels containing sodium dodecyl sulphate reveals equal amounts of two different types of subunit, termed alpha (Mrabout 61000) and beta (Mrabout 60000); thus the oligomer has the composition a α6β6. The alpha and beta subunits have been separated; their amino-terminal sequences are different, and antibodies raised against one subunit do not cross-react with the other subunit. Antibodies raised against the binding protein do not cross-react with the Rubisco large subunit, but do cross-react with polypeptides ofMrabout 60000 in extracts of chloroplasts from wheat, barley and tobacco, and in extracts of leucoplasts from castor-bean endosperm. The binding protein is made as a higher-molecular-mass precursor whan leaf polysomes are translated in a wheatgerm extract containing chloramphenicol, but is not synthesized by isolated intact chloroplasts. Thus the binding protein subunits are synthesized by cytoplasmic ribosomes and hence are likely to be encoded by nuclear genes. EtiolatedPisumplants contain binding protein, but exposure to light does not cause the same dramatic increase in amount that is seen in the case of Rubisco. Treatment of stromal extracts with Mg-ATP in the range 0.1-5.0 mM causes dissociation of the binding protein oligomer into monomeric subunits; CTP, GTP, UTP, AMP and cyclic AMP do not have this effect. Mg2+is required for dissociation but can be replaced by Ca2+. Newly synthesized large subunits are released when the binding protein oligomer is dissociated, but re-attach when the dissociation is reversed by removal of ATP.