The chloroplastic glutamine synthetase (GS-2) of tobacco is phosphorylatedand associated with 14-3-3 proteins inside the chloroplast

The chloroplastic isoform of glutamine synthetase (GS-2, EC 6.3.1.2) from N icotiana tabacum L. is phosphorylated at the serine residues. At least thre e of the six GS-2 subunits separated by two-dimensional polyacrylamide gel electrophoresis cross-reacted with an antibody raised against phosphoserine . This provoked the question as to whether 14-3-3 proteins might be present in the chloroplast and bind to chloroplastic GS-2. Although two different 14-3-3 proteins of 32 and 30 kDa were present in total leaf extracts, in th e soluble fraction of chloroplasts, only the 32-kDa 14-3-3 protein was immu nodetected with an antibody raised against a conserved region of 14-3-3 pro tein from corn. This demonstrates the presence of a chloroplast-located iso form of 14-3-3 proteins in tobacco. To examine a putative binding of GS-2 t o these 14-3-3 proteins in vivo the native GS-2 holoenzyme was probed with a 14-3-3 antibody. The strong cross-reaction between GS-2 and the 14-3-3 an tibody clearly points to a binding of GS-2 and 14-3-3 in tobacco chloroplas ts. Only those oligomers of GS-2 that were strongly associated with 14-3-3 proteins were catalytically active.