Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide.

Phosphorylation of the transit peptide of several chloroplast-targeted proteins enables the binding of 14-3-3 proteins. The complex that forms, together with Hsp70, has been demonstrated to be an intermediate in the chloroplast protein import pathway in vitro[May, T. & Soll, J. (2000) Plant Cell 12, 53-63]. In this paper we report that mutagenesis (in order to remove the phosphorylation site) of the transit peptide of the small subunit of ribulose bisphosphate carboxylase/oxygenase did not affect its ability to target green fluorescent protein to chloroplasts in vivo. We also found no mistargeting to other organelles such as mitochondria. Similar alterations to the transit peptides of histidyl- or cysteinyl-tRNA synthetase, which are dual-targeted to chloroplasts and mitochondria, had no effect on their ability to target green fluorescent protein in vivo. Thus, phosphorylation of the transit peptide is not responsible for the specificity of chloroplast import.