Interaction of a plant 14-3-3 protein with the signal peptide of a thylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma

dThe 14-3-3 proteins are acidic, dimeric proteins that have been implicated in many eukaryotic cellular processes because of direct protein associatio n with enzymes and other metabolic and regulatory proteins. 14-3-3 proteins are largely considered to be cytoplasmic, but a search for proteins that s pecifically interact with a plant 14-3-3 resulted in the isolation of a nuc lear-encoded, thylakoid-targeted chloroplast precursor, the full-length Ara bidopsis photosystem I N-subunit At pPSI-N (P.C. Sehnke, R.J. Ferl [1995] P lant Physiol 109: 1126). Using precursor truncations in the two-hybrid syst em, it was determined that the leader sequence is the site of PSI-N that as sociates with 14-3-3. This suggested the novel possibility that 14-3-3 woul d be found within chloroplasts. Immunoelectron microscopy of leaf tissue an d western analysis of chloroplast fractions with monoclonal anti-14-3-3 ant ibodies localized 14-3-3 proteins to the chloroplast stroma and the stromal side of thylakoid membranes. Using peptide-generated, isoform-specific ant ibodies, GF14 nu, CF14 epsilon, CF14 mu, and GF14 upsilon were shown to be present in the chloroplast stromal extract. These isoforms represent two di stinct phylogenetic 14-3-3 groupings. These data suggest a novel interorgan ellar role for these phylogenetically distinct 14-3-3 proteins.