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

摘要

The 14-3-3 proteins are acidic, dimeric proteins that have been implicated in many eukaryotic cellular processes because of direct protein association with enzymes and other metabolic and regulatory proteins. 14-3-3 proteins are largely considered to be cytoplasmic, but a search for proteins that specifically interact with a plant 14-3-3 resulted in the isolation of a nuclear-encoded, thylakoid-targeted chloroplast precursor, the full-length Arabidopsis photosystem I N-subunit At pPSI-N (P.C. Sehnke, R.J. Ferl [1995] Plant Physiol 109: 1126). Using precursor truncations in the two-hybrid system, it was determined that the leader sequence is the site of PSI-N that associates with 14-3-3. This suggested the novel possibility that 14-3-3 would be found within chloroplasts. Immuno-electron microscopy of leaf tissue and western analysis of chloroplast fractions with monoclonal anti-14-3-3 antibodies localized 14-3-3 proteins to the chloroplast stroma and the stromal side of thylakoid membranes. Using peptide-generated, isoform-specific antibodies, GF14ν, GF14ε, GF14μ, and GF14υ were shown to be present in the chloroplast stromal extract. These isoforms represent two distinct phylogenetic 14-3-3 groupings. These data suggest a novel interorganellar role for these phylogenetically distinct 14-3-3 proteins.