IDENTIFICATION OF A TRANSLOCATION INTERMEDIATE OCCUPYING FUNCTIONAL PROTEIN IMPORT SITES IN THE CHLOROPLASTIC ENVELOPE MEMBRANE

We used complexes of avidin and biotinylated precursors to generate tr anslocation intermediates that occupy functional transport sites and t hereby block the transport of other precursor proteins into pea chloro plasts. Cysteine residues of purified precursor to the small subunit o f rubisco (prSS) were modified with the biotinylation reagent [-4'-(ma leimidomethyl)-cyclohexane-carboxamido]but Chemically biotinylated prS S was readily imported into chloroplasts. The addition of avidin, howe ver, resulted in the formation of an avidin-biotinylated precursor com plex that could not be imported into chloroplasts even when precursors had already engaged the transport apparatus before avidin was added. On fractionation, the avidin-biotinylated precursor complex associated with envelope membranes. Titration of transport sites with avidin-bio tinylated precursor complexes revealed that saturation was reached at 2,000 molecules/chloroplast. Even with less than saturating levels of complexes, a sufficient number of translocation sites could be occupie d with avidin precursor complexes so that the import rate of freshly a dded radiolabeled prSS was reduced by 35%. From these observations we conclude that the trapped intermediates were blocking functional trans location sites. These biotinylated translocation intermediates should be useful in future efforts to purify and characterize the chloroplast ic protein import machinery.