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 translocation intermediates that occupy functional transport sites and thereby block the transport of other precursor proteins into pea chloroplasts. Cysteine residues of purified precursor to the small subunit of rubisco (prSS) were modified with the biotinylation reagent biotin-1-biotinamido-4-[-4'-(maleimidomethyl)-cyclohexane-carboxamido]but Chemically biotinylated prSS was readily imported into chloroplasts. The addition of avidin, however, resulted in the formation of an avidin-biotinylated precursor complex 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-biotinylated 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 occupied with avidin precursor complexes so that the import rate of freshly added radiolabeled prSS was reduced by 35%. From these observations we conclude that the trapped intermediates were blocking functional translocation sites. These biotinylated translocation intermediates should be useful in future efforts to purify and characterize the chloroplastic protein import machinery.