ERD1 import, binding, and fractionation using pea
chloroplasts. A, Import of ERD1 into pea chloroplasts was performed as
described in Methods. Aliquots were removed at
given times, and import was terminated by reisolating intact
chloroplasts by sedimentation through a 40% Percoll cushion. Fractions
were resuspended in sample buffer and subsequently analyzed by SDS-PAGE
and fluorography. B, Binding of ERD1 to pea chloroplasts under low-ATP
conditions. The binding reaction was equally divided into two
fractions. One sample was treated with thermolysin (+) and the other
sample was not (−). Protease digestion was allowed to continue for an
additional 30 min on ice in the dark. Proteolysis was terminated by
adding EDTA to a final concentration of 5 mm, and intact
chloroplasts were reisolated by sedimentation through a 40% Percoll
cushion with 5 mm EDTA present. C, ERD1 was imported into
pea chloroplasts and fractionated as described in Methods. All fractions were analyzed by SDS-PAGE and fluorography.
TP, Ten percent of translation product added to the import reaction; M,
crude membrane pellet; S, supernatant fraction; U, unprocessed form of
ERD1; P, processed form of ERD1; MW, Mr
standards.