FIGURE 1.

Increasing BSA concentration and adding reducing agents to the homogenization buffer increases the import of prRBCS and prTic40 into leucoplasts. (A) Roots of 4-days-old pea seedlings grown in the dark were harvested and homogenized in homogenization buffer (see Materials and Methods) containing 0.1, 0.5, or 1% BSA. Isolated leucoplasts (500 μg protein) were then incubated with in vitro-translated [³⁵S]Met-prRBCS or [³⁵S]Met-prTic40 under import conditions for 25 min. After import, intact leucoplasts were re-isolated and analyzed by SDS-PAGE. The gels were stained with Coomassie blue and dried for fluorography. Twenty micrograms of proteins were loaded. Tr, 1% equivalent of the in vitro-translated proteins used in each import reaction. (B) Same as (A) except the homogenization buffer was supplemented with 1% BSA, and with or without 2 mM ascorbic acid, 0.1 mM DTT, and 1.2 mM glutathione (ADG). pr, precursor form; int, imported intermediate form; m, imported mature form.