Figure 5.

Preprotein translocation across the outer membrane is not affected by decreasing atToc159 GTPase activity. (A) In vitro–translated [³⁵S]preSSu was incubated with isolated chloroplasts from WT:ppi2 or A864R:ppi2 plants in the presence of 0.1 mM ATP and 0.1 mM GTP to initiate translocation across the outer membrane (early import intermediate), but preclude complete translocation across the envelope. Where indicated, 1 mM GTPγS was substituted for GTP. The graph presents the quantification of results from triplicate experiments. (B) WT:ppi2 and A864R:ppi2 chloroplasts containing bound early import intermediates (Inter.; lanes 3 and 6) were reisolated and resuspended under protein import conditions in the presence of 5 mM ATP (Chase; lanes 4, 7, and 8–11). Where indicated, 1 mM GTPγS was added to the chase reactions (lanes 9 and 11). (C) Quantification of the levels of early import intermediate formed in B from triplicate experiments. (D) Quantification of the effects of GTPγS on the chase of the early intermediate (B) from triplicate experiments. Dashed lines indicate that the figures were generated from different regions of the same SDS-PAGE gel using samples from the same experiment.