Table 2.

Measurement of translocation ATPase activity with RTS in vitro translated [³H] tp22-GFP as substrate

Experiment	Signal ATPase activity (nmol ATP hydrolyzed ⋅mg Chl−1⋅h−1)	Import rate (pmol tp22-GFP imported ⋅mg Chl−1⋅h−1)	Translocation ATPase activity (ATP hydrolyzed per tp22-GFP imported)
1	18.5	117.0	158
2	11.8	92.1	128
3	33.6	97.7	344
Average			210 ± 117

Chloroplasts were incubated at room temperature in the dark for 30 min in IB containing 5 μM tentoxin and 10 μM DCMU. ATP mixed with [γ-³²P]-ATP was then added to the chloroplasts forming a final concentration of 1 mM. Reactions were started by adding 36 pmol [³H]-tp22-GFP (final concentration, 0.60 μM) or [³H]-GUS (containing equal amounts of wheat germ as that in [³H]-tp22-GFP), both of which were translated in the RTS wheat germ system (Materials and Methods). Samples were withdrawn at 0 and 60 min. ATPase activity analysis and import assay were as described in Table 1 and in Materials and Methods.