Abstract
Protein import into chloroplasts is initiated by a binding interaction between a precursor protein and the surface of the outer envelope. The binding step was previously shown to be energy-dependent (Olsen, L. J., Theg, S. M., Selman, B. R., and Keegstra, K. (1989) J. Biol. Chem. 264, 6724-6729). We took advantage of the broad nucleotide specificity of the energy requirement for binding to investigate the site of the nucleoside triphosphate (NTP) requirement. GTP supported precursor binding to chloroplasts. It was not converted to ATP, as determined by direct ATP measurements, and was not transported across the inner envelope. Thus, GTP supported binding from either the intermembrane space or outside the outer membrane. To distinguish between an intermembrane space and an external NTP requirement, we experimentally manipulated the NTP levels inside and outside chloroplasts. Internally generated ATP was able to support binding in the presence of an external membrane-impermeant ATP trap. Therefore, since GTP supported binding from either the intermembrane space or outside the chloroplast, and ATP supported binding from either the intermembrane space or the stroma, we concluded that the site of NTP utilization for precursor binding to chloroplasts was the intermembrane space between the two envelope membranes.
Highlights
Protein import into chloroplasts is initiatedby a largely unknown
Pain and Blobel [13], from either the intermembrane space or outside the using approaches similar to the othegrroups, concluded that chloroplast, and ATP supported binding from either protein transportrequires the hydrolysis of ATP inside chlothe intermembrane space or thestroma, we concluded that the site of NTP utilization for precursor binding to chloroplasts was the intermembrane space between the two envelope membranes
N T P Requirement for Precursor Binding to Chloroplasts brane space or stroma to support binding, and GTPsupported binding from the intermembrane space or outside the outer envelope, we conclude that the intermembrane space is the most likely site of NTP utilization for the binding of precursor proteins to pea chloroplasts
Summary
Nucleotide Specificity for Binding-The binding of precurbuffer (330mM sorbitol, 50 mM Hepes/KOH, p H 8)a t a concentration sor proteins to chloroplasts was initially assumed to occur of approximately 1mg of chlorophyll/ml (1-3 X IO9chloroplasts/ml) and kept onice in the dark untiul se. Neither the inability of ATP, GTP, and UTP to restore analog supported binding or translocation even at thehighest binding in the presence of the analogs provides evidence that concentrations tested, as seen in Fig. 2 (lanes 5 and 9). Itseemed possible were incubatedwith 10-15 p1 of gel-filtered 3H-labeled prSS and that athigh GTP levels, the GTPwas acting as a chelatorfor either ATP, ADP, or AMP (100 or 1000 p ~ as, M e salts or with equimolar Mg(OAc), added to the buffer) for 10 min in the dark. Samples were prepared for analysis by SDS-PAGE andfluorography, and quantitation of the results was performed as described under "Experimental Procedures." The number of molecules bound (prSS) and translocated (SS) per chloroplast are presented from a represome required cation. Precursor proteinswhich had been translocated but not processed should be resistant
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