The effect of amino acid-modifying reagents on chloroplast protein import and the formation of early import intermediates

In order to identify functionally important amino acid residues in the chloroplast protein import machinery, chloroplasts were preincubated with amino-acid-modifying reagents and then allowed to import or form early import intermediates with precursor proteins. Incubation of chloroplasts with N-ethyl maleimide, diethyl pyrocarbonate, phenylglyoxal, 4,4'-di-isothiocyanatostilbene 2,2'-disulphonic acid (DIDS), dicyclohexylcarbodiimide (DCCD), and 1-ethyl- 3-dimethylaminopropylcarbodiimide (EDC) inhibited both import and formation of early import intermediates with precursor proteins by chloroplasts. This suggests that one or more of the binding components of the chloroplast protein import machinery contains functionally important solvent-exposed cysteine, histidine, arginine, and aspartate/glutamate residues, as well as functionally important lysine and aspartate/ glutamate residues in a hydrophobic environment.