Synthesis and Uptake of Cytoplasmically Synthesized Pyruvate, Pi Dikinase Polypeptide by Chloroplasts

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Abstract
Polyadenylated RNA was isolated from maize leaves and translated in vitro. In agreement with a previous report by others, we found among the translation products a 110-kilodalton pyruvate orthophosphate dikinase (PPDK) precursor that is about 16 kilodaltons larger than the polypeptide isolated from cells. This maize PPDK precursor polypeptide was taken up from the translation product mixture by intact spinach chloroplasts and yielded a mature PPDK polypeptide (94 kilodaltons). The uptake and processing support the proposal that the extra 16-kilodalton size of the polypeptide from in vitro translation of maize leaf mRNA represents a transit sequence which is cleaved after its entry into chloroplasts. Moreover, these results provide additional evidence that in vivo in maize leaf cells PPDK polypeptide is synthesized in the cytoplasm and is transported into the chloroplasts. Location of PPDK in C3 plant leaves was investigated by immunochemical analysis. Intact chloroplasts were isolated from leaves of spinach [Spinacia oleracea], wheat [Triticum aestivum], and maize [Zea mays]. A protein blot of stromal protein in each case gave rise to bands corresponding to authentic PPDK polypeptide. This results indicates that PPDK is present in chloroplasts of C3 plant leaves as it is in the case of C4 plants.