The problem of organellar targeting in eukaryotic cells is addressed by studying the sorting of closely related, cytoplasmically synthesized polypeptides to mitochondria and chloroplasts in Pisum sativum, the pea. The hypotheses to be tested are that there exists some sort of discriminatory mechanism which prevents the import of mitochondrial proteins into chloroplasts, and vice versa, and that the discrimination involves specific sequences present in mitochondrial but not chloroplast transit peptides. To test these hypotheses, the genes for organellar protein synthesis factors (specifically, EF-Tu and EF-G) of pea mitochondria and chloroplasts will be cloned and sequenced. From a comparison of the transit peptides of these closely related mitochondrial and chloroplast polypeptides, sequences which may specify the destination of the polypeptide will be predicted. Segments of the mitochondrial and chloroplast transit peptides will be deleted, switched back and forth, and specific sequences will be altered by site-directed mutagenesis in vitro. Normal and mutagenized proteins will be synthesized by in vitro transcription/translation and import into purified organelles will be studied. The effects of the altered transit peptides in vitro, and in some cases, in vivo, in transgenic plants, will serve to establish which sequences within the transit peptides act as targeting signals. These studies will lead to an improved understanding of how polypeptides are targeted to specific organelles in eukaryotes. In the long term, this work will also add to the general knowledge of organellar biogenesis and of organellar protein synthesis.
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