The Identification and Characterization of Cytosolic FactorsInvolved in Targeting of Nuclear-Encoded Precursors to Higher Plant Chloroplasts

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9401840 Bruce, Barry, D. This is a start-up grant for a Post Doctoral Fellow in Plant Biology. In plants and other eukaryotes, the translation of nuclear encoded genes can be divided into two diverging pathways: one, where proteins are translated on ribosomes associated with the endoplasmic reticulum and are destined either for the ER/Golgi complex for secretion, or for the plasma membrane; and a second pathway, where proteins are synthesized on free ribosomes in the cytoplasm. In plants, proteins synthesized on free cytoplasmic ribosomes can either remain in the cytoplasm, or being imported into one of four different organelles: the nucleus, the peroxisome, the mitochondria or the chloroplast via a post translational pathway. This post translational import requires a precursor form of the protein containing a targeting sequence which directs it to a specific organelle. It has been proposed that one prerequisite for the import of these precursors is that they maintain an import competent configuration. It is commonly accepted today that import of proteins into mitochondria requires that they be in a destabilized conformation. The role of protein conformation on import into chloroplasts is much less certain. The goal of this proposal is to determine what defines the import competence of chloroplast targeted precursors, and also what cytosolic factors are involved in maintaining this competence. The small subunit of ribulose bisphosphate carboxylase has been chosen as a model to address this question. The investigation will focus on purification of cytosolic factors that interact with the transit peptide for chloroplastic transport; on identification of trans-acting chloroplast import factors by the two-hybrid cloning technique, that allows specific protein-protein interactions to be identified in vivo through the reconstitution of a transport activator; and assaying in vitro the soluble factors in increasing import competence of the precursor of the sma ll subunit of ribulose bisphosphate carboxylase. %%% The PI made good progress during his post doctoral research on the problems concerning protein import into organelles, and obtained results which suggest that the conformational requirements for chloroplast import and mitochondrial import may be different. The goal of this project is to investigate the specific requirements for protein transport into chloroplasts. This is the topic that the PI started during his post doctoral research in the Keegstra laboratory. He will be taking the project with him to the University of Tennessee at Knoxville, where he starts a tenure track position in January 1994. This start-up grant for NSF Post Doctoral fellows in Plant Biology therefore will be essential to help him establish his own laboratory, and also scientific status. ***