The experiments in this proposal combine in vivo studies in Arabidopsis thaliana, where Dr. Schnell will study the influence of mutants of the TOC complex of chloroplast biogenesis, with biochemical studies aimed to elucidate the functional role of various subunits of the TOC complex in membrane translocation. There are 5 specific aims proposed in this grant application.
The first aim i nvolves the study of null mutants of Toc34,75 and 159 in A. thaliana and consequences of expressing mutated forms of these proteins. This work is part of an ongoing collaboration with Dr. Kessler at ETH, Zurich, an expert in A. thaliana genetics.
The second aim i s related to the first one, in that it will use similar methods to explore the function of Toc 132 and Toc 120, two genes they have discovered that appear to be members of the same family of proteins.
The third aim focuses on the functional dissection of Toc 159. Specifically it is proposed to study the role of this GTPase in the recognition of pre-proteins that are imported from the cytosol to the chloroplast. It is proposed to search for other proteins that facilitate this recognition by using established methods of protein-protein interaction. Other proteins are part of the TOC complex, and aims 4 and 5 will use in vitro assays to study their potential regulatory role in protein import and their contact map.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cell Development and Function Integrated Review Group (CDF)
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Shapiro, Bert I
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University of Massachusetts Amherst
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Richardson, Lynn G L; Small, Eliana L; Inoue, Hitoshi et al. (2018) Molecular Topology of the Transit Peptide during Chloroplast Protein Import. Plant Cell 30:1789-1806
Richardson, Lynn G L; Singhal, Rajneesh; Schnell, Danny J (2017) The integration of chloroplast protein targeting with plant developmental and stress responses. BMC Biol 15:118
O'Neil, Patrick K; Richardson, Lynn G L; Paila, Yamuna D et al. (2017) The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts. Proc Natl Acad Sci U S A 114:E4868-E4876
Nevarez, P Andrew; Qiu, Yongjian; Inoue, Hitoshi et al. (2017) Mechanism of Dual Targeting of the Phytochrome Signaling Component HEMERA/pTAC12 to Plastids and the Nucleus. Plant Physiol 173:1953-1966
Paila, Yamuna D; Richardson, Lynn Gl; Inoue, Hitoshi et al. (2016) Multi-functional roles for the polypeptide transport associated domains of Toc75 in chloroplast protein import. Elife 5:
Paila, Yamuna D; Richardson, Lynn G L; Schnell, Danny J (2015) New insights into the mechanism of chloroplast protein import and its integration with protein quality control, organelle biogenesis and development. J Mol Biol 427:1038-1060
Okawa, Kumiko; Inoue, Hitoshi; Adachi, Fumi et al. (2014) Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments. J Exp Bot 65:5257-65
Inoue, Hitoshi; Li, Ming; Schnell, Danny J (2013) An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts. Proc Natl Acad Sci U S A 110:3173-8
Oreb, Mislav; Höfle, Anja; Koenig, Patrick et al. (2011) Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33. Biochem J 436:313-9
Inoue, Hitoshi; Wang, Fei; Inaba, Takehito et al. (2011) Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions. Methods Mol Biol 774:307-20

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