title: Non-native, N-terminal Hsp70 molecular motor recognition elements in transit peptides support plastid protein translocation.
abstract: Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences.
authors: From the Graduate School of Genome Science and Technology.
authors: Prakitchai
authors: P
authors: Chotewutmontri
authors: From the Graduate School of Genome Science and Technology, Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996 bbruce@utk.edu.
authors: Barry D
authors: BD
authors: Bruce
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D018087:Plastids / Q000378:metabolism*; D021381:Protein Transport; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D029681:Arabidopsis Proteins; D018840:HSP70 Heat-Shock Proteins
keywords: 70-Kilodalton Heat Shock Protein (Hsp70); Arabidopsis thaliana; Cell Biology; Chloroplast; Molecular Motor; Protein Translocation; Transit Peptide
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