title: Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.
abstract: Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.
authors.affiliation: Horticultural Sciences Department and Plant Molecular and Cellular Biology, University of Florida, Gainesville, Florida 32611, USA.
authors.forename: Xianyue
authors.initials: X
authors.lastname: Ma
authors.forename: Kenneth
authors.initials: K
authors.lastname: Cline
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D010449:Peptide Mapping / Q000379:methods*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021382:Protein Sorting Signals; D021381:Protein Transport; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D060431:Thylakoid Membrane Proteins / Q000235:genetics / Q000378:metabolism; D020524:Thylakoids / Q000235:genetics / Q000378:metabolism
chemical_list: C109236:Hcf106 protein, plant; D008565:Membrane Proteins; D010940:Plant Proteins; D021382:Protein Sorting Signals; C432107:TatC protein, plant; D060431:Thylakoid Membrane Proteins; D001120:Arginine; D003545:Cysteine