Positive charges determine the topology and functionality of the transmembrane domain in the chloroplastic outer envelope protein Toc34.
The chloroplastic outer envelope protein Toc34 is inserted into the membrane by a COOH-terminal membrane anchor domain in the orientation Ncyto-Cin. The insertion is independent of ATP and a cleavable transit sequence. The cytosolic domain of Toc34 does not influence the insertion process and can be replaced by a different hydrophilic reporter peptide. Inversion of the COOH-terminal, 45-residue segment, including the membrane anchor domain (Toc34Cinv), resulted in an inverted topology of the protein, i.e., Nin-Ccyto. A mutual exchange of the charged amino acid residues NH2- and COOH-proximal of the hydrophobic alpha-helix indicates that a double-positive charge at the cytosolic side of the transmembrane alpha-helix is the sole determinant for its topology. When the inverted COOH-terminal segment was fused to the chloroplastic precursor of the ribulose-1,5-bisphosphate carboxylase small subunit (pS34Cinv), it engaged the transit sequence-dependent import pathway. The inverted peptide domain of Toc34 functions as a stop transfer signal and is released out of the outer envelope protein translocation machinery into the lipid phase. Simultaneously, the NH2-terminal part of the hybrid precursor remained engaged in the inner envelope protein translocon, which could be reversed by the removal of ATP, demonstrating that only an energy-dependent force but no further ionic interactions kept the precursor in the import machinery.
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Botanisches Institut, Christian-Albrechts-Universität Kiel, D-24118 Kiel, Germany.
Soll
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000378:metabolism* / Q000648:ultrastructure; D003600:Cytosol / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000378:metabolism*; D018515:Plant Leaves; D010940:Plant Proteins; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis
D008565:Membrane Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C112475:Toc34 protein, plant; D000255:Adenosine Triphosphate; D012273:Ribulose-Bisphosphate Carboxylase