Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34.
Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved in transit peptide recognition. The cytosolic region of Toc34 reveals 34% alpha-helical and 26% beta-strand structure and is stabilized by intramolecular electrostatic interaction. Toc34 binds both chloroplast preproteins and isolated transit peptides in a guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that the soluble, cytosolic domain of Toc34 (Toc34deltaTM) functions as receptor in vitro and is capable to compete with the import of the preprotein of the small subunit (preSSU) of ribulose-1,5-bisphosphate carboxylase-oxygenase into chloroplasts in a GTP-dependent manner. We have developed a biosensor assay to study the interaction of Toc34deltaTM with purified preproteins and transit peptides. The results are compared with the interactions of both a full-size preprotein and the transit peptide of preSSU with the translocon of the outer envelope of chloroplasts (Toc complex) in situ. Several mutants of the transit peptide of preSSU were evaluated to identify amino acid segments that are specifically recognized by Toc34. We present a model of how Toc34 may recognize the transit peptide and discuss how this interaction may facilitate interaction and translocation of preproteins via the Toc complex in vivo.
Schleiff
E
E
Institut of Botany, University Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.
Soll
J
J
Sveshnikova
N
N
Tien
R
R
Wright
S
S
Dabney-Smith
C
C
Subramanian
C
C
Bruce
B D
BD
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry; D004792:Enzyme Precursors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010940:Plant Proteins; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D017384:Sequence Deletion
D002352:Carrier Proteins; D004792:Enzyme Precursors; D008565:Membrane Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C112475:Toc34 protein, plant; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D012273:Ribulose-Bisphosphate Carboxylase