Topology studies of the chloroplast protein import channel Toc75.
A major goal in understanding protein transport across membranes is the investigation of the structure and regulation of the translocon subunits. We analysed Toc75, a pore-forming subunit of the translocon of the outer envelope of chloroplasts. Toc75 was overexpressed and reconstituted into liposomes. Immunoprecipitation of liposome-reconstituted Toc75 indicates an N(in)-C(in) orientation of Toc75. Limited proteolytic digestion of Toc75 present in outer envelope vesicles with specific proteases combined with amino acid sequencing was used to study the topology of Toc75. Finally, computer modelling based on known protein structures indicates that Toc75 traverses the outer envelope with 16 amphiphilic beta sheets and the topology model is presented.
Sveshnikova
N
N
Botanisches Institut, Universität Kiel, Germany.
Grimm
R
R
Soll
J
J
Schleiff
E
E
D000465:Algorithms; D002352:Carrier Proteins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D008567:Membranes, Artificial; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry; D011233:Precipitin Tests; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry*; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D020539:Sequence Analysis, Protein
D002352:Carrier Proteins; D008081:Liposomes; D008565:Membrane Proteins; D008567:Membranes, Artificial; C103933:OEP75 protein precursor, plant; D010446:Peptide Fragments; D010940:Plant Proteins; D011498:Protein Precursors; D021122:Protein Subunits; D010447:Peptide Hydrolases