In vitro assays to analyze translocation of the model secretory preprotein alkaline phosphatase.
Almost one-third of the proteins synthesized in the cytosol of cells ends up in membranes or outside the cell. Secretory polypeptides are synthesized as precursor proteins that carry N-terminal signal sequences. Secretion is catalyzed by the "translocase" that comprises a channel-clamp protein and an ATPase motor. Translocase activities have been fully reconstituted in vitro. This provided powerful tools to examine the role of each component in the reaction. Here we describe protocols for the purification of the secretory preprotein alkaline phosphatase and a series of in vitro assays developed in order to examine the binding of alkaline phosphatase to the translocase, its ability to stimulate ATP hydrolysis, and finally its transfer across the membrane. The assays are applicable to any similar study of secretory preproteins.
Gouridis
Giorgos
G
Department of Biology, University of Crete and Institute of Molecular Biology and Biotechnology-Foundation for Research and Technology Hellas, Heraklion, Crete, Greece.
Karamanou
Spyridoula
S
Koukaki
Marina
M
Economou
Anastassios
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins