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.
Key wordsE. coli translocase alkaline phosphatase preprotein translocasion mature domain targeting
We are grateful to D. Boyd (Beckwith Lab) and S. Schulman (Rapoport Lab) for gifts of strains, plasmids, and protocols. The research leading to these results has received funding from the European Community’s Sixth Framework Programme (FP6/2002–2007) under grant agreement no. LSHC-CT-2006-037834/Streptomics (to A.E.) and the Greek General Secretariat of Research and the European Regional Development Fund (01AKMON46 and PENED03ED623; to A.E.). G.G. is an Onassis Foundation predoctoral fellow.
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