Title: In vitro assays to analyze translocation of the model secretory preprotein alkaline phosphatase
Authors: Gouridis, Giorgos ×
Karamanou, Spyridoula
Koukaki, Marina
Economou, Anastassios #
Issue Date: 2010
Series Title: Methods in Molecular Biology vol:619 pages:157-72
Article number: 10.1007/978-1-60327-412-8_10
Abstract: 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.
ISSN: 1064-3745
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Non-KU Leuven Association publications
× corresponding author
# (joint) last author

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