Abstract
Many nonenzymatic cellular proteins exert their functions by interacting with other proteins or macromolecules. Analysis of the physical interactions of proteins is an important step to understand their functions, and the information obtained is helpful for predicting the roles of the proteins in cells. Here we describe three biochemical and immunological methods for the detection of interactions between ALG-2 (a penta-EF-hand Ca2+-binding protein, also known as PDCD6) and its target proteins: (1) glutathione-S-transferase (GST) pulldown assay, (2) co-immunoprecipitation assay, and (3) Far Western blot analysis using biotinylated ALG-2. Dependency of Ca2+ for interaction is examined by inclusion of CaCl2 or EGTA in buffers used for binding assays.
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References
Maki M, Narayana S, Hitomi K (1997) A growing family of the Ca2+-binding proteins with five EF-hand motifs. Biochem J 328:718–720
Maki M, Kitaura Y, Satoh H, Ohkouchi S, Shibata H (2002) Structures, functions and molecular evolution of the penta-EF-hand Ca2+-binding proteins. Biochim Biophys Acta 1600:51–60
Maki M, Yamaguchi K, Kitaura Y, Satoh H, Hitomi K (1998) Calcium-induced exposure of a hydrophobic surface of mouse ALG-2, which is a member of the penta-EF-hand protein family. J Biochem 124:1170–1177
Maki M, Suzuki H, Shibata H (2011) Structure and function of ALG-2, a penta-EF-hand calcium-dependent adaptor protein. Sci China Life Sci 54:770–779
Missotten M, Nichols A, Rieger K, Sadoul R (1999) Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein. Cell Death Differ 6:124–129
Vito P, Pellegrini L, Guiet C, D’Adamio L (1999) Cloning of AIP1, a novel protein that associates with the apoptosis-linked gene ALG-2 in a Ca2+-dependent reaction. J Biol Chem 274:1533–1540
Suzuki H, Kawasaki M, Inuzuka T, Okumura M, Kakiuchi T, Shibata H, Wakatsuki S, Maki M (2008) Structural basis for Ca2+ -dependent formation of ALG-2/Alix peptide complex: Ca2+/EF3-driven arginine switch mechanism. Structure 16:1562–1573
Shibata H, Suzuki H, Kakiuchi T, Inuzuka T, Yoshida H, Mizuno T, Maki M (2008) Identification of Alix-type and non-Alix-type ALG-2-binding sites in human phospholipid scramblase 3. J Biol Chem 283:9623–9632
Shibata H, Inuzuka T, Yoshida H, Sugiura H, Wada I, Maki M (2010) The ALG-2 binding Site in Sec31A influences the retention kinetics of Sec31A at the endoplasmic reticulum exit sites as revealed by live-cell time-lapse imaging. Biosci Biotechnol Biochem 74:1819–1826
Katoh K, Suzuki H, Terasawa Y, Mizuno T, Yasuda J, Shibata H, Maki M (2005) The penta-EF-hand protein ALG-2 interacts directly with the ESCRT-I component TSG101, and Ca2+-dependently co-localizes to aberrant endosomes with dominant-negative AAA ATPase SKD1/Vps4B. Biochem J 391:677–685
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, vol 3, 3rd edn. Cold Spring Harbor Laboratory Press, New York. Chapter 16
Shibata H, Suzuki H, Yoshida H, Maki M (2007) ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in a Ca2+-dependent manner. Biochem Biophys Res Commun 353:756–763
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, vol 3, 3rd edn. Cold Spring Harbor Laboratory Press, New York. Appendix 8.40
Shibata H, Yamada K, Mizuno T, Yorikawa C, Takahashi H, Satoh H, Kitaura Y, Maki M (2004) The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix/AIP1. J Biochem 135:117–128
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research (B) (to MM). We thank members of the Laboratory of Molecular and Cellular Regulation for valuable suggestions.
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Osugi, K., Shibata, H., Maki, M. (2013). Biochemical and Immunological Detection of Physical Interactions Between Penta-EF-Hand Protein ALG-2 and Its Binding Partners. In: Heizmann, C. (eds) Calcium-Binding Proteins and RAGE. Methods in Molecular Biology, vol 963. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-230-8_12
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DOI: https://doi.org/10.1007/978-1-62703-230-8_12
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Publisher Name: Humana Press, Totowa, NJ
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