Advertisement

Biochemical and Immunological Detection of Physical Interactions Between Penta-EF-Hand Protein ALG-2 and Its Binding Partners

  • Kanae Osugi
  • Hideki Shibata
  • Masatoshi MakiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 963)

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.

Key words

Protein–protein interaction Calcium-binding protein GST-pulldown Co-immuno-precipitation Far Western Biotinylation, EF-hand 

Notes

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.

References

  1. 1.
    Maki M, Narayana S, Hitomi K (1997) A growing family of the Ca2+-binding proteins with five EF-hand motifs. Biochem J 328:718–720PubMedGoogle Scholar
  2. 2.
    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 1600:51–60PubMedCrossRefGoogle Scholar
  3. 3.
    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 124:1170–1177PubMedCrossRefGoogle Scholar
  4. 4.
    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 54:770–779PubMedCrossRefGoogle Scholar
  5. 5.
    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 6:124–129PubMedCrossRefGoogle Scholar
  6. 6.
    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 274:1533–1540PubMedCrossRefGoogle Scholar
  7. 7.
    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 16:1562–1573PubMedCrossRefGoogle Scholar
  8. 8.
    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 283:9623–9632PubMedCrossRefGoogle Scholar
  9. 9.
    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 74:1819–1826PubMedCrossRefGoogle Scholar
  10. 10.
    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 391:677–685PubMedCrossRefGoogle Scholar
  11. 11.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, vol 3, 3rd edn. Cold Spring Harbor Laboratory Press, New York. Chapter 16Google Scholar
  12. 12.
    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 353:756–763PubMedCrossRefGoogle Scholar
  13. 13.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, vol 3, 3rd edn. Cold Spring Harbor Laboratory Press, New York. Appendix 8.40Google Scholar
  14. 14.
    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 135:117–128PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of Applied Molecular Biosciences, Graduate School of Bioagricultural SciencesNagoya UniversityNagoyaJapan

Personalised recommendations