Related Experiment Videos
Biochemical characterization of mapmodulin, a protein that binds microtubule-associated proteins
N Ulitzur1, C Rancaño, S R Pfeffer
1Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA. pfeffer@cmgm.stanford.edu
Abstract:
Mapmodulin is a 31-kDa protein that stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. We have shown previously that it binds the microtubule binding domains of the microtubule-associated proteins, MAP2, MAP4, and tau. We also showed that mapmodulin is identical to a protein named PHAPI (Vaesen, M., Barnikol-Watanabe, S. , Götz, H., Awni, L.A., Cole, T., Zimmermann, B., Kratzin, H.D. and Hilschmann, N. (1994) Biol. Chem. Hoppe-Seyler 375, 113-126). We report here that mapmodulin is a phosphoprotein that is predominantly cytosolic but is also found peripherally associated with endoplasmic reticulum and Golgi membranes in mammalian cells. The protein occurs as a trimer in cytosol, and phosphorylation is required for its microtubule-associated protein-binding activity. Heat treatment of nonphosphorylated mapmodulin can render it competent for binding to microtubule-associated proteins, suggesting that phosphorylation induces a conformational change in mapmodulin. Finally, despite identity in polypeptide sequence with a protein reported to act as an inhibitor of protein phosphatase 2A, native mapmodulin was not able to inhibit protein phosphatase 2A in Chinese hamster ovary cell cytosol.
Insights
Mapmodulin, a protein regulating Golgi complex localization, is a phosphoprotein. Phosphorylation is essential for its microtubule-binding activity, suggesting a conformational change.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mapmodulin (PHAPI) is a 31-kDa protein involved in Golgi complex localization.
- It binds microtubule-binding domains of MAP2, MAP4, and tau.
- Previous studies identified mapmodulin and its association with microtubule-associated proteins.
Purpose of the Study:
- To investigate the phosphorylation status of mapmodulin.
- To determine the role of phosphorylation in mapmodulin's function.
- To clarify mapmodulin's cellular localization and its relationship with protein phosphatase 2A.
Main Methods:
- Cellular fractionation to determine mapmodulin localization.
- Biochemical assays to assess microtubule-associated protein-binding activity.
- Phosphorylation studies and heat treatment experiments.
Main Results:
- Mapmodulin is a phosphoprotein found in the cytosol and associated with endoplasmic reticulum and Golgi membranes.
- Phosphorylation is required for mapmodulin's microtubule-associated protein-binding activity.
- Heat treatment of nonphosphorylated mapmodulin restored binding activity, indicating a conformational change.
Conclusions:
- Mapmodulin's phosphorylation state regulates its interaction with microtubule-associated proteins.
- Phosphorylation likely induces a conformational change necessary for function.
- Despite sequence homology, mapmodulin does not inhibit protein phosphatase 2A activity.