Rapid Purification of Native Dynamin I and Colorimetric GTPase Assay

doi:10.1016/S0076-6879(05)04049-8

Methods in Enzymology

Volume 404, 2005, Pages 556-569

https://doi.org/10.1016/S0076-6879(05)04049-8 Get rights and content

Abstract

Dynamin I is a large GTPase enzyme required in membrane constriction and fission during multiple forms of endocytosis. The first method described here is for the rapid purification of native dynamin from peripheral membrane extracts of sheep brain using ammonium sulfate precipitation and affinity purification on recombinant SH3 domains. The method greatly enriches for dynamin I at high purity and allows for large‐scale biochemical and functional studies. The second method is a nonradioactive, high‐throughput colorimetric GTPase assay for dynamin activity. The approach is based on terminating incubations with EDTA and the use of malachite green for high‐sensitivity detection of inorganic phosphate release. The two methods will facilitate high‐throughput screens for potential dynamin inhibitors or activators.

Introduction

Dynamin is a 96‐kDa GTPase enzyme involved in membrane constriction and fission during receptor‐mediated endocytosis (RME) and synaptic vesicle endocytosis (SVE). At a late stage of the process, dynamin assembles into rings to form a collar or helix around the neck of the invaginated vesicles. Upon GTP hydrolysis it pinches them from the plasma membrane. Dynamin is also needed for most, but not all, forms of non‐clathrin‐dependent endocytosis, such as phagocytosis, caveolae internalization, and endocytosis of cytokine receptors. Dynamin contains four functional domains: an N‐terminal GTPase domain, a pleckstrin homology (PH) domain, a proline‐arginine rich domain (PRD), and an assembly domain (Cousin and Robinson, 2001). In a Drosophila strain called shibire, mutations in dynamin's GTPase domain allow assembly of dynamin helices at the nascent vesicle neck and block a late stage of synaptic vesicle fission (Koenig and Ikeda, 1989). The mutations do not block GTP binding, but block GTP hydrolysis. Overexpression of GTPase‐defective dynamin mutants inhibits both RME and SVE in a variety of cells (Marks et al., 2001). Thus the GTPase activity of dynamin is a potentially attractive candidate for development of a specific endocytosis inhibitor. We describe a method for the large‐scale rapid purification of dynamin I and a sensitive colorimetric high‐throughput GTPase assay that together will facilitate the screening of large chemical libraries for dynamin inhibitors.

Section snippets

Purification of Native Dynamin I

Purification of native dynamin I from sheep brain using affinity purification provides a rapid and reproducible technique for isolation of high levels of biologically active native dynamin I without the use of large‐scale ion exchange columns, detergents, or guanosine nucleotides. The affinity ligand is bacterially expressed glutathione S‐transferase (GST) fused to the amphiphysin II‐SH3 domain, which is easy to produce in large quantities. The McMahon lab demonstrated the utility of using

Colorimetric GTPase Assay

We and others have developed a sensitive, nonradioactive malachite green based colorimetric assay to measure inorganic phosphate released by dynamin I's GTPase activity (Hill 2004, Song 2004, Song 2004). The most sensitive colorimetric assays for inorganic phosphate are based on the formation of a phosphomolybdate complex at low pH (Hohenwallner and Wimmer, 1973). The colorless phosphomolybdate complex is converted to a colored complex in the presence of an enhancer, which is a basic pH

Acknowledgments

We thank Timothy Hill and Adam McCluskey (The University of Newcastle, Australia) for advice and assistance in developing the malachite green colorimetric assay. We thank Victor Anggono (Children's Medical Research Institute) for Fig. 1A and Ngoc Chau (Children's Medical Research Institute) for her technical assistance. We also thank Byeong Doo Song and Sandra Schmid (The Scripps Research Institute, La Jolla, CA) for initial discussions on the colorimetric assay.

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In experiments studying the effect of CaM, 1.2 mM CaCl2 and 1.0 mM EGTA were added to the reaction buffer (0.2 mM free [Ca2+]). The reaction was stopped with the addition of EDTA (final 20 mM) and liberated Pi was assayed using the malachite green method [19,22]. Since both NAP-22 (prepared from brain and expressed in bacteria) showed basically same inhibitory effect on dynamin GTPase, bacteria-expressed NAP-22 was used throughout the inhibition assay.
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Intellectual disability (ID) is a prevalent developmental disorder of cognition that remains incurable. Here, we report that knockdown of the X-linked ID (XLID) protein polyglutamine-binding protein 1 (PQBP1) in neurons profoundly impairs the morphogenesis of the primary cilium, including in the mouse cerebral cortex in vivo. PQBP1 is localized at the base of the neuronal cilium, and targeting its WW effector domain to the cilium stimulates ciliary morphogenesis. We also find that PQBP1 interacts with Dynamin 2 and thereby inhibits its GTPase activity. Accordingly, Dynamin 2 knockdown in neurons stimulates ciliogenesis and suppresses the PQBP1 knockdown-induced ciliary phenotype. Strikingly, a mutation of the PQBP1 WW domain that causes XLID disrupts its ability to interact with and inhibit Dynamin 2 and to induce neuronal ciliogenesis. These findings define PQBP1 and Dynamin 2 as components of a signaling pathway that orchestrates neuronal ciliary morphogenesis in the brain.
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Dynamin was then eluted from the column with buffer 4 (20 mM Hepes-KOH, 1.2 M NaCl, 1 mM DTT, pH 6.5), dialyzed to buffer 5 (10 mM Hepes-KOH, 50 mM NaCl, 1 mM MgCl2, 0.2 mM EGTA, 1 mM DTT, pH 7.6) and used immediately or stored at −80°C after the addition of glycerol to 20%. The amount of dynamin II and III in this fraction is known to be less than 1% of dynamin I [17]. Primary culture of neonatal rat cortex neuron was performed as described [22].
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Phosphorylation and nitration of protein tyrosine residues are thought to play a role in signaling pathways at the nerve terminal and to affect functional properties of proteins involved in the synaptic vesicle (SV) exo-endocytotic cycle. We previously demonstrated that the tyrosine residues in the C-terminal domain of the SV protein Synaptophysin (SYP) are targets of peroxynitrite (PN). Here, we have characterized the association between SYP and c-src tyrosine kinase demonstrating that phosphorylation of Tyr₂₇₃ in the C-terminal domain of SYP is crucial in mediating SYP binding to and activation of c-src. SYP forms a complex with Dynamin I (DynI), a GTPase required for SV endocytosis, which may be regulated by tyrosine phosphorylation of SYP. We here report that, in rat brain synaptosomes treated with PN, the formation of SYP/DynI complex was impaired. Noteworthy, we found that DynI was also modified by PN. DynI tyrosine phosphorylation was down-regulated in a dose-dependent manner, while DynI tyrosine nitration increased. Using mass spectrometry analysis, we identified Tyr₃₅₄ as one nitration site in DynI. In addition, we tested DynI self-assembly and GTPase activity, which are enhanced by c-src-dependent tyrosine phosphorylation of DynI, and found that both were inhibited by PN. Our results suggest that the site-specific tyrosine residue modifications may modulate the association properties of SV proteins and serve as a regulator of DynI function via control of self-assembly, thus influencing the physiology of the exo-endocytotic cycle.

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Rapid Purification of Native Dynamin I and Colorimetric GTPase Assay

Abstract

Introduction

Section snippets

Purification of Native Dynamin I

Colorimetric GTPase Assay

Acknowledgments

Methods Enzymol.

Methods Enzymol.

Anal. Biochem.

Trends Neurosci.

Anal. Biochem.

Bioorg. Med. Chem. Lett.

Clin. Chim. Acta

J. Biol. Chem.

J. Biol. Chem.