Dynamin methods

Dynamin GTPase activity was measured essentially by the method of Barylko (2001). GTPase assay was performed in cytosolic buffer (25 mM Hepes-KOH, pH 7.2, 25 mM KCl, 2.5 mM magnesium acetate, 100 mM potassium glutamate) in 2.0 ml microcentrifuge tubes. One hundred /ul of reaction mixture contained protein and lipid at the following concentrations. 

We describe here methods for analyzing the interaction of fragments of Tuba with lipids and with dynamin. The property of the DH-BAR module of Tuba to bind liposomes in a cosedimentation essay (Fig. 1C) supports the hypothesis that the BAR domain of Tuba localizes and/or activates its DH domain at the membrane. The juxtaposition of four SH3 domains that bind dynamin generates a module with a striking avidity for this GTPase. Thus, the NH2-terminus of Tuba is a very convenient tool to deplete dynamin from tissue extracts as well as to affinity-purify this protein from tissue and cell extracts to near purity in one step. 

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... 

Dynamin hydrolyzes GTP into GDP and inorganic phosphate (Pi). Its GTPase activity can be stimulated by a number of protein or lipid effectors, or by its own self assembly. This method uses sonicated L-a-phosphatidyl-L-serine (PS, 100%) liposomes. PS liposomes facihtate dynamin self assembly (Lin et al, 1997 Tuma et al, 1993) and an optimal concentration of PS liposomes will stimulate maximal dynamin GTPase activity in vitro (Barylko et al, 2001) at very low cost. The protocol offers advantages in speed and... 

Barylko, B., Binns, D. D., and Albanesi, J. P. (2001). Activation of dynamin GTPase activity by phosphoinositides and SH3 domain-containing proteins. Methods Enzymol. 329, 486 96. 

Damke, H., Muhlberg, A. B., Sever, S., Sholly, S., Warnock, D. E., and Schmid, S. L. (2001). Expression, purification, and functional assays for self-association of dynamin-1. Methods Enzymol 329, 447 57. 

Recent studies have demonstrated that peroxisome division requires at least one dynamin-like protein, Vpslp, in the yeast Saccharomyces cerevi-siae and DLPl (DRPl) in mammalian cells. Although the requirement for these proteins in peroxisome division is supported by many lines of evidence, their roles in peroxisome division have yet to be identified. Given the independence of peroxisomes from other organelle systems, the peroxisome system appears to have unique attributes for studying the function of dynamin-like proteins in organelle division. Here, we present methods that have been used for studying the role of DLPl in peroxisome biogenesis and division. 

Method 2 Analysis of Conformational Changes of Dynamin on Preformed Lipid Nanotubes... 

We propose that lipid nanotnbes will be important for analysis of the conformational states of other members of the dynamin superfamily. However, it should be noted that these tubules are conformationally stable and may not be as easily squeezed as liposomes. Therefore conformational changes perpendicular to the membrane plane may be underestimated. Thus (as with the tubulation assay described in Method 1) conclusions must be verified in live cells, where mutants can be expressed and trafficking monitored (Marks et al, 2001). 

Opal, also known as Mgml in yeast, is a mitochondrial member of the dynamin family. Unlike other dynamin family members. Opal has an N-terminal mitochondrial targeting sequence, suggesting that this protein is imported into mitochondria. Here, we describe biochemical techniques, such as mitochondrial isolation, digitonin extraction, a protease protection assay, and carbonate extraction, that were used to determine that mammalian Opal resides in the intermembrane space where it is tightly bound to the inner membrane. In addition, we describe bacterial expression of the Opal GTPase domain, methods for purification, and an in vitro assay for GTP hydrolysis. 

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