DYNAMIN FORMS POLYMERIC COMPLEXES IN THE PRESENCE OF LIPID VESICLES -CHARACTERIZATION OF CHEMICALLY CROSS-LINKED DYNAMIN MOLECULES

Dynamin is a GTP-binding protein that is involved in the release of co ated endocytic vesicles from the plasma membrane, We have been charact erizing the enzymatic properties of purified rat brain dynamin to bett er understand how GTP binding and hydrolysis relate to its proposed fu nction, Previously, we have demonstrated that activation of dynamin GT Pase results from positive cooperative associations between dynamin mo lecules as they are bound to a polymeric surface, Our present report h as extended these studies and has examined the structural features of dynamin self association. After treatment with the zero length protein cross-linking reagent, 1-ethyl-3-[3-(dimethylamino)propyl]carbo ide, dynamin in solution was found cross linked into dimers, This homodimer likely reflects the native soluble state of the molecule, After bindi ng to brain vesicles, dynamin was cross-linked into higher order oligo mers of greater than 800 kDa, Dynamin, copurified on brain membranous organelles, also formed multimeric complexes when cross-linked suggest ing dynamin exists in polymeric form in vivo. No cross linked species other than homo-oligomers were observed, providing no evidence for clo se interactions between dynamin and membrane proteins, From experiment s examining the effects of GTP, GDP, guanosine 5'-3-O-(thio)triphospha te, and 5'-guanylyl-beta,gamma-imidodiphosphate on cross-linking, we h ave determined that both dynamin membrane binding and self-association occur independently from the nucleotide-bound state of the enzyme, An 80-kDa dynamin fragment that is lacking its carboxyl-terminal domain is not cross-linked into higher order oligomers, suggesting that this domain is required for binding of dynamin to membranes and the subsequ ent enhancement of oligomerization, However, the dynamin fragment was found to form dimers indicating that this domain is not required for d ynamin dimerization, Cross linked dynamin was able to cooperatively bi nd microtubules, but did not exhibit GTPase activation, We propose tha t intramolecular cross links in the dynamin monomer impart structural constraints that prevent the enhancement of GTP hydrolysis, We describ e a model of the dynamin activation process to be considered in furthe r investigations of the role for dynamin in endocytic vesicle formatio n.