THE PLECKSTRIN HOMOLOGY DOMAINS OF DYNAMIN ISOFORMS REQUIRE OLIGOMERIZATION FOR HIGH-AFFINITY PHOSPHOINOSITIDE BINDING

The dynamins are 100-kDa GTPases involved in the scission event requir ed for formation of endocytotic vesicles. The two main described mamma lian dynamins (dynamin-1 and dynamin-2) both contain a pleckstrin homo logy (PH) domain, which has been implicated in dynamin binding to (and activation by) acidic phospholipids, most notably phosphoinositides. We demonstrate that the PH domains of both dynamin isoforms require ol igomerization for high affinity phosphoinositide binding. Strong phosp hoinositide binding was detected only when the PH domains were dimeriz ed by fusion to glutathione S-transferase, or via a single engineered intermolecular disulfide bond. Phosphoinositide binding specificities agreed reasonably with reported effects of different phospholipids on dynamin GTPase activity. Although they differ in their ability to inhi bit rapid endocytosis in adrenal chromaffin cells, the dynamin-1 and d ynamin-2 PH domains showed identical phosphoinositide binding specific ities. Since oligomerization is required for binding of the dynamin PH domain to phosphoinositides, it follows that PH domain mediated phosp hoinositide binding will favor oligomerization of intact dynamin (whic h has an inherent tendency to self-associate). We propose that the dyn amin PH domain thus mediates the observed cooperative binding of dynam in to membranes containing acidic phospholipids and promotes the self- assembly that is critical for both stimulation of its GTPase activity and its ability to achieve membrane scission.