| Dynamin is an essential factor in many endocytic processes, including receptor-mediated endocytosis (RME). The Pleckstrin Homology (PH) domain is a common protein domain that interacts with membrane phospholipids, and in some cases can mediate membrane association of its host protein. My work addresses the role of the PH domain of dynamin in endocytosis. The question of whether the dynamin PH domain binds with phosphoinositides was examined, as well as the affinity and specificity of binding. The role of phosphoinositide binding and the function of the dynamin PH domain in endocytosis were studied. Methods employed for the studies include dot blot, vesicle centrifugation assay, analytical ultracentrifugation experiment, transferrin uptake assay, and immunofluorescence microscopy. We found that the dynamin PH domain binds with low affinity and specificity to phosphoinositides, and it is unlikely to mediate membrane association for its host protein. However, phosphoinositide binding by the dynamin PH domain is crucial to RME, because a dynamin mutant with a phosphoinositide-binding deficient PH domain inhibits RME in a dominant negative fashion. Oligomerization of the dynamin PH domain increases its affinity for phosphoinositides by an avidity effect. This is relevant to dynamin and its action in RME because dynamin self-assembles into tetramers and assembles into rings/spirals, which are required for the scission of the necks of coated pits. Although the dynamin PH domain does not appear to have a role in the initial recruitment of dynamin to the coated pits, it is likely to be involved in the redistribution of dynamin to the necks of the coated pits. |
