Dominant-negative inhibition of receptor-mediated endocytosis by a dynamin-1 mutant with a defective pleckstrin homology domain

The dynamins are 100 kDa GTPases involved in the scission of endocytic vesi cles from the plasma membrane [1], Dynamin-1 is present in solution as a te tramer [2], and undergoes further self-assembly following its recruitment t o coated pits to form higher-order oligomers that resemble 'collars' around the necks of nascent coated buds [1,3], GTP hydrolysis by dynamin in these collars is thought to accompany the 'pinching off' of endocytic vesicles [ 1,4]. Dynamin contains a pleckstrin homology (PH) domain that binds phospho inositides [5,6], which in turn enhance both the GTPase activity [5,7,8] an d self-assembly [9,10] of dynamin. We recently showed that the dynamin PH d omain binds phosphoinositides only when it is oligomeric [6]. Here, we demo nstrate that interactions between the dynamin PH domain and phosphoinositid es are important for dynamin function in vivo. Full-length dynamin-1 contai ning mutations that abolish phosphoinositide binding by its PH domain was a dominant-negative inhibitor of receptor-mediated endocytosis. Mutated dyna min-1 with both a defective PH domain and impaired GTP binding and hydrolys is also inhibited receptor-mediated endocytosis. These findings suggest tha t the role of the PH domain in dynamin function differs from that seen for other PH domains. We propose that high avidity binding to phosphoinositide rich regions of the membrane by the multiple PH domains in a dynamin oligom er is critical for dynamin's ability to complete vesicle budding. (C) Elsev ier Science Ltd ISSN 0960-9822.