Mutation of key lysine residues in the Insert B region of the yeast dynamin Vps1 disrupts lipid binding and causes defects in endocytosis.

The yeast dynamin-like protein Vps1 has roles at multiple stages of membrane trafficking including Golgi to vacuole transport, endosomal recycling, endocytosis and in peroxisomal fission. While the majority of the Vps1 amino acid sequence shows a high level of identity with the classical mammalian dynamins, it does not contain a pleckstrin homology domain (PH domain). The Dyn1 PH domain has been shown to bind to lipids with a preference for PI(4,5)P2 and it is considered central to the function of Dyn1 in endocytosis. The lack of a PH domain in Vps1 has raised questions as to whether the protein can function directly in membrane fusion or fission events. Here we demonstrate that the region Insert B, located in a position equivalent to the dynamin PH domain, is able to bind directly to lipids and that mutation of three lysine residues reduces its capacity to interact with lipids, and in particular with PI(4,5)P2. The Vps1 KKK-AAA mutant shows more diffuse staining but does still show some localization to compartments adjacent to vacuoles and to endocytic sites suggesting that other factors are also involved in its recruitment. This mutant selectively blocks endocytosis, but is functional in other processes tested. While mutant Vps1 can localise to endocytic sites, the mutation results in a significant increase in the lifetime of the endocytic reporter Sla2 and a high proportion of defective scission events. Together our data indicate that the lipid binding capacity of the Insert B region of Vps1 contributes to the ability of the protein to associate with membranes and that its capacity to interact with PI(4,5)P2 is important in facilitating endocytic scission.