Investigating the Role of the Yeast Dynamin-like Protein Vps1 in Endocytosis

Endocytosis is a highly regulated process in most eukaryotic cells, and is essential for the cell to appropriately respond to changes in its environment. It is critical for recycling plasma membrane proteins and lipids and for the uptake/down-regulation of cell-surface receptors and cargo molecules ensuring appropriate membrane composition. Clathrin-mediated endocytosis (CME) is the best-studied form of endocytosis occurring in both mammals and in yeast. Stages of CME involve the formation of a clathrin-coated pit (CCP), which undergoes progressive invagination before scission occurs forming a clathrin-coated vesicle (CCV). Dynamins are a conserved family of large GTPases whose function is best understood in the context of CME specifically in the vesicle scission process. Mutations in dynamin have been implicated in various neurological disorders such as Epilepsy, Charcot-Marie Tooth and many others. There are no classical dynamins in yeast however, Vps1, a dynamin-like-protein has been shown to perform similar functions to the classical mammalian dynamins. The work in this thesis aimed to use yeast as a model system to increase our understanding of dynamins and their endocytic function. Specifically, mutations corresponding to disease causing mutations in dynamin1 and 2 were generated and studied in yeast Vps1. Attempts were also made to investigate whether mammalian Dynamin-2 can complement deletion of vps1 in any of its membrane trafficking roles. A chimeric construct was generated comprising vps1 with the InsertB region replaced by the Dynamin-2 PH domain to determine whether this was able to localize and function in yeast. Finally, the generation of stable cells with integrated Vps1 endocytic mutations allowed a screen to be carried out to identify genes, which when overexpressed rescue the temperature sensitivity associated with one of these endocytic mutations and thereby indicate cell processes or pathways that might impact on Vps1 function during endocytosis.