How is vesicle uncoating achieved? Dissecting early events in clathrin disassembly by the molecular chaperone, Hsc70

Cells use the movement of vesicles as their primary transport system for many important compounds. This method of transportation is termed endocytosis and vesicle trafficking . Substances are brought into the cell via endocytosis, during which a vesicle is formed from the cell membrane which encloses specific molecules. A specialised protein coat is required around the forming vesicle which helps select the contents and also directs the time and place of formation and detachment. The coat assembles and disassembles on the vesicle in a precisely controlled manner which is vital to the correct functioning of the cell. We wish to study in molecular detail how the coat is removed from the vesicle and what events are important for triggering this uncoating event. We will use a range of different physical methods to see which proteins bind at which point in time and whether their shape alters during uncoating. We will also use special mutant forms of Hsc70 which only perform certain steps along this pathway to dissect out the order of these steps and their importance in uncoating. Hence these studies will produce a picture of the initiation of uncoating of the vesicle coat and allow us to see how this intriguing molecular machine works.

The aim of this project is to investigate the early events of clathrin disassembly by the molecular chaperone, Hsc70, so as to understand what events cause the uncoating of clathrin-coated vesicles. We will view the process using biophysical methods which report on changes in protein conformation, protein-substrate binding events, ATP hydrolysis and clathrin disassembly. By monitoring disassembly with these parallel techniques, we will obtain a detailed picture of clathrin disassembly which will relate to the dynamics of the vesicle uncoating process. We will use well-studied mutants of Hsc70 which are specifically impaired in certain functions of the chaperone, e.g. ATP hydrolysis, to pinpoint when, in relation to other steps, these events occur and which binding events and conformational changes are associated with a particular step. Initially we will work on the simplest system available, clathrin cages, Hsc70 and auxilin401-910. Once this has been thoroughly characterized, we will move towards the more complex cellular situation by introducing further components, such as adaptor proteins, which are essential components of clathrin-coated vesicles and have been implicated in uncoating. Thus we will determine in molecular detail the nature of the events which cause uncoating of clathrin-coated vesicles and so shed light on how the process is regulated in the cell.