Tail-anchored protein biogenesis: defining the ATP dependent route

Living things are made up of cells, and cells need membranes to provide barriers inside the cell and to protect them from the outside world. Whilst the biological membranes that form these barriers are incredibly complex and diverse in nature, they are generally characterised by a lipid bilayer that is studded with many proteins. Cells must normally replenish and renew their membranes by making new ones, and a key part of this process is the insertion or stitching of new proteins into the lipid bilayer. In more complex cells, such as those that make up our body, this integration of membrane proteins is one of the key functions of a specialised compartment known as the endoplasmic reticulum. As is the case for many biological processes, it has become clear that there are different ways of integrating a protein into the lipid bilayer of the endoplasmic reticulum. One mechanism is now fairly well understood and can be described as the 'classical' route for membrane proteins synthesis at the endoplasmic reticulum. Whilst the precise details of this classical pathway remain to be fully determined, we know most of the cellular factors and machineries that mediate it. In contrast, a second quite different pathway has been far less well studied so we know very little about the cellular components that are responsible for it or exactly what they do. This second pathway is used to make an important class of molecules called tail-anchored membrane proteins that are characteristic of more complex cells such as our own. The aim of this project is to identify the cellular machines that are responsible for putting tail-anchored proteins into the membrane of the endoplasmic reticulum. We will make use of the fact that we can now replicate this second pathway in a test tube, allowing us to compare how well different combinations of cellular components can carry out tail-anchored protein insertion. We are particularly interested in looking at the contribution of cellular machines known as molecular chaperones, since we have good reason to believe they play a major role in putting tail-anchored proteins into the endoplasmic reticulum. There are many different chaperones, and our work will tell us which particular chaperones are important for the second pathway and what exactly it is that they do during this process.

The primary objective of this project is to investigate the role of cytosolic factors during the biosynthesis of tail-anchored membrane proteins at the endoplasmic reticulum (ER). Tail-anchored proteins are unusual in that their membrane integration at the ER is post-translational, and is quite distinct from the biosynthesis of most other membrane proteins. There is now a substantive body of evidence indicating that there are in fact two alternative pathways that can support tail-anchored protein integration. We have shown that one of these routes is mediated by the signal recognition particle, acting in a novel and previously uncharacterised post-translational fashion. The second route is poorly defined and its major characteristic is that it requires ATP and one or more cytosolic factors to operate efficiently. On the basis of such observations, it is widely assumed that one or more molecular chaperones facilitate this ATP dependent route. However, there is no direct experimental evidence that this is truly the case. We have now developed an experimental system that will allow us to identify the cytosolic factors that mediate the ATP dependent integration of tail-anchored proteins at the ER, and the aim of this project is to exploit this system in full. With hindsight it is clear that different tail-anchored proteins use different pathways, i.e. signal recognition particle dependent or ATP dependent, and some precursors can use both. We will therefore begin by clearly identifying a set of model tail-anchored proteins that are exclusively or primarily integrated via the ATP dependent route. We have already shown that cytochrome b5 is integrated via the ATP dependent pathway, and it will then be taken forward with the other selected tail-anchored proteins to define the cytosolic factors that mediate this process. A key feature of our assay is that we can purify the tail-anchored proteins away from the cytosol used to synthesise them. Having separated the tail-anchored proteins from these cytosolic factors, we are then in a position to add back defined components and reconstitute the post-translational membrane integration of tail-anchored proteins into ER membranes. By using variously treated cytosols, different combinations of purified chaperones, and selective inhibitors we will be able to identify the proteins that mediate the ATP dependent pathway. We can also exploit membrane integration as a read out in order to identify any novel cytosolic components that stimulate tail-anchored protein insertion by purifying them from fractionated cytosol. Having identified the molecular chaperones and cytosolic factors that facilitate the ATP pathway for tail-anchored protein integration, we will use a variety of strategies to determine the mechanism that underlies this process. In particular, we will seek to establish whether the role of the chaperones is purely to prevent aggregation and keep the tail-anchored proteins in an 'insertion competent' conformation, or whether the chaperones interact directly with ER specific receptors and actively deliver the tail-anchored proteins to the site of membrane insertion. As a whole this project will enable us to identify the cytosolic factors that mediate the ATP dependent pathway for tail-anchored protein integration at the ER, and establish the molecular basis by which this process is achieved.