Substrates of the N-end rule of targeted protein degradation

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Proteins are the workhorses of the cell and perform a fascinatingly wide range of tasks. To perform their jobs correctly, proteins need to be in the right place, at the right time and in the right amount. There are several ways in which protein amounts can be regulated in space and in time. Much attention has been focussed on different processes which control protein synthesis, but the importance of protein breakdown has only relatively recently been appreciated. Previously, protein breakdown (known as proteolysis) was seen in a negative light, associated with cell damage and death. However, it is now known that controlled proteolysis is an extremely important mechanism which enables protein abundance to be modulated with elegance and precision.In almost all organisms, there is a specialised form of controlled, targeted proteolysis, called the N-end rule pathway. The pathway has been elucidated using artificial "reporter" proteins and it has been shown that a given protein can be cleaved (broken by an enzyme) to reveal a new front end (the "N-terminus"). The identity of the amino acid at the new N-terminus determines the fate of the protein in the cell: whether it is targeted for proteolysis or not, hence the name, N-end rule. In animals, proteins involved in the development of nerves and the heart are controlled in this manner and the N-end rule pathway also plays important roles in fertility and DNA repair. Recent evidence has shown that this pathway regulates key stages of plant development and metabolism. We know this because plants display a range of dramatic defects if the specific enzymes needed for proteolysis are absent: seeds cannot germinate properly, seedlings cannot break down stored oil and grow into adult plants, seedlings cannot respond appropriately to altered oxygen levels and leaves do not develop normally. This tells us that the N-end rule pathway controls the levels of important proteins, including proteins which regulate plant development. However, the identities of the proteins which are broken down by the pathway (the "substrates") are unknown and difficult to discover by conventional methods. This proposal seeks to solve this problem by adapting three complementary methods for protein identification to compare normal plants with plants which lack N-end rule enzymes.The methods developed in this project and the information obtained can be more widely useful in studying a range of different organisms. In the longer term, this project will also provide important information about plant physiology which can be applied to help develop better crops.