Substrates of the N-end rule of targeted protein degradation

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.

Targeted protein degradation is a quantitatively and qualitatively important mechanism for the control of metabolism and development, especially in plants. The N-end rule pathway (NERP) of targeted proteolysis associates the fate of a protein substrate with the identity of its N-terminus. A destabilising N-terminal residue which commits a protein for proteasomal degradation is created through a specific initial proteolytic cleavage, but can also be generated via successive enzymatic or chemical modifications to the N-terminus, for example, arginylation by Arg-tRNA protein transferases (ATE). Genetic studies using artificial substrates have revealed that NERP architecture is conserved between yeast, mammals and plants, but that the pathway plays a range of different important physiological roles in different organisms. However, only very few protein substrates have been identified.

This proposal seeks to identify Arabidopsis thaliana NERP substrates using three, complementary proteomics strategies and mutants defective in the arginine branch of the NERP (which lack arginyl transferases, ATE 1 ATE 2 and the E3 ligase, PRT6). Firstly, we will adopt a comparative, positional proteomics approach to identify and quantify N-termini in WT and mutant backgrounds. In parallel, we will isolate PRT6 binding proteins using a pull-down strategy. Since studies in mammals have shown that not all post-translationally arginylated proteins are NERP substrates, we will identify ATE1/2-interacting proteins via tandem affinity purification, to learn more about the role and interaction of these enzymes in the pathway. Finally, we will test whether the identified proteins are indeed substrates by following their fate in the plant and will attempt to link this information to physiology by producing forms of the proteins that cannot be degraded by the N-end rule to see if they recapitulate any of the defects seen in mutant plants.

This proposal is focused on fundamental research, deploying new methods to solve a biological problem: identification of substrates for the N-end rule pathway of targeted protein degradation. As such, this research is expected to benefit researchers in the first instance. The knowledge base and technology developed can benefit projects across the range of BBSRC's strategic priorities but especially 'food security', 'bioenergy and industrial biotechnology' and "synthetic biology". Although this project employs the model plant, Arabidopsis thaliana, knowledge and techniques are applicable to other plant species. Academic beneficiaries not only include plant scientists and researchers with an interest in proteomics and protein degradation but also synthetic biologists. The N-end rule has a number of potential applications, including the development of transgenic lines permitting the conditional removal of key proteins. A better understanding of the substrates and molecular mechanisms of this pathway will facilitate development and use of such resources. Knowledge and resources generated in this project are also of potential interest to plant breeders, either via an improved understanding of plant development and/or through potential transgenic routes to crop improvement. Routes by which outcomes will be communicated to potential beneficiaries are outlined in "Pathways to Impact". Finally, one of the most important outcomes of this project will be an experienced postdoctoral scientist trained in a range of state-of-the art proteomics techniques.