Substrates of the N-end rule of targeted protein degradation

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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.