CLPS1-CLPF ADAPTORS FOR PROTEASE SUBSTRATE SELECTION IN CHLOROPLASTS

Cornell University, Ithaca NY

Investigators

Abstract

Proteolysis, the breakdown of proteins or peptides to amino acids, is critical for removal of unwanted or damaged proteins and regulation of cellular processes such as metabolism. Chloroplasts are essential plant organelles that are important in biomass productivity and agriculture. However, the determinants of chloroplast protein life-time and degradation are poorly understood. Cells and organelles contain hundreds of proteolytic systems. This research will provide insight into the role of the central chloroplast Clp protease system, consisting of protein chaperones, a protease core complex and several proteins involved in selection and delivery of specific Clp protease substrates. The research findings can also be implemented in molecular farming and synthetic biology (e.g. through tunable protein degradation), since cellular compartments such as chloroplasts are favored for overexpression of products with nutritional or pharmaceutical value. The outcome of this research will allow more rational protein design for stable accumulation of chloroplast proteins, thus directly impacting these applications. This project will provide training in proteomics, mass spectrometry, molecular genetics, biochemistry, structural biology at the undergraduate, graduate and post-doctoral levels. Summer internships will be offered through our NSF-sponsored REU programs. This project is built on extensive investments in biological materials such as Arabidopsis protease mutants, previously identified candidate Clp substrate or Clp adaptors, as well as expertise in mass spectrometry, proteomics and structural biology. The central objective of this proposal is to unravel the functional, structural, and mechanistic details of a novel ClpS1-ClpF adaptor system, including substrate selection, substrate half-life, and interactions with Clp chaperones. It may also lead to the discovery of additional Clp adaptors. We will use our in vivo tools to test the physiological role of ClpS1 and ClpF in substrate selection. In vivo degradation assays will test the impact of Clp components on the half-life of candidate substrates. High-resolution structural information through X-ray crystallography or CryoEM will be obtained for ClpS1 and ClpF, with their functional partners. In vitro and in planta interaction assays and functional complementation will determine the role of protein domains. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.