Plastids are essential organelles that are present in every plant cell. Plastids include chloroplasts, the sites of photosynthetic energy generation in plants and algae, amyloplasts, which store starch in roots and tubers, and chromoplasts, which accumulate carotenoids in fruits. Around 14% percent of genes in the plant genome are predicted to encode plastid proteins, underlining the importance of the plastid for plant biology. This project concerns the function and substrates of the essential plastid localized Clp protease machinery in the model plant Arabidopsis. The ClpPR protease is a double ring shaped complex containing 10 different ClpPR proteins, most of which are essential for embryogenesis or seedling development. Attached to the barrel are small ClpS1,2 proteins with unknown function and unique to photosynthetic organisms. Intracellular proteases have many functions and must be highly selective in recognizing their substrates. The plastid-localized Clp proteins are clearly important for plastid biogenesis and differentiation, but their mechanism of action in plastids, including Clp substrates and substrate recognition mechanisms, are unknown. The objectives of the project are to identify Clp protease substrates and their degradation signals, to determine the role of ClpS in substrate delivery to the Clp protease system, to determine the function of ClpT1,T2 as a specific plastid component of the Clp system, and to test potential substrates and degradation signals (degrons) identified in objectives 1, 2 and 3 (or in new literature) in vivo.
Broader impacts: This project will help to determine the molecular function and organization of the Clp protease system, as well as its substrates and substrate recognition mechanisms. Understanding the role of the Clp proteolytic machinery and substrate recognition signals is also critical to control protein stability with the objective to improve the plastid as the site for overproduction of foreign proteins with nutritional or other added value. The proposed research will provide multi-disciplinary training in plant molecular genetics and biochemistry at the undergraduate, graduate and post-doctoral levels. The PI will participate in curriculum development workshops for high school teachers to develop genomics-based modules for the classroom.
Proteases are enzymes that cut other proteins into smaller fragments. This cleavage can help to activate proteins or to remove proteins that are unwanted, e.g. because there are damaged or not needed anymore. Proteases are found in all species on earth including bacteria, mammals and plants. The objective of this project was to study the role of a specific and abundant protease system in the chloroplasts of plants. Chloroplasts carry out photosynthesis through absorption of sunlight and using this energy to convert CO2 into carbohydrates, while releasing oxygen into the atmosphere. Chloroplasts contain several thousands of different proteins in one or more copies. For a functional chloroplast and thus a healthy plant, it is essential that the protein population in the chloroplast is of the correct composition without accumulation of damaged or otherwise unwanted proteins. In this project, we studied the abundant plastid-localized Clp protease system involving at least 15 different proteins. We have demonstrated that the Clp system is essential for growth and development of the plant Arabidopsis; from these results we predict that the Clp system is equally important in other plant species on earth. Biochemical and genetic analyses showed that the plastid Clp system consists of a barrel-shaped ClpPR core complex as well as ATP-dependent Clp chaperones which receive, unfold and transfer substrates into the ClpPR cavity. In the project we have determined that each of the different ClpP and ClpR proteins that make up the Clp protease complex have unique contributions to the Clp protease function. Furthermore, we discovered a new chloroplast protein that we named ClpS1. We show that ClpS1 interacts with a specific set of chloroplast proteins most likely with the purpose to deliver them to the Clp chaperones for unfolding and subsequent degradation. Unique to plants, there are two essential chloroplast ClpT proteins that interact with the ClpPR core. In collaboration with others, we determined the high resolution structures of these ClpT proteins by crystallography. Based on this structural information, specific amino acid residues with ClpT were tested in Arabidopsis plants for their predicted role in the interaction with the ClpPR protease complex. This showed that the ClpT proteins help to stabilize the Clp protease complex and likely have an additional, but as yet unknown, function. Several undergraduate and graduate students, as well as postdoctoral researchers carried out this project and received multi-disciplinary training in molecular genetics, biochemistry and proteomics. Results of this project have been published in several international journals and further disseminated through poster and oral presentations are (inter)national conferences and institutes.
