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The assembly and structure of the chloroplast protein import machinery in plants

Project description

Chloroplast logistics and the role of protein import complexes

Cells are complex factories with individual membrane-bound organelles that help compartmentalise jobs and the resources needed to do them, enhancing efficiency. Cellular logistics can get pretty complicated given that proteins are coded in the nucleus, assembled on ribosomes and trafficked to their place of work throughout the cell. Chloroplasts are the photosynthetic organelles in plant cells, and they have specialised molecular machines that translocate proteins across their double-walled membrane envelope. The so-called translocon apparatus at the outer envelope membrane of chloroplasts (TOC) has at least two main configurations corresponding to the pre-protein cargo imported. The EU-funded TOC-maker project is investigating the details of the assembly process and the final structural organisation of the TOC complexes.

Objective

Plants convert solar energy into chemical energy by the process called photosynthesis in a specialized compartment of the cell known as chloroplasts. Chloroplasts are majorly enriched with nucleus-encoded proteins and to import them, chloroplast outer and inner envelope membranes are equipped with apparatus called the TOC and TIC translocons, respectively. For the TOC apparatus, there are two major configurations, TOC-P and TOC-H, reported so far, which import highly abundant, Photosynthetic and Housekeeping pre-proteins, respectively. TOC-P and TOC-H are multiprotein complexes which must be specifically assembled for proper development and homeostasis of the chloroplast. Due to the dynamic nature of the translocons, component synthesis and assembly must be rapid and tightly coupled, making the process difficult to investigate. Thus, understanding the mechanisms of the assembly process is both challenging and exciting. Biogenesis of TOC complexes is rapidly enhanced during chloroplast development or de-etiolation, and I will exploit this process to investigate the assembly of different TOC configurations, using the model plant Arabidopsis thaliana. For this purpose, transgenic plants expressing epitope-tagged TOC components and cells expressing nascent polypeptides of TOC components with stalled ribosomes will be generated. Proteins transiently interacting with new TOC components, which are predicted to assist integration and assembly of the TOC complex, will be studied by using affinity purification, pulse-chase experiments, and other biochemical techniques, and thus a sequence of assembly events will be elucidated. Although the molecular composition of the TOC protein import machinery has been well studied, the detailed structural organization of TOC complexes has not yet been elucidated. I will address this knowledge gap by analysing affinity-purified TOC complexes from mature chloroplasts at high resolution by cryo-electron microscopy.

Coordinator

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
Net EU contribution
€ 224 933,76
Address
WELLINGTON SQUARE UNIVERSITY OFFICES
OX1 2JD Oxford
United Kingdom

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Region
South East (England) Berkshire, Buckinghamshire and Oxfordshire Oxfordshire
Activity type
Higher or Secondary Education Establishments
Links
Total cost
€ 224 933,76