Analysis of LLPS in Plant Intracellular Protein Sorting

Plant communities can regulate plant growth and developmental processes through liquid-liquid phase separation (LLPS). CD BioSciences is committed to studying the emerging role of plant phase separation with the aim to provide comprehensive analytical services to explore LLPS in plant intracellular protein sorting, with a particular focus on intrachloroplast cargo sorting.

Introduction to LLPS Phenomenon on Protein Sorting Within Chloroplasts

A typical plant or animal cell contains up to 10,000 different types of proteins, and each of these many proteins must be precisely targeted to function properly. Chloroplasts contain 3,000 proteins, only a small fraction of which are encoded by the chloroplast genome. Most of the nuclear-encoded chloroplast proteins are synthesized in the cytoplasm as precursors to N-terminal targeting signal transport peptides and are imported post-translationally into different organelles. However, the mechanism by which imported nuclear-encoded chloroplast proteins migrate across the crowded stroma to the vesicle-like membrane is poorly understood. Interestingly, LLPS was found in chloroplasts to facilitate the formation of droplets within the organelle to sort proteins into the vesicle-like lumen. The formation of phase-separated droplets becomes a novel mechanism for cargo sorting in chloroplasts.

Fig. 1. Liquid-liquid phase transition drives intrachloroplast cargo sorting. (Pardi SA, et al., 2021)

Customized Services

Our technical team focuses on analyzing LLPS for cargo sorting in chloroplasts to help you understand the mechanisms of chloroplast protein sorting and targeting.

CD BioSciences offers an integrated approach of high-resolution real-time imaging and biophysics to analyze the role of LLPS in intrachloroplast protein sorting. We can identify and characterize two Arabidopsis anchor protein repeat proteins, STT1 and STT2, that specifically mediate the sorting of chloroplast double arginine translocation (cpTat) pathway proteins to the vesicle-like membrane via LLPS. Our strategies are as follows:

• We provide cutting-edge computational tools and protein prediction tools to analyze the intrinsically disordered regions of STT1 and STT2.
• Our crystal structure studies help you identify the structural domains of STT1 and STT2.
• We provide isothermal titration calorimetry and pull-down analysis to investigate the importance of weak multivalent interactions between the STT complex and cpTat signal peptide for phase separation.
• We have further investigated STT function by detecting STT1-STT2 interactions using the bimolecular fluorescence complementation (BiFC) technique of Arabidopsis protoplasts.
• We provide delayed differential interference contrast imaging (DIC), fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP) to analyze STT1 and STT2 LLPS in vitro and in vivo.

Based on high-resolution microscopy, proteomics, genomics, structural, computational, and biophysical approaches, CD BioSciences aims to help you understand the emerging mechanisms by which STTs drive LLPS as cpTat substrate sorting during plant evolution. Our experts are also developing phylogenetic analyses to search for the origin of STTs and to identify molecular chaperones of the cpTat pathway in LLPS. If you have any special requirements for our services, please feel free to contact us.