Functional characterization of the PRAF family in plant endomembrane trafficking
Creators: name: Li, Dongmeng
Publisher: name: No Publisher Supplied
Year: 2023

description: Polarity (unequal distribution of substances) is a fundamental feature of a cell and cell polarization underlies cellular and developmental processes in multicellular organisms. Cell polarization is manifested by the asymmetric distribution of biological molecules, such as proteins and lipids, much of which heavily rely on the precisely controlled actions of endomembrane trafficking. In the Arabidopsis thaliana stomatal lineage cells, the polarization machinery and mechanism underlying the intrinsic scaffold protein BASL (BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE) remain largely unknown, in particular from the perspective of endomembrane-based vesicle trafficking.In Chapter 1, I summarize current research advances on the plant endomembrane trafficking system with highlights on the commonalities and uniqueness of the plant endomembrane system. I discuss the key components and regulators in the plant endomembrane trafficking, with an emphasis on the Rab GTPases and their activators, Rab GEFs. Chapter 2 is a reformatted publication, on which I was co-first authored. Our collaborative effort demonstrates the four members from a plant specific endomembrane trafficking regulator PRAF (PH, RCC1 and FYVE) protein family are required for the establishment of BASL polarization. Simultaneous mutations of these four PRAF members (4/5/8/9) lead to severe defects in stomatal development and plant growth, phenocopying those of a gnom mutant. GNOM is an evolutionary conserved Arf GEF and has been shown to play critical roles in establishing both embryonic and post-embryonic cell polarity. Our data prove that PRAFs and GNOM physically interact in vitro and in vivo. In addition, I find that PRAF8 is localized to specific post-Golgi endosomes. Thus, we propose that the connected function of PRAF-GNOM in the post-Golgi endosomal trafficking controls the establishment of cell polarity in the stomatal lineage.In Chapter 3, I demonstrate that individual PRAF members show preferential binding activities with specific Rab GTPases via the RCC1 domain, a putative Rab GEF domain. These protein-protein interactions are supported by yeast two-hybrid, BiFC, and co-localization in plant cells, collectively suggesting PRAF4 interacts with RabD2 and RabE1 in the post-Golgi to plasma-membrane secretion pathway. Furthermore, I find the PRAF4-RabE interaction complex specifically recruits SEC5A, an exocyst component, but not VAMP721, a SNARE protein, indicating a specific functional connection that directs PRAF4-mediated protein secretion. Finally, genetic complementation assays show the overexpression of constitutively active forms of Rab GTPases partially complements the dwarf and sterile defects of the praf mutants, providing direct genetic evidence that Rab GTPases function downstream of PRAFs in membrane trafficking. In Chapter 4, I propose future directions for the PRAF-Rab GTPase studies based on the previous data. Also, I highlight several recent technical breakthroughs in the plant endomembrane research and discuss possibilities of these applications in the PRAF-Rab project.Overall, my work on PRAF family members 1) contributed to the understanding of the novel regulation of BASL polarization from the perspective of endomembrane-mediated trafficking processes in plant cells; 2) revealed the function of PRAF proteins in regulating the plant endomembrane trafficking, especially the post-Golgi trafficking; 3) identified specific Rab members from a large 57-member containing Rab GTPase family that function with specific PRAFs and their RCC1 domain; 4) proposed potential functions of PRAF-Rab GTPase in the post-TGN/EE to PM secretion pathway.