Identification and characterization of unconventional membrane protein trafficking regulators in Arabidopsis: A genetic approach

Proper trafficking and subcellular localization of membrane proteins are essential for plant growth and development. The plant endomembrane system contains several membrane-bound organelles with distinct functions including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN) or early endosome, prevacuolar compartment (PVC) or multivesicular body (MVB) and vacuole. Multiple approaches have been successfully used to identify and study the regulators and components important for signal transduction, growth and development, as well as membrane trafficking in the endomembrane system in plants. These include the homologous characterization of the counterparts in mammals or yeast employing both reverse genetic as well as the forward genetic screen approaches. However, the deletion or mutation of membrane trafficking related proteins usually leads to seedling lethality due to their essential roles in plant development and organelle biogenesis. To overcome the limitation of lethal phenotype of the target proteins, we used DEX-inducible RNAi knock-down lines to study their function in plants. More recently, we developed and used both RNAi knock-down and T-DNA insertional lines as starting materials to screen for mutations that could suppress and rescue the lethal phenotype, or a suppressor screening. Further characterization of the newly identified suppressor mutants has resulted in the identification of novel negative regulators in mediating membrane trafficking and organelle biogenesis in plants. In this review, we summarize the current approaches in studying protein trafficking in the endomembrane system. We then describe three examples of suppressor screening with distinct starting materials (i.e. FREE1, MON1, and SH3P2 that are regulators of MVB, vacuole, and autophagosomes, respectively) to discuss the rationale, procedures, advantages and disadvantages, and possible outcomes of such a suppressor screening. We finally propose that these novel screening approaches will lead to the identification of new unconventional players in regulating protein trafficking and organelle biogenesis in plants and discuss their impact on plant cell biology research.