Dissect the PSIs' Interaction Network Involved in Conventional and Unconventional Sorting Routes

In plants, there are several thousands of different types of proteins with different functions that must be correctly located to a specific subcellular compartment. The conventional vacuolar sorting route is already well described and research teams are now more interested in understanding mechanisms behind how unconventional sorting routes work. Our laboratory has been studying the plant-specific insert (PSI), a domain shown to be both sufficient and necessary for correct vacuolar sorting, for a long time. Even though different PSI domains (PSI A and PSI B) present high similarity, they mediate different routes: PSI A has Golgi bypass ability, directly delivering proteins from the endoplasmic reticulum to the vacuole; while PSI B mediates a conventional ER–Golgi–vacuole pathway. The main goal of this study was to identify intermediate players in PSI sorting processes. We purified both PSIs and several endomembrane reporters involved in specific events of protein transport and tested their interactions through pulldown assays. Furthermore, purified PSIs were also used as bait for co-immunoprecipitation in tobacco and Arabidopsis extracts. The data obtained will set the basis for a broader objective aimed at mapping the PSI network of interactions, which will help the characterization of unconventional trafficking.