In plants, there are several thousands of different types of proteins. All and each have different functions and, therefore, must be correctly located to a specific subcellular compartment. This study focuses on plant vacuolar proteins and their sorting pathways to the vacuole. 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 working for a long time with two very similar aspartic proteinases from Cynara cardunculus: cardosin A and cardosin B. Recently, we found that a 100 amino acid domain inside cardosins’ sequence is sufficient and necessary for correct vacuolar sorting - the plant specific insert (PSI). Even though both PSI domains from cardosin A (PSI A) and cardosin B (PSI B) present high similarity between them, their sorting mediated routes are very different: PSI A is able to mediate a Golgi bypass route, directly delivering proteins from the Endoplasmic Reticulum (ER) to the Vacuole. In contrast, PSI B mediated sorting route follows the conventional vacuolar sorting pathway, ER - Golgi apparatus - Pre-Vacuolar Compartment - Vacuole. The main goal of this study is to identify intermediate playersin such trafficking pathways and involved in PSIs sorting processes We expressed, purified and isolated from bacterial cultures both PSIs and several endomembrane reporters involved in specific events of protein transport and will study their interaction through pulldown assays. Furthermore, purified PSIs were also used as bait for co-Immunoprecipitation in Tobacco and Arabidopsis extracts. Preliminary results using yeast-two hybrid assays already showed positive interactions between sorting related proteins and the PSIs, that need to be confirmed. The data retrieved from this work will set the basis of a broader objective aiming at mapping the PSIs network of interactions, that will help the characterization and define intermediate players of unconventional trafficking.