Functional Analysis of Plant FYVE Domain Proteins in Endosomal Trafficking

The FYVE domain is a double zinc finger-like domain that predominantly binds phosphatidylinositol 3-phosphate. The FYVE domain is usually found in proteins primarily involved in regulating various aspects of endomembrane homeostasis, including endosome tethering, endocytic recycling, membrane protein sorting, and autophagosome maturation. Whereas FYVE domain proteins have been extensively studied in mammals and yeast, only a few FYVE domain proteins have been identified and characterized in plants. Here, by using as an example FREE1 (FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1), a protein previously identified by us as a critical factor for endosomal trafficking, we describe methods to determine its lipid binding properties and endosomal localization. In addition, we also demonstrate a method to quickly test whether an FYVE domain protein is involved in endosomal sorting in plant cells.