Biochemical and Imaging Analysis of ALIX Function in Endosomal Trafficking of Arabidopsis Protein Cargoes

ALIX/Bro1 proteins are conserved in eukaryotes where they enable targeted trafficking of membrane-associated proteins through the late endosome route to the vacuole. For this, ALIX/Bro1 proteins associate with the endosomal sorting complex required for transport (ESCRT) machinery acting as ubiquitin receptors that recognize and sort protein cargoes by binding to ubiquitin–cargo conjugates. However, recent findings show direct interaction of ALIX and protein cargoes, pointing to the existence of different mechanisms for specific target recognition by ALIX. The catalogue of proteins that interact with the Arabidopsis homologue of ALIX is increasing, including both protein cargoes and regulatory proteins that mediate or modulate ALIX function. In this context, we describe a toolkit of techniques to analyze the effect of ALIX function in the endosomal trafficking of specific cargoes, which could be easily extended to other components of the plant ESCRT machinery