Abstract
Plant vacuole is a enormous organelle that accumulates a variety of solutes such as nutrients, proteins, second metabolites, and toxic ions. Vacuole not only conserves solutes but also contributes to dynamic events, such as cytosolic pH control, Ca2+ signaling and stomatal movement. These functions are supported by a great number of tonoplast proteins and their interactions. In this study, we investigated protein complexes of vacuolar membrane comprehensively.
We prepared vacuolar membrane from suspension-cultured cell of Arabidopsis thaliana. Solubilized membranes were subjected to 2D- blue native- SDS- PAGE to separate complexes of membrane protein. We detected approximately 20 candidates for hetero-oligomeric protein complex and about 20 candidates for homo-oligomeric protein. Using antibody library for vacuolar-membrane transporters of about 40, we analyzed apparent molecular masses of protein (complex) under blue native-PAGE. We also screened protein complexes in the vacuolar membrane by co-immunoprecipitation method using the antibody library.
