Expression and characterization of two functional vacuolar sorting receptor (VSR) proteins, BP-80 and AtVSR4 from culture media of transgenic tobacco BY-2 cells

Abstract

Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins that mediate protein transport from late Golgi or trans-Golgi network (TGN) to vacuole in plant cells. The N-terminus of VSR is believed to be important for cargo binding while its transmembrane domain (TMD) and cytoplasmic tail (CT) are essential for its correct subcellular localization. In this study, we have developed and tested an expression system using transgenic tobacco BY-2 cells to produce truncated VSR (VSRNT) proteins lacking the TMD/CT into the culture media. The expressed truncated VSRs (BP80NT and AtVSR4NT) are properly secreted into the culture media and bind specifically to the known vacuolar sorting determinants (VSDs) of various vacuolar proteins in a calcium-dependent manner in vitro. Therefore, since VSR cargo proteins are likely secreted into the culture media along with the truncated VSRs, potential applications of such expression system include identification of native VSR cargo proteins from the culture media of transgenic cells via LC–MS/MS analysis and large-scale purification of truncated VSR proteins for structural study to understand cargo–receptor interaction mechanisms in plants.