Fluorescent Reporters for Ubiquitin-Dependent Proteolysis in Plants

Ubiquitin is a small protein commonly used as a signal molecule which upon attachment to the proteins affects their function and their fate in the cells. For example, it can be used as a degradation marker by the cell. Ubiquitin plays a significant role in regulation of numerous cellular processes. Therefore, monitoring of ubiquitin-dependent proteolysis can provide important information. Here, we describe construction of YFP-based proteasome substrates containing modified ubiquitin and the protocol for their transient expression in plant cells for functional analysis of the ubiquitin/proteasome system. To facilitate further subcloning all plasmids generated by us are based on the Gateway(®) Cloning Technology and are compatible with the Gateway(®) destination vectors.