Analysis of Ubiquitylation and SUMOylation of Yeast Nuclear Pore Complex Proteins.

Posttranslational modifications and in particular ubiquitylation and SUMOylation of the nuclear pore complex (NPC), have been shown to regulate some of its functions, particularly in response to diverse stress signals.Although proteomic approaches are extremely powerful to identify substrates and modification sites, dissecting specific mechanisms and regulation functions of ubiquitylation and SUMOylation of the diverse NPC proteins, in different genetic backgrounds or cell environmental conditions, requires specific biochemical assays based on purification and precise analysis of 6His-tagged ubiquitylated or SUMOylated protein of interest. Here we describe an approach that can be easily employed without specific equipment. It allowed to successfully analyze yeast NPC proteins but can easily be adapted to the study of the mammalian NPC.