yeast membrane proteomics using Leu3 labelling

The yeast signal peptide peptidase homologue Ypf1 functionally interacts with the ER-associated degradation (ERAD) factors Dfm1 and Doa10 to control the abundance of the zinc transporter Zrt1. Here we report that Ctr1 is another substrate for this degradation mechanism with deletion of Ypf1 leading to an accumulation of Ctr1. We describe in detail the usage of leucine metabolic labelling in yeast in order to monitor quantitative proteome alterations, e.g. upon removal of a protease. Since laboratory yeast strains are typically leucine auxotroph, metabolic labelling with trideuterated leucine (d3-leucine) is a straightforward, cost-effective, and ubiquitously applicable strategy for quantitative proteomic studies similar to the widely used arginine/lysine SILAC method for mammalian cells. We showcase the usage of advanced peptide quantification using the FeatureFinderMultiplex algorithm (part of the OpenMS software package) for robust and reliable quantification. Furthermore, we present an OpenMS bioinformatics data analysis workflow that combines accurate quantification with high proteome coverage. In order to enable visualization, peptide-mapping, and sharing of quantitative proteomic data, especially for membrane-spanning and cell-surface proteins, we further developed the web-application Proteator. Due to its simplicity and robustness, we expect metabolic leucine labelling in yeast to be of great interest to the research community.