Toolbox: Creating a systematic database of secretory pathway proteins uncovers new cargo for COPI

A third of yeast genes encode for proteins that function in the endomembrane system. However, the precise localization for many of these proteins is still uncertain. Here, we visualized a collection of ~500 N‐terminally, green fluorescent protein (GFP), tagged proteins of the yeast Saccharomyces cerevisiae. By co‐localizing them with 7 known markers of endomembrane compartments we determined the localization for over 200 of them. Using this approach, we create a systematic database of the various secretory compartments and identify several new residents. Focusing in, we now suggest that Lam5 resides in contact sites between the endoplasmic reticulum and the late Golgi. Additionally, analysis of interactions between the COPI coat and co‐localizing proteins from our screen identifies a subset of proteins that are COPI‐cargo. In summary, our approach defines the protein roster within each compartment enabling characterization of the physical and functional organization of the endomembrane system and its components. In this work, we systematically mapped the subcellular localization of hundreds of N‐terminally green fluorescent protein (GFP) tagged proteins to endomembrane compartments of yeast. Our analysis describes previously uncharacterized proteins in each of the endomembrane compartments and suggests a function for Lam5 in endoplasmic reticulum (ER)/Golgi contact sites. In addition, we identify novel proteins that are exclusively enriched in a COPI‐labeled compartment and demonstrate that some of them interact with the coat, suggesting that these are cargo proteins. Our analysis helps to better define the distribution of proteins across the endomembrane system, which may shed new light on its organization and functionality.