Abstract
A significant challenge in cell biology is to uncover the function of uncharacterized proteins. Surprisingly, a quarter of the proteome is still poorly understood even in the most well-studied model organisms. Systematic methodologies, including the use of tagged protein collections, have emerged as a powerful approach to address this gap. Despite the availability of proteome-wide collections featuring various fused proteins, the impact of different tags on protein function highlights the need for diversifying the tags used for functional genomic studies. To rise to this challenge, we created a proteome-wide collection of yeast strains in which proteins are N-terminally tagged with the broadly utilized and compact hemagglutinin (HA) epitope. We showcase the potential uses of our library for systematically evaluating protein size, abundance and localization using an in vivo labeling approach. Our characterization underscores the potential utility of a proteome-wide HA-tagged library in revealing novel aspects of cell biology, providing an additional powerful tool for functional genomics.