ALIBY: ALFA Nanobody-Based Toolkit for Imaging and Biochemistry in Yeast

Specialized epitope tags continue to be an integral component in various biochemical and cell biological applications such as fluorescence microscopy, immunoblotting, immunoprecipitation, and protein purification. However, until recently, no single tag could offer this complete set of functionalities on its own. Here, we present a plasmid-based toolkit named ALIBY ( AL FA Toolkit for I maging and B iochemistry in Y east) that provides a universal workflow to adopt the versatile ALFA tag/ Nb ALFA system within the well-established model organism Saccharomyces cerevisiae. The kit comprises of tagging plasmids for labelling a protein-of-interest with the ALFA tag, and detection plasmids encoding a fluorescent protein-tagged Nb ALFA for live-cell imaging purposes. We demonstrate the suitability of ALIBY for visualizing the spatiotemporal localization of yeast proteins (i.e., cytoskeleton, nucleus, centrosome, divisome and exocyst) in live cells. Our approach has yielded an excellent signal-to-noise ratio without off-targeting or effect on cell growth. In summary, our yeast-specific toolkit aims to simplify and further advance the live-cell imaging of differentially abundant yeast proteins while also being suitable for biochemical applications. Importance In yeast research, conventional fluorescent protein tags and small epitope tags are widely used to study the spatiotemporal dynamics and activity of proteins. Though proven to be efficient, these tags lack the versatility for usage across different cell biological and biochemical studies of a given protein-of-interest. Therefore, there is an urgent need for a unified platform for visualization, biochemical, and functional analyses of proteins-of-interest in yeast. Herein, we have engineered ALIBY, a plasmid-based toolkit which expands the benefits of the recently developed ALFA tag/ Nb ALFA system to studies in the well-established model organism Saccharomyces cerevisiae. We demonstrate that ALIBY provides a simple and versatile strain construction workflow for long duration live-cell imaging and biochemical applications in yeast.