Evaluation of protein-based phosphoinositide probes in Saccharomyces cerevisiae

Phosphoinositides are low-abundance membrane lipids serving as critical regulators of organelle identity, membrane trafficking, and signal transduction in eukaryotic cells. Live-cell imaging using genetically encoded, protein-based fluorescent probes has revolutionized the study of these lipids. Yet many existing probes were developed and analyzed in isolation across different experimental systems. This study presents a comprehensive evaluation of 37 published protein-based phosphoinositide probes within the budding yeast Saccharomyces cerevisiae . The specificity of candidate probes was tested through the disruption of their corresponding lipid kinase activities. By comparing the performance of multiple probes under uniform conditions, we identified seven constructs based on the following domains that reliably detect the four primary phosphoinositide species in yeast: PX NCF4 and PX Vam7 for PtdIns(3)P; 2PX SnxA for PtdIns(3,5)P 2 ; 2PH Osh2 , PH FAPP1 , and PH OSBP for PtdIns(4)P; and TubbyCT for PtdIns(4,5)P 2 . Furthermore, we systematically mapped the subcellular distribution of phosphoinositides and their producing kinases, providing an updated atlas of their localization. This work offers a validated toolkit and a selection guide for investigating phosphoinositide dynamics in vivo . IMPORTANCE Cells use tiny lipid molecules called phosphoinositides as “zip codes” to organize their internal compartments and direct traffic between them. Because these molecules are scarce and constantly changing, scientists rely on special glowing protein probes to watch them in living cells. However, most of these probes were invented in separate labs under different conditions, making it hard to know which ones truly work. This study tested 37 published probes side-by-side in yeast and found seven reliable “trackers” for the four most important phosphoinositide types. By mapping exactly where these lipids and the enzymes that make them reside, the work provides a standardized toolkit and clear guide that helps researchers confidently study how cells organize their inner life.