Carl A. Denard

Enzymes that catalyze posttranslational modifications (PTMs) of peptides and proteins (PTM–enzymes)—proteases, protein ligases, oxidoreductases, kinases, and other transferases—are foundational to our understanding of health and disease and empower applications in chemical biology, synthetic biology, and biomedicine. To fully harness the potential of PTM–enzymes, there is a critical need to decipher their enzymatic and biological mechanisms, develop molecules that can probe and modulate them, and endow them with improved and novel functions. These objectives are contingent upon implementation of high‐throughput functional screens and selections that interrogate large sequence libraries to isolate desired PTM–enzyme properties. This review discusses the principles of Saccharomyces cerevisiae organelle sequestration to study and engineer PTM–enzymes. These include outer membrane sequestration, specifically methods that modify yeast surface display, and cytoplasmic sequestration based on enzyme‐mediated transcription activation. Furthermore, we present a detailed discussion of yeast endoplasmic reticulum sequestration for the first time. Where appropriate, we highlight the major features and limitations of different systems, specifically how they can measure and control enzyme catalytic efficiencies. Taken together, yeast‐based high‐throughput sequestration approaches significantly lower the barrier to understanding how PTM–enzymes function and how to reprogram them.