Genetically encoded lockdown of SERCA in the endoplasmic reticulum membrane arrests Ca 2+ signaling through proximity‐covalent crosslinking

Reversible conformational dynamics of membrane proteins are essential for intracellular signaling, but no method enables their irreversible arrest in living cells. Here, we developed a genetically encoded proximity‐based lockdown enzyme derived from an engineered transglutaminase catalytic core (TGC) that covalently crosslinks membrane proteins. By fusing TGC to the endoplasmic reticulum (ER)‐resident microprotein ALN encoded by a short open reading frame (sORF), we created an organelle‐specific module that selectively catalyzes covalent crosslinking within the SERCA Ca 2+ pump, strongly suppressing its ATP‐dependent pump activity and arresting ER Ca 2+ signaling. This engineered lockdown enzyme remodels ER membrane protein architecture and restricts conformational dynamics, providing a versatile platform for long‐term covalent control of intracellular signaling and a foundation for future therapeutic cellular applications. Impact statement Our proximity‐based lockdown enzyme, engineered from microbial transglutaminase, provides a new strategy to covalently arrest the conformational states of organelle‐resident membrane proteins in living cells, enabling long‐term control of intracellular signaling and establishing a foundation for next‐generation cellular therapeutics.