Rapid optogenetic blockade of autophagy reveals that nuclear pore complex proteins are robust autophagy substrates

Autophagy, a conserved recycling process, manages intracellular quality control to mitigate stress. To determine the rapid effects of autophagy perturbation, we developed the first optogenetic tool to rapidly inhibit autophagy, termed ASAP. Our approach leverages light-induced proteolytic cleavage to selectively inhibit autophagy within 5 minutes of light exposure, providing a precise and dynamic approach to study autophagy regulation. Proteomic profiling with ASAP revealed the most tightly regulated autophagy substrates along with novel, previously unidentified substrates, including nuclear pore complex (NPC) proteins. Interestingly, autophagy regulates quality control of cytoplasmic complexes of nucleoporins via specific LC3-interacting regions (LIRs), sparing nuclear pore complex proteins embedded in the nuclear envelope. Upon rapid autophagy inhibition, incomplete nucleoporin complexes accumulate and instead of undergoing autophagic degradation, cytoplasmic nucleoporin complexes aggregate in processing bodies (P bodies). Using ASAP, we demonstrate rapid and specific inhibition of autophagy, revealing that nuclear pore complex proteins are tightly regulated autophagy substrates.