Abstract
Chaperone-mediated autophagy (CMA) is a selective autophagic pathway responsible for degrading cytoplasmic proteins within lysosomes. Monitoring CMA flux is essential for understanding its functions and molecular mechanisms but remains technically complex and challenging. In this study, we developed a pH-resistant probe, KFERQ-Gamillus, by screening various green fluorescent proteins. This probe is activated under conditions known to induce CMA, such as serum starvation, and relies on LAMP2A and the KFERQ motif for lysosomal localization and degradation, demonstrating its specificity for the CMA pathway. It enables the detection of CMA activity in living cells through both microscopy and image-based flow cytometry. Additionally, we created a dual-reporter system, KFERQ-Gamillus-Halo, by integrating KFERQ-Gamillus with the Halo-tag system. This probe not only distinguishes between protein synthesis and degradation but also facilitates the detection of intracellular CMA flux via immunoblotting and the rapid assessment of CMA activity using flow cytometry. Together, the KFERQ-Gamillus-Halo probe provides quantitative and time-resolved monitoring for CMA activity and flux in living cells. This tool holds promising potential for high-throughput screening and biomedical research related to CMA.