ATG conjugation system -dependent and -independent mechanisms underlie lysosomal stress-induced TFEB regulation

Transcription factor EB (TFEB), well characterized as a master regulator of autophagy and lysosomal biogenesis, is translocated to the nucleus and activated by varieties of cellular stresses including starvation and lysosomal damage. However, compared to the starvation condition, the molecular mechanism of TFEB activation by other stress conditions is poorly understood. Previously, we have shown that TFEB activation during lysosomal damage but not starvation condition depends on a subset of autophagy regulators, collectively called ATG conjugation system, whose function is essential for the lipidation of ATG8 proteins. In this study, by time-lapse imaging, we newly identified the presence of ATG conjugation system -independent TFEB regulation which precedes the ATG conjugation system-dependent regulation, designated mode I and mode II, respectively. Consistent with the presence of different modes, our time course transcriptome analysis revealed two different sets of TFEB downstream. Comprehensive interactome analysis of TFEB and subsequent functional screening identified unique regulars of TFEB in each mode: APEX1 for Mode I and CCT7 and/or TRIP6 for Mode II, respectively. APEX1 interacted with TFEB and was required for its protein stability in a manner independent of ATG conjugation system. On the other hand, both CCT7 and TRIP6 were accumulated on lysosomes during lysosomal damage and interacted with TFEB mainly in ATG conjugation system deficient cells, presumably blocking TFEB nuclear translocation. Moreover, we further revealed that TFEB regulatory mechanisms by other cellular stresses such as oxidative stress, proteasome inhibition, mitochondria depolarization, and DNA damage can be classified into either APEX1-mediated Mode I or TRIP6-mediated Mode II. Our results pave the way for a unified understanding TFEB regulatory mechanisms from the perspective of ATG conjugation system under varieties of cellular stresses.