Mitochondrial energetic stress triggers AMPK/FNIP1-dependent TFEB nuclear translocation, inducing transcriptional waves of lysosomal and mitochondrial biogenesis.

Cells respond to mitochondrial energetic stress with rapid activation of the AMP-activated protein kinase (AMPK), which acutely inhibits anabolism and stimulates catabolism. AMPK also induces sustained transcriptional reprogramming of metabolism. The TFEB transcription factor is a major effector of AMPK signals, inducing lysosome genes following energetic stress. Yet the molecular mechanism underlying how AMPK activates TFEB remains unresolved. We demonstrate here that AMPK directly phosphorylates five conserved serine residues in FNIP1, which suppresses the function of the FLCN/FNIP1 RagC GAP complex, in turn controlling TFEB lysosomal localization. We demonstrate that FNIP1 phosphorylation is required for AMPK to induce nuclear translocation of TFEB, which is fully separable from AMPK control of canonical mTORC1 signaling. Using a non-phosphorylatable allele of FNIP1, we show that in parallel to lysosomal biogenesis, AMPK induces mitochondrial biogenesis via TFEB-dependent induction of PGC1a mRNA. This signaling from mitochondrial stress is also independent of amino-acid control of the Rags and TFEB, which still proceed normally in cells bearing the AMPK-non phosphorylatable allele of FNIP1. Taken together, mitochondrial energetic stress triggers AMPK/FNIP1-dependent TFEB nuclear translocation, inducing transcriptional waves of lysosomal and mitochondrial biogenesis. Overall design: We generated FNIP1 knock out HEK 293T cells, which were reconstituted with WT FNIP1 or mutant FNIP1 cDNA, in which all 5 AMPK phosphosites had been mutated to alanine (SA5). Following treatment with the synthetic AMPK activator 991 for 0-16 hours, RNAseq was RNAseq was carried out to examine mitochondrial and lysosomal gene transcription under these conditions. Additionally, WT or AMPK KO HEK 293T cells were treated with 991 or the mitochondrial poisons CCCP, Phenformin or Rotenone to observe effects on global gene transcription and more specifically on lysosomal and mitochondrial gene sets. Similarly, RNAseq was carried out following 991 treatments in WT and TFEB/TFE3 double knock out. HEK 293T; or WT and ERRa knock out HEK 293T cells to examine the role of TFEB/TFE3 and ERRa on lysosomal and mitochondrial gene transcription. RNAseq was also carried out following PGC1A knockdown by siRNA and compared with control siRNA conditions following 991 treatments for 0-16 hours to assess. effects on mitochondrial gene transcription.