ABSTRACT
Sensing nutrient availability is essential for appropriate cellular growth, and mTORC1 is a major regulator of this process. Mechanisms causing mTORC1 activation are, however, complex and diverse. We report here an additional important step in the activation of mTORC1, which regulates the efflux of amino acids from lysosomes into the cytoplasm. This process requires DRAM-1, which binds the membrane carrier protein SCAMP3 and the amino acid transporters SLC1A5 and LAT1, directing them to lysosomes and permitting efficient mTORC1 activation. Consequently, we show that loss of DRAM-1 also impacts pathways regulated by mTORC1, including insulin signaling, glycemic balance, and adipocyte differentiation. Interestingly, although DRAM-1 can promote autophagy, this effect on mTORC1 is autophagy independent, and autophagy only becomes important for mTORC1 activation when DRAM-1 is deleted. These findings provide important insights into mTORC1 activation and highlight the importance of DRAM-1 in growth control, metabolic homeostasis, and differentiation.
Subject(s)
Amino Acids/metabolism , Autophagy-Related Protein 7/metabolism , Energy Metabolism , Lysosomes/enzymology , Mechanistic Target of Rapamycin Complex 1/metabolism , Membrane Proteins/metabolism , 3T3-L1 Cells , Adipocytes/enzymology , Adipogenesis , Amino Acid Transport System ASC/genetics , Amino Acid Transport System ASC/metabolism , Amino Acid Transport System y+L/genetics , Amino Acid Transport System y+L/metabolism , Animals , Autophagy-Related Protein 7/genetics , Blood Glucose/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Enzyme Activation , HEK293 Cells , HeLa Cells , Humans , Insulin/blood , Large Neutral Amino Acid-Transporter 1/genetics , Large Neutral Amino Acid-Transporter 1/metabolism , Male , Mechanistic Target of Rapamycin Complex 1/genetics , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Minor Histocompatibility Antigens/genetics , Minor Histocompatibility Antigens/metabolism , Protein TransportABSTRACT
(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.
Subject(s)
Autophagy , DNA Repair/genetics , Genes, Lethal , Animals , Base Sequence , Cells, Cultured , DNA Primers , Homologous Recombination , Mice , Real-Time Polymerase Chain ReactionABSTRACT
(Macro)autophagy is a membrane-trafficking process that serves to sequester cellular constituents in organelles termed autophagosomes, which target their degradation in the lysosome. Autophagy operates at basal levels in all cells where it serves as a homeostatic mechanism to maintain cellular integrity. The levels and cargoes of autophagy can, however, change in response to a variety of stimuli, and perturbations in autophagy are known to be involved in the aetiology of various human diseases. Autophagy must therefore be tightly controlled. We report here that the Drosophila cyclin-dependent kinase PITSLRE is a modulator of autophagy. Loss of the human PITSLRE orthologue, CDK11, initially appears to induce autophagy, but at later time points CDK11 is critically required for autophagic flux and cargo digestion. Since PITSLRE/CDK11 regulates autophagy in both Drosophila and human cells, this kinase represents a novel phylogenetically conserved component of the autophagy machinery.