RESUMO
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.
Assuntos
Aminoácidos/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Metabolismo Energético , Lisossomos/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/metabolismo , Células 3T3-L1 , Adipócitos/enzimologia , Adipogenia , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Sistema y+L de Transporte de Aminoácidos/genética , Sistema y+L de Transporte de Aminoácidos/metabolismo , Animais , Proteína 7 Relacionada à Autofagia/genética , Glicemia/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ativação Enzimática , Células HEK293 , Células HeLa , Humanos , Insulina/sangue , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Transporte ProteicoRESUMO
(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.