Abstract
The GABARAPL1 protein belongs to the ATG8 family whose members are involved in autophagy. Our laboratory previously demonstrated that GABARAPL1 associates with autophagic vesicles, regulates autophagic flux and acts as a tumor suppressor protein in breast cancer. In this study, we aimed to determine whether GABARAPL1 conjugation to autophagosomes is necessary for its tumor suppressive functions using the MCF-7 breast cancer cell line overexpressing GABARAPL1 or a G116A mutant, which is unable to be lipidated and associated to autophagosomes. We show that the G116A mutation impaired GABARAPL1 function in autophagosome/lysosome fusion and inhibited lysosome activity but did not alter MTOR and ULK1 activities or tumor growth in vivo. Our results demonstrate for the first time that GABARAPL1 plays different regulatory functions during early and late stages of autophagy, independently or not of its conjugation to autophagosomes, but its tumor suppressive function appeared to be independent of its conjugation to autophagic vesicles.
Highlights
IntroductionMacroautophagy (hereafter called autophagy) is a cellular degradation process in which damaged proteins, organelles and other cytoplasmic constituents are degraded and recycled to provide nutrients and energy [1, 2]
Macroautophagy is a cellular degradation process in which damaged proteins, organelles and other cytoplasmic constituents are degraded and recycled to provide nutrients and energy [1, 2]
We observed that GABARAPL1 but not GABARAPL1 G116A colocalized with LAMP1 during autophagy, suggesting that the G116A mutation impaired the localization of GABARAPL1 to lysosomes, which is consistent with the fact that this protein could not be linked to autophagosomes anymore. These results suggested that GABARAPL1 might increase autophagosome/lysosome fusion depending on its conjugation to autophagosomes, which could explain the increased lysosomal acidification observed in MCF-7 GABARAPL1 cells
Summary
Macroautophagy (hereafter called autophagy) is a cellular degradation process in which damaged proteins, organelles and other cytoplasmic constituents are degraded and recycled to provide nutrients and energy [1, 2]. The phagophore elongates and closes to generate a doublemembrane organelle called the autophagosome which fuses with the lysosome to form the autophagolysosome, leading to the degradation of its content by lysosomal hydrolases [3, 4] This mechanism occurs at low basal levels to maintain cellular homeostasis but can be induced by different stresses such as hypoxia or nutrient starvation to allow cell survival. These stresses induce different signaling pathways involving MTORC1 (mechanistic target of rapamycin complex), an autophagy inhibitor, or AMPK (AMP-activated protein kinase), an inducer of autophagy. During nutrient or energy starvation, AMPK can inhibit MTORC1 and activate ULK1 through phosphorylation www.impactjournals.com/oncotarget at Ser 317, 555 and 777 leading to the induction of autophagy [7,8,9]
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