Abstract
Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.
Highlights
Macroautophagy, here referred to as autophagy, is a major pathway used by cells to degrade large cytoplasmic cargoes in lysosomes
In order to study whether forced targeting of Beclin 1 to endoplasmic reticulum (ER) or mitochondria affect autophagy, we generated constructs of N-terminally epitope-tagged Beclin 1 with C-terminal targeting peptides (Figure 1A)
We first studied the subcellular localization of wild-type Beclin 1 in human embryonic kidney 293 (HEK293)
Summary
Macroautophagy, here referred to as autophagy, is a major pathway used by cells to degrade large cytoplasmic cargoes in lysosomes. Defects in autophagy have been implicated in several diseases including cancer, neurodegeneration and muscle disorders [1,2]. A flat membrane cistern called the phagophore grows around the cytoplasmic cargo, forming a closed, double-membraned autophagosome. The completed autophagosome fuses with a lysosome forming an autolysosome, where the cytoplasmic cargo is degraded. This releases macromolecules back to the cytoplasm, and the cell can use them for biosynthesis and energy production [3]
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