In situ structural analysis reveals membrane shape transitions during autophagosome formation

Bieber, Anna; Capitanio, Cristina; Erdmann, Philipp; Fiedler, Fabian; Beck, Florian; Lee, Chia‐Wei; Li, Delong; Hummer, Gerhard; Schulman, Brenda A.; Baumeister, Wolfgang; Wilfling, Florian

doi:10.1073/pnas.2209823119

Cited by 65 publications

(88 citation statements)

References 70 publications

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“…Third, we clearly showed the single-membrane characteristic of APs after they entered vacuoles by Cryo-FIB and Cryo-ET analysis (Fig 4). Indeed, similar clear images showing the single-membrane characteristic of ABs and double-membrane characteristic of APs or phagophores (obtained using the same techniques) were reported in a bioRxiv reprint during the final preparation of this manuscript [35].…”

Section: Plos Geneticssupporting

confidence: 69%

The entry of unclosed autophagosomes into vacuoles and its physiological relevance

Xu²,

Wang³

et al. 2022

PLoS Genet

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It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy.

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Section: Plos Geneticssupporting

confidence: 69%

The entry of unclosed autophagosomes into vacuoles and its physiological relevance

Xu²,

Wang³

et al. 2022

PLoS Genet

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“…This may account for the retention of WT‐like phagophore size upon depletion of PC as observed in our study. A recent report by Baumeister and coworkers (Bieber et al , 2022 ), has indicated that the phagophore rim has a complex architecture that may be particularly prone to phospholipid imbalance, thereby compromising membrane closure while maintaining expansion of the relatively flat majority of the phagophore surface.…”

Section: Discussionmentioning

confidence: 99%

Phospholipid imbalance impairs autophagosome completion

et al. 2022

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Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single-membrane cisternae called phagophores. While the role of phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well-studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy. We reveal for the first time that genetic loss of PC biosynthesis via the CDP-DAG pathway leads to changes in lipid composition of autophagic membranes, specifically replacement of PC by phosphatidylserine (PS). This impairs closure of the autophagic membrane and autophagic flux. Consequently, we show that choline-dependent recovery of de novo PC biosynthesis via the CDP-choline pathway restores autophagosome formation and autophagic flux in PC-deficient cells. Our findings therefore implicate phospholipid metabolism in autophagosome biogenesis.

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“…The ER plays a key role in the lipid biosynthesis as it serves as a central cellular hub for intracellular communication, organelle trafficking and macromolecule synthesis. Therefore, it forms MCSs with all intracellular organelles, including the phagophore whose extremities are tethered with the ER in both yeast and mammalian cells [ 31 , 32 , 33 , 49 , 50 , 51 , 52 , 53 ]. In mammals, it appears that the phagophores also have some MCSs with the ER distributed elsewhere on their surface [ 54 , 55 ].…”

Section: Mcss During Autophagosome Biogenesismentioning

confidence: 99%

“…While these latter MCSs remain to be characterized, work in yeast has identified some of the components and the mechanism establishing the MCSs between the extremities of the phagophore and the ER ( Figure 3 A). This association seems to be less strong than the one with other contacts (e.g., the nucleus) since the level of visible deformation on both organelles is much lower [ 31 ]. The fact that the phagophore is very frequently tethered via part of its rim to the ER or the nucleus is consistent with ongoing lipid transfer at these MCSs [ 31 , 56 ].…”

Section: Mcss During Autophagosome Biogenesismentioning

confidence: 99%

“…Two well-conserved key regulators of autophagy are mechanistic target of rapamycin kinase complex 1 (mTORC1) and AMP-activated kinase (AMPK), which through phosphorylation, negatively and positively regulate autophagy induction in response to amino acids and ATP levels, respectively [ 23 , 24 , 25 , 26 ]. Autophagosomes are generated at specific subcellular locations called phagophore assembly site or pre-autophagosomal site (PAS) in yeast or omegasomes in mammalian cells ( Figure 2 B,C) [ 27 , 28 , 29 , 30 , 31 ]. The PAS is formed in between the vacuole and the ER, while the omegasomes are a subdomain of the ER enriched in phosphatidylinositol-3-phosphate (PtdIns3P) ( Figure 2 B,C) [ 29 , 32 , 33 ].…”

Section: Introductionmentioning

confidence: 99%

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Membrane Contact Sites in Autophagy

Zwilling

Reggiori

2022

Cells

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Eukaryotes utilize different communication strategies to coordinate processes between different cellular compartments either indirectly, through vesicular transport, or directly, via membrane contact sites (MCSs). MCSs have been implicated in lipid metabolism, calcium signaling and the regulation of organelle biogenesis in various cell types. Several studies have shown that MCSs play a crucial role in the regulation of macroautophagy, an intracellular catabolic transport route that is characterized by the delivery of cargoes (proteins, protein complexes or aggregates, organelles and pathogens) to yeast and plant vacuoles or mammalian lysosomes, for their degradation and recycling into basic metabolites. Macroautophagy is characterized by the de novo formation of double-membrane vesicles called autophagosomes, and their biogenesis requires an enormous amount of lipids. MCSs appear to have a central role in this supply, as well as in the organization of the autophagy-related (ATG) machinery. In this review, we will summarize the evidence for the participation of specific MCSs in autophagosome formation, with a focus on the budding yeast and mammalian systems.

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In situ structural analysis reveals membrane shape transitions during autophagosome formation

Cited by 65 publications

References 70 publications

The entry of unclosed autophagosomes into vacuoles and its physiological relevance

The entry of unclosed autophagosomes into vacuoles and its physiological relevance

Phospholipid imbalance impairs autophagosome completion

Membrane Contact Sites in Autophagy

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