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- Daumke, Oliver, et al.
(författare)
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Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling
- 2007
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 449:7164, s. 923-927
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Tidskriftsartikel (refereegranskat)abstract
- The ability to actively remodel membranes in response to nucleotide hydrolysis has largely been attributed to GTPases of the dynamin superfamily, and these have been extensively studied. Eps15 homology (EH)-domain-containing proteins (EHDs/RME-1/pincher) comprise a less-well-characterized class of highly conserved eukaryotic ATPases implicated in clathrin-independent endocytosis, and recycling from endosomes. Here we show that EHDs share many common features with the dynamin superfamily, such as a low affinity for nucleotides, the ability to tubulate liposomes in vitro, oligomerization around lipid tubules in ring-like structures and stimulated nucleotide hydrolysis in response to lipid binding. We present the structure of EHD2, bound to a non-hydrolysable ATP analogue, and provide evidence consistent with a role for EHDs in nucleotide-dependent membrane remodelling in vivo. The nucleotide-binding domain is involved in dimerization, which creates a highly curved membrane-binding region in the dimer. Oligomerization of dimers occurs on another interface of the nucleotide-binding domain, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for understanding membrane deformation.
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| 3. |
- Knyazeva, Anastasia, 1995-
(författare)
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Non-canonical ATG8 conjugation in ESCRT-driven membrane remodeling processes
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- ATG8 family proteins have the unique ability to conjugate to membrane lipids. Initially identified as a hallmark of autophagy, ATG8 lipidation is emerging as an important regulator of a growing list of non-degradative cellular functions. In this thesis we developed and applied novel chemical genetic approaches to perturb dynamic membrane remodeling processes and induce non-canonical ATG8 conjugation in cells. We investigated novel roles of ATG8 in membrane deformation processes carried out by the Endosomal Sorting Complex Requiredfor Transport (ESCRT) machinery.In Paper I, using a high-throughput phenotypic screening assay, we developed a collection of pseudo-natural product-based compounds which potently induce ATG8 lipidation in mammalian cells. The most potent compound, Tantalosin, induces ATG8 lipidation which is insensitive to simultaneous inhibition of autophagosome-lysosome fusion, suggesting a non-canonical function ofTantalosin-induced ATG8 conjugation.In Paper II we investigated the molecular target of Tantalosin. We found that Tantalosin targets the ESCRT-III protein IST1 and inhibits IST1-CHMP1B copolymer formation. This inhibition results in the impairment of transferrin receptor (TfR) recycling resulting in the rapid accumulation of the receptor inearly/sorting endosomes. At the same time, Tantalosin induces non-canonical ATG8 conjugation on stalled sorting endosomes containing TfR. This conjugation is dependent on the ATG16L1-ATG5-ATG12 complex which is recruited to stalled endosomes via ATG16L1-V-ATPase interaction.In Paper III and Paper IV we studied the induction of non-canonical ATG8 lipidation in response to endolysosomal membrane damage. We used two established membrane damaging agents: V. Cholerae cytotoxin MakA and the lysosomotropic compound, LLOMe. In Paper III we demonstrated that, at lowpH, MakA assembles into small pores in endosomal membranes which arerecognized by the ESCRT membrane repair machinery. Non-canonical ATG8 lipidation in response to MakA-induced pore formation is mediated by V-ATPase activity. In Paper IV we identified a novel player in the lysosomal damage response – TECRP1. TECPR1 is recruited to damaged membranes where it forms an alternative ATG16L1-independent E3 ligase complex with the ATG5-ATG12 conjugate and plays a role in the restoration of lysosomal integrity after damage.
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