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1.
Nat Commun ; 14(1): 5503, 2023 09 07.
Article in English | MEDLINE | ID: mdl-37679347

ABSTRACT

Autophagosome formation, a crucial step in macroautophagy (autophagy), requires the covalent conjugation of LC3 proteins to the amino headgroup of phosphatidylethanolamine (PE) lipids. Atg3, an E2-like enzyme, catalyzes the transfer of LC3 from LC3-Atg3 to PEs in targeted membranes. Here we show that the catalytically important C-terminal regions of human Atg3 (hAtg3) are conformationally dynamic and directly interact with the membrane, in collaboration with its N-terminal membrane curvature-sensitive helix. The functional relevance of these interactions was confirmed by in vitro conjugation and in vivo cellular assays. Therefore, highly curved phagophoric rims not only serve as a geometric cue for hAtg3 recruitment, but also their interaction with hAtg3 promotes LC3-PE conjugation by targeting its catalytic center to the membrane surface and bringing substrates into proximity. Our studies advance the notion that autophagosome biogenesis is directly guided by the spatial interactions of Atg3 with highly curved phagophoric rims.


Subject(s)
Autophagy , Phosphatidylethanolamines , Humans , Macroautophagy , Chemical Phenomena , Membranes
2.
Nat Commun ; 12(1): 374, 2021 01 14.
Article in English | MEDLINE | ID: mdl-33446636

ABSTRACT

During autophagy the enzyme Atg3 catalyzes the covalent conjugation of LC3 to the amino group of phosphatidylethanolamine (PE) lipids, which is one of the key steps in autophagosome formation. Here, we have demonstrated that an N-terminal conserved region of human Atg3 (hAtg3) communicates information from the N-terminal membrane curvature-sensitive amphipathic helix (AH), which presumably targets the enzyme to the tip of phagophore, to the C-terminally located catalytic core for LC3-PE conjugation. Mutations in the putative communication region greatly reduce or abolish the ability of hAtg3 to catalyze this conjugation in vitro and in vivo, and alter the membrane-bound conformation of the wild-type protein, as reported by NMR. Collectively, our results demonstrate that the N-terminal conserved region of hAtg3 works in concert with its geometry-selective AH to promote LC3-PE conjugation only on the target membrane, and substantiate the concept that highly curved membranes drive spatial regulation of the autophagosome biogenesis during autophagy.


Subject(s)
Autophagy-Related Proteins/chemistry , Autophagy-Related Proteins/metabolism , Autophagy , Cell Membrane/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , Autophagy-Related Proteins/genetics , Biocatalysis , Cell Membrane/genetics , Humans , Microtubule-Associated Proteins/metabolism , Protein Conformation, alpha-Helical , Protein Domains , Ubiquitin-Conjugating Enzymes/genetics
3.
Methods Mol Biol ; 1688: 99-109, 2018.
Article in English | MEDLINE | ID: mdl-29151206

ABSTRACT

Mechanistic studies of protein-membrane interactions can be complicated by the limitations of the membrane model system chosen. Many of these limitations can be overcome by using a spherical silica nanoparticle to support the membrane. In this chapter, we present a detailed protocol for the construction of spherical nanoparticle supported lipid bilayers (SSLBs), with discussion of methods to improve production.


Subject(s)
Lipid Bilayers/metabolism , Nanoparticles/chemistry , Proteins/metabolism , Lipid Bilayers/chemistry , Magnetic Resonance Spectroscopy , Proteins/chemistry
4.
Cell Syst ; 5(5): 518-526.e3, 2017 11 22.
Article in English | MEDLINE | ID: mdl-29102609

ABSTRACT

In Bacillus subtilis, sporulation requires that the 26-amino acid protein SpoVM embeds specifically into the forespore membrane, a structure with convex curvature. How this nanometer-sized protein can detect curves on a micrometer scale is not well understood. Here, we report that SpoVM exploits a "dash-and-recruit" mechanism to preferentially accumulate on the forespore. Using time-resolved imaging and flow cytometry, we observe that SpoVM exhibits a faster adsorption rate onto membranes of higher convex curvature. This preferential adsorption is accurately modeled as a two-step process: first, an initial binding event occurs with a faster on rate, then cooperative recruitment of additional SpoVM molecules follows. We demonstrate that both this biochemical process and effective sporulation in vivo require an unstructured and flexible SpoVM N terminus. We propose that this two-pronged strategy of fast adsorption followed by recruitment of subsequent molecules is a general mechanism that allows small proteins to detect subtle curves with a radius 1,000-fold their size.


Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Amino Acids/metabolism , Bacillus subtilis/metabolism , Spores, Bacterial/metabolism
5.
J Am Chem Soc ; 137(44): 14031-14034, 2015 Nov 11.
Article in English | MEDLINE | ID: mdl-26488086

ABSTRACT

Many essential cellular processes including endocytosis and vesicle trafficking require alteration of membrane geometry. These changes are usually mediated by proteins that can sense and/or induce membrane curvature. Using spherical nanoparticle supported lipid bilayers (SSLBs), we characterize how SpoVM, a bacterial development factor, interacts with differently curved membranes by magic angle spinning solid-state NMR. Our results demonstrate that SSLBs are an effective system for structural and topological studies of membrane geometry-sensitive molecules.


Subject(s)
Bacterial Proteins/chemistry , Cell Membrane/chemistry , Lipid Bilayers/chemistry , Nanoparticles/chemistry , Bacterial Proteins/metabolism , Cell Membrane/metabolism , Lipid Bilayers/metabolism , Magnetic Resonance Spectroscopy , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Molecular Structure
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