title: Lipid Geometry and Bilayer Curvature Modulate LC3/GABARAP-Mediated Model Autophagosomal Elongation.
abstract: Autophagy, an important catabolic pathway involved in a broad spectrum of human diseases, implies the formation of double-membrane-bound structures called autophagosomes (AP), which engulf material to be degraded in lytic compartments. How APs form, especially how the membrane expands and eventually closes upon itself, is an area of intense research. Ubiquitin-like ATG8 has been related to both membrane expansion and membrane fusion, but the underlying molecular mechanisms are poorly understood. Here, we used two minimal reconstituted systems (enzymatic and chemical conjugation) to compare the ability of human ATG8 homologs (LC3, GABARAP, and GATE-16) to mediate membrane fusion. We found that both enzymatically and chemically lipidated forms of GATE-16 and GABARAP proteins promote extensive membrane tethering and fusion, whereas lipidated LC3 does so to a much lesser extent. Moreover, we characterize the GATE-16/GABARAP-mediated membrane fusion as a phenomenon of full membrane fusion, independently demonstrating vesicle aggregation, intervesicular lipid mixing, and intervesicular mixing of aqueous content, in the absence of vesicular content leakage. Multiple fusion events give rise to large vesicles, as seen by cryo-electron microscopy observations. We also show that both vesicle diameter and selected curvature-inducing lipids (cardiolipin, diacylglycerol, and lyso-phosphatidylcholine) can modulate the fusion process, smaller vesicle diameters and negative intrinsic curvature lipids (cardiolipin, diacylglycerol) facilitating fusion. These results strongly support the hypothesis of a highly bent structural fusion intermediate (stalk) during AP biogenesis and add to the growing body of evidence that identifies lipids as important regulators of autophagy.
authors.affiliation: Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
authors.forename: Ane
authors.initials: A
authors.lastname: Landajuela
authors.affiliation: Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
authors.forename: Javier H
authors.initials: JH
authors.lastname: Hervás
authors.affiliation: Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
authors.forename: Zuriñe
authors.initials: Z
authors.lastname: Antón
authors.affiliation: Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
authors.forename: L Ruth
authors.initials: LR
authors.lastname: Montes
authors.affiliation: Structural Biology Unit, Center for Cooperative Research in Biosciences, CIC bioGUNE, Derio, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Gil
authors.affiliation: Structural Biology Unit, Center for Cooperative Research in Biosciences, CIC bioGUNE, Derio, Spain.
authors.forename: Mikel
authors.initials: M
authors.lastname: Valle
authors.affiliation: Departmento de Biología Molecular y Celular, Centro Nacional de Biotecnología-CSIC, Cantoblanco, Madrid, Spain.
authors.forename: J Francisco
authors.initials: JF
authors.lastname: Rodriguez
authors.affiliation: Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
authors.forename: Felix M
authors.initials: FM
authors.lastname: Goñi
authors.affiliation: Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain. Electronic address: alicia.alonso@ehu.es.
authors.forename: Alicia
authors.initials: A
authors.lastname: Alonso
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins; D001343:Autophagy; D000071190:Autophagy-Related Protein 8 Family; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D008561:Membrane Fusion; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010588:Phagosomes / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D000071190:Autophagy-Related Protein 8 Family; C116741:GABARAP protein, human; C498698:GABARAPL2 protein, human; D008051:Lipid Bilayers; C477012:MAP1LC3A protein, human; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins