title: Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins.
abstract: Autophagy is a cellular surveillance pathway that balances metabolic and energy resources and transports specific cargos, including damaged mitochondria, other broken organelles, or pathogens for degradation to the lysosome. Central components of autophagosomal biogenesis are six members of the LC3 and GABARAP family of ubiquitin-like proteins (mATG8s). We used phage display to isolate peptides that possessbona fideLIR (LC3-interacting region) properties and are selective for individual mATG8 isoforms. Sensitivity of the developed sensors was optimized by multiplication, charge distribution, and fusion with a membrane recruitment (FYVE) or an oligomerization (PB1) domain. We demonstrate the use of the engineered peptides as intracellular sensors that recognize specifically GABARAP, GABL1, GABL2, and LC3C, as well as a bispecific sensor for LC3A and LC3B. By using an LC3C-specific sensor, we were able to monitor recruitment of endogenous LC3C toSalmonelladuring xenophagy, as well as to mitochondria during mitophagy. The sensors are general tools to monitor the fate of mATG8s and will be valuable in decoding the biological functions of the individual LC3/GABARAPs.
authors.affiliation: Institute of Biochemistry II Goethe University, Frankfurt am Main, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Stolz
authors.affiliation: Institute of Biochemistry II Goethe University, Frankfurt am Main, Germany.|Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology TMP, Frankfurt am Main, Germany.
authors.forename: Mateusz
authors.initials: M
authors.lastname: Putyrski
authors.affiliation: Buchmann Institute for Molecular Life Sciences, Frankfurt am Main, Germany.
authors.forename: Ivana
authors.initials: I
authors.lastname: Kutle
authors.affiliation: Institute of Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Huber
authors.affiliation: Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Chunxin
authors.identifier: 0000-0001-6015-6806
authors.initials: C
authors.lastname: Wang
authors.affiliation: Institute of Biochemistry II Goethe University, Frankfurt am Main, Germany.
authors.forename: Viktória
authors.initials: V
authors.lastname: Major
authors.affiliation: Banting and Best Department of Medical Research, The Donnelly Centre, University of Toronto, Toronto, ON, Canada.|Department of Molecular Genetics, The Donnelly Centre, University of Toronto, Toronto, ON, Canada.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.affiliation: Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Youle
authors.affiliation: Institute of Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Rogov
authors.affiliation: Institute of Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.affiliation: Institute of Biochemistry II Goethe University, Frankfurt am Main, Germany ernst@biochem2.de dikic@biochem2.uni-frankfurt.de.|Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology TMP, Frankfurt am Main, Germany.
authors.forename: Andreas
authors.identifier: 0000-0001-5122-5005
authors.initials: A
authors.lastname: Ernst
authors.affiliation: Institute of Biochemistry II Goethe University, Frankfurt am Main, Germany ernst@biochem2.de dikic@biochem2.uni-frankfurt.de.|Buchmann Institute for Molecular Life Sciences, Frankfurt am Main, Germany.
authors.forename: Ivan
authors.identifier: 0000-0001-8156-9511
authors.initials: I
authors.lastname: Dikic
mesh_terms: D001343:Autophagy; D000071190:Autophagy-Related Protein 8 Family / Q000032:analysis*; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D005453:Fluorescence; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D012475:Salmonella / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000071190:Autophagy-Related Protein 8 Family; D019151:Peptide Library
keywords: ATG8; LC3; immunofluorescence; phage display; selective autophagy