Fluorescence‐based ATG8 sensors monitor localization and function of LC3/GABARAP proteins. (27th December 2016)

• Record Type: Journal Article
• Authors: Stolz, Alexandra; Putyrski, Mateusz; Kutle, Ivana; Huber, Jessica; Wang, Chunxin; Major, Viktória; Sidhu, Sachdev S; Youle, Richard J; Rogov, Vladimir V; Dötsch, Volker; Ernst, Andreas; Dikic, Ivan
• Is Part Of: EMBO journal. Volume 36:Number 4(2017)
• Page Start: 549
• Page End: 564
• Publication Date: 2016-12-27
• Subjects: ATG8 -- immunofluorescence -- LC3 -- phage display -- selective autophagy; Molecular biology -- Periodicals; 572.805
• DOI: 10.15252/embj.201695063
• Languages: English
• ISSNs: 0261-4189

Abstract

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 possess bona fide LIR (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 to Salmonella during 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. Synopsis: New peptide sensors have been developed to monitor localization and fate of individual LC3s and GABARAPs of the mATG8 family, which serve as a central platform for autophagosome biogenesis. Phage display allowed isolation of peptides with LIR (LC3‐interacting region) properties that are selective for individual mATG8 family members. These sensors were optimized by peptide multiplication, charge distribution, and fusion with a membrane recruitment domain (FYVE) or oligomerization domain (PB1). mATG8 sensors that recognize specifically GABARAP, GABL1, GABL2, and LC3C, as well as a combined sensor for LC3A and LC3B, were successfully used in cells to localize the respective mATG8s. Endogenous LC3C and LC3C‐specific sensor mCh‐PB1‐AS3_67 are recruited to Salmonella during xenophagy and to mitochondria during mitophagy. Abstract : New peptide sensors have been developed to monitor localization and fate of individual LC3s and GABARAPs of the mATG8 family, which serve as a central platform for autophagosome biogenesis.