Characterization of ATG8 Family Protein Binding to TRIM23 and TRIM31
Macroautophagy (hereafter called autophagy) is a process where proteins, organelles and intracellular micro-organisms are degraded by the lysosome. The autophagosome engulfs a part of the cytoplasm where the cargo is, and transports it to the endosomal-lysosomal system for degradation. Autophagy can also be selective, where cargos are recognized directly by the autophagy receptors or by other proteins in which target a specific cargo for degradation and can bind to for example ATG8s via a LIR domain. There are many proteins involved in autophagy, and TRIM proteins represent some of them. TRIM proteins are classified in a family of E3 ligases because of their RING domain, even though not all TRIM proteins contain this RING domain. Some TRIM proteins have been shown to be of great importance in the process of autophagy and the selectivity of autophagy by binding to autophagy-related proteins. Recent studies show that TRIM23 and TRIM31 are important in autophagy. ATG8 family proteins have hydrophobic pockets which bind LIR motifs in other proteins. Here, the interaction of TRIM23 and TRIM31 with the ATG8 family proteins is studied, and moreover their interaction with human ATG8 family proteins and if the binding is LIR dependent. GST pulldown, and co-localization assays with confocal fluorescence microscope were methods used for this purpose. Furthermore, the mCherry-EYFP double-tag was used to monitor if TRIM23 and TRIM31 are degraded in the lysosome. The lysosomal inhibitor Bafilomycin A1 (Baf) and the proteosomal inhibitor MG132 were included in the assays performed in HeLa cells to determine the degradation pathway. The results showed that both TRIM23 and TRIM31 bind ATG8s in a LIR-dependent manner. Neither TRIM23 nor TRIM31 were found to be degraded by autophagy using the double-tag assay, even though both were degraded upon starvation. Confocal imaging showed that TRIM31 co-localizes with LC3A, LC3B and GABARAP in HeLa cells.
PublisherUiT Norges arktiske universitet
UiT The Arctic University of Norway
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