Selective Binding Of Mitophagy Receptor Protein Bcl-rambo To LC3/GABARAP Family Protein

Mitophagy regulates the metabolic level and destiny of cells by specifically degrading damaged or redundant mitochondria.Abnormalities in the mitophagy pathway may lead to a range of diseases,such as neurodegenerative diseases and cancer.The key step in the process of mitophagy is the specific recognition of the degraded mitochondria by autophagosomes,including the recognition between LC3 and the adaptor proteins through the ubiquitin-dependent or non-dependent pathway,and the direct recognition between LC3 and the receptor.Bcl-rambo is a homologous protein of mitophagy receptor Atg32 in yeast,which has been shown to cause mitochondrial fragmentation and mitophagy by its WXXI motif binding to LC3s/GABARAPs.Mammals Atg8 proteins are grouped into two subfamilies,LC3 s and GABARAPs.In selective autophagy,Atg8 protein selectively removes protein aggregates and organelles by recruiting autophagy receptors or adaptor proteins containing LIR(LC3 interacting region)motif to drive the formation of autophagosomes.The interaction between LIR motif and LC3s/GABARAPs is highly selective,but the molecular mechanism of this selective binding is not very clear.This study mainly investigated the selective binding of mitophagy specific receptor Bclrambo to LC3s/GABARAPs and its molecular recognition mechanism,and further assayed the effect of selective binding on the mitophagy mediated by Bcl-rambo.In this study,we first found that Bcl-rambo selectively bound to different LC3s/GABARAPs through ITC,and further verified that the selective binding of Bclrambo to LC3s/GABARAPs depended on the LIR motif.On this basis,through sequence alignment and molecular docking,key sites for the selective binding were found and verified by ITC of mutants.In cells,through co-localization and immunoprecipitation,we found that the selective binding of Bcl-rambo to LC3s/GABARAPs would affect the mitophagy it mediates.