Abstract
Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with—and regulation of its ubiquitination and stability by—RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5−/− MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5−/− using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5−/− mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5−/− macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.
Highlights
Autophagy is an intracellular catabolic process by which cellular components are degraded through the lysosomal machinery
RNF5 interacts with and ubiquitinates ATG4B Given the various effects of RNF5 on endoplasmic reticulum (ER) stress and innate immune pathways, processes that are influenced by autophagy, we examined the possibility that RNF5 may play a direct role in the control of autophagy
ATG4B associated with the RING mutant (RM) form of RNF5, indicating that the ubiquitin ligase activity of RNF5 was not required for this interaction
Summary
Autophagy is an intracellular catabolic process by which cellular components are degraded through the lysosomal machinery. Conserved from yeast to humans, autophagy is fundamental to eukaryotic cell homeostasis [1,2]. Autophagy functions in diverse cellular processes such as growth and development, cancer, and inflammation [3,4,5], and is implicated in both cell survival and death, depending on the cell type and stress conditions. While certain viruses and bacteria can subvert and manipulate autophagic pathways during establishment of infection, autophagy plays a protective role against intracellular replication of several pathogens including group A Streptococcus (GAS) [8,9]. Given the broad importance of autophagy in cell biology, it is of great interest to define the mechanisms underlying its control under normal and stress-related conditions
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