Limits...
Regulation of ATG4B stability by RNF5 limits basal levels of autophagy and influences susceptibility to bacterial infection.

Kuang E, Okumura CY, Sheffy-Levin S, Varsano T, Shu VC, Qi J, Niesman IR, Yang HJ, López-Otín C, Yang WY, Reed JC, Broday L, Nizet V, Ronai ZA - PLoS Genet. (2012)

Bottom Line: RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 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.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction and Cell Death Programs, Sanford-Burnham Medical Research Institute, La Jolla, California, USA.

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.

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RNF5 co-localizes with—and limits—ATG4B at the membrane compartment.(A) RNF5/LC3 immunofluorescence images in HeLa cells expressing GFP-sec61b. Magnified view of the white square region is shown on the bottom right panels; white arrows denote LC3 foci that colocalized with ER-localized RNF5. Scale bar, 10 microns. (B) ATG4B and RNF5 co-fractionate in ER and autophagosomes. 293T cells were maintained under normal medium or HBSS starvation (3 h) were collected and passed through a 25-gauge needle 10 times and the membrane fractions were enriched and loaded to 10–50% linear sucrose gradient and subjected to ultracentrifugation (160,000 g for 2.5 h). Twelve fractions (1 ml each) were collected from the top to the bottom, and analyzed by western blots as indicated. Calnexin, ER membrane marker. (C) 293T cells were starved using media supplemented with HBSS, and the cells were collected at the indicated time points. Membrane fractions were separated by sucrose gradient ultracentrifugation. Proteins (40 µg/lane) were resolved and immunoblotted with the indicated antibodies. Top, ER-associated GFP-LC3 puncta; Middle, cytosolic GFP-LC3 puncta. (D) PC3 cells stably expressing shRNF5 were maintained in HBSS (3 h) or regular medium. Membrane fractions were separated by sucrose gradient ultracentrifugation. Proteins (40 µg/lane) were resolved and immunoblotted with the indicated antibodies.
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pgen-1003007-g005: RNF5 co-localizes with—and limits—ATG4B at the membrane compartment.(A) RNF5/LC3 immunofluorescence images in HeLa cells expressing GFP-sec61b. Magnified view of the white square region is shown on the bottom right panels; white arrows denote LC3 foci that colocalized with ER-localized RNF5. Scale bar, 10 microns. (B) ATG4B and RNF5 co-fractionate in ER and autophagosomes. 293T cells were maintained under normal medium or HBSS starvation (3 h) were collected and passed through a 25-gauge needle 10 times and the membrane fractions were enriched and loaded to 10–50% linear sucrose gradient and subjected to ultracentrifugation (160,000 g for 2.5 h). Twelve fractions (1 ml each) were collected from the top to the bottom, and analyzed by western blots as indicated. Calnexin, ER membrane marker. (C) 293T cells were starved using media supplemented with HBSS, and the cells were collected at the indicated time points. Membrane fractions were separated by sucrose gradient ultracentrifugation. Proteins (40 µg/lane) were resolved and immunoblotted with the indicated antibodies. Top, ER-associated GFP-LC3 puncta; Middle, cytosolic GFP-LC3 puncta. (D) PC3 cells stably expressing shRNF5 were maintained in HBSS (3 h) or regular medium. Membrane fractions were separated by sucrose gradient ultracentrifugation. Proteins (40 µg/lane) were resolved and immunoblotted with the indicated antibodies.

Mentions: Because RNF5 is a membrane-bound E3 ubiquitin ligase, and since it requires its membrane-anchor to affect ATG4B and autophagy we assessed whether RNF5 activity towards ATG4B may preferentially take place at membranal domains. The autophagy markers LC3 and the ER membrane marker Sec61 were used to determine degree of RNF5-colocalization with LC3 at the membranal fraction. Analysis was performed in both MEFs and HeLa cells grown under normal conditions. In HeLa cells we found that 74% of the LC3/RNF5 colocalized spots were within the sec61b structures, whereas in the MEFs this percentage was found to be 83% (Figure 5A). This data suggest that RNF5 effect on LC3 processing does take place within the ER domain.


Regulation of ATG4B stability by RNF5 limits basal levels of autophagy and influences susceptibility to bacterial infection.

Kuang E, Okumura CY, Sheffy-Levin S, Varsano T, Shu VC, Qi J, Niesman IR, Yang HJ, López-Otín C, Yang WY, Reed JC, Broday L, Nizet V, Ronai ZA - PLoS Genet. (2012)

RNF5 co-localizes with—and limits—ATG4B at the membrane compartment.(A) RNF5/LC3 immunofluorescence images in HeLa cells expressing GFP-sec61b. Magnified view of the white square region is shown on the bottom right panels; white arrows denote LC3 foci that colocalized with ER-localized RNF5. Scale bar, 10 microns. (B) ATG4B and RNF5 co-fractionate in ER and autophagosomes. 293T cells were maintained under normal medium or HBSS starvation (3 h) were collected and passed through a 25-gauge needle 10 times and the membrane fractions were enriched and loaded to 10–50% linear sucrose gradient and subjected to ultracentrifugation (160,000 g for 2.5 h). Twelve fractions (1 ml each) were collected from the top to the bottom, and analyzed by western blots as indicated. Calnexin, ER membrane marker. (C) 293T cells were starved using media supplemented with HBSS, and the cells were collected at the indicated time points. Membrane fractions were separated by sucrose gradient ultracentrifugation. Proteins (40 µg/lane) were resolved and immunoblotted with the indicated antibodies. Top, ER-associated GFP-LC3 puncta; Middle, cytosolic GFP-LC3 puncta. (D) PC3 cells stably expressing shRNF5 were maintained in HBSS (3 h) or regular medium. Membrane fractions were separated by sucrose gradient ultracentrifugation. Proteins (40 µg/lane) were resolved and immunoblotted with the indicated antibodies.
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Related In: Results  -  Collection

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pgen-1003007-g005: RNF5 co-localizes with—and limits—ATG4B at the membrane compartment.(A) RNF5/LC3 immunofluorescence images in HeLa cells expressing GFP-sec61b. Magnified view of the white square region is shown on the bottom right panels; white arrows denote LC3 foci that colocalized with ER-localized RNF5. Scale bar, 10 microns. (B) ATG4B and RNF5 co-fractionate in ER and autophagosomes. 293T cells were maintained under normal medium or HBSS starvation (3 h) were collected and passed through a 25-gauge needle 10 times and the membrane fractions were enriched and loaded to 10–50% linear sucrose gradient and subjected to ultracentrifugation (160,000 g for 2.5 h). Twelve fractions (1 ml each) were collected from the top to the bottom, and analyzed by western blots as indicated. Calnexin, ER membrane marker. (C) 293T cells were starved using media supplemented with HBSS, and the cells were collected at the indicated time points. Membrane fractions were separated by sucrose gradient ultracentrifugation. Proteins (40 µg/lane) were resolved and immunoblotted with the indicated antibodies. Top, ER-associated GFP-LC3 puncta; Middle, cytosolic GFP-LC3 puncta. (D) PC3 cells stably expressing shRNF5 were maintained in HBSS (3 h) or regular medium. Membrane fractions were separated by sucrose gradient ultracentrifugation. Proteins (40 µg/lane) were resolved and immunoblotted with the indicated antibodies.
Mentions: Because RNF5 is a membrane-bound E3 ubiquitin ligase, and since it requires its membrane-anchor to affect ATG4B and autophagy we assessed whether RNF5 activity towards ATG4B may preferentially take place at membranal domains. The autophagy markers LC3 and the ER membrane marker Sec61 were used to determine degree of RNF5-colocalization with LC3 at the membranal fraction. Analysis was performed in both MEFs and HeLa cells grown under normal conditions. In HeLa cells we found that 74% of the LC3/RNF5 colocalized spots were within the sec61b structures, whereas in the MEFs this percentage was found to be 83% (Figure 5A). This data suggest that RNF5 effect on LC3 processing does take place within the ER domain.

Bottom Line: RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 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.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction and Cell Death Programs, Sanford-Burnham Medical Research Institute, La Jolla, California, USA.

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.

Show MeSH
Related in: MedlinePlus