Figure 1.
Potential Interaction stages of p62 with LC3 species in the course of LC3 lipidation.
The known stages for LC3 lipidation are boxed. After translation pro-LC3B is processed by Atg4B by removing the last five amino acids to expose the G120 residue (LC3-I). A portion of LC3-I forms a complex with the E1-like enzyme Atg7 and subsequently generates a LC3-Atg7 conjugate. LC3-Atg7 then interacts with the E2-like enzyme Atg3 to form a LC3-Atg3 conjugate which interacts with Atg16L1:Atg5-Atg12 complex to convert to LC3-II under autophagic stimulation. p62 could potentially bind any LC3 specie at any stage of this process.
Figure 2.
Validation of recombinant Atg gene expression.
(A) Expression plasmids were transfected into HEK293 cells for 24 h. Cell lysates were used to detect the fusion genes’ expression by Western blot using antibodies as indicated. (B) Flag-LC3 or Flag-LC3A120 was transfected into HEK293 cells for 24 h. 50 µM of Chloroquine (CQ) was used to induce Flag- LC3-II accumulation for 2 h before harvesting the cells. Flag-LC3 is normally lipidated as demonstrated by Flag-LC3-II accumulation after CQ treatment. No such accumulation was seen in Flag-LC3A120 mutant after CQ treatment demonstrating that this mutant does not pass through the lipidation process. (C) MEFwt cells were transfected with indicated expression plasmids for 24 h before they were recorded by fluorescence microscopy. GFP-Atg4B, GFP-Atg7 and GFP-Atg3 were mainly distributed in the cytosol. No puncta was observed at any given time. Scale bar = 25micron.
Figure 3.
p62 aggregates do not sequester Atg4B.
GFP-Atg4B and mRFP-p62 expression plasmids (A) or with Flag-LC3 (B) were co-transfected into MEFwt cells for 24h. GFP-Atg4BC74A and mRFP-p62 with Flag-LC3 vectors (C) or with Flag-LC3A120 vector (D) were co-transfected into MEFwt cells for 24 h. GFP-Atg4B or GFP-Atg4BC74A expression vector was co-transfected with Flag-LC3 (E) or Flag-LC3A120 (F) into HEK293 cells for 48 h. Immunoprecipitation (IP) was performed using total cell lysates. Immunoblotting was conducted using the antibodies as indicated. (G) mRFP-p62, GFP-Atg4BC74A and Flag-LC3 expression vectors were co-transfected into MEFwt cells for 24 h. Colocalization of Flag-LC3 within mRFP-p62 aggregates were detected by immunofluorescence staining (IF) using Flag M2 antibody. Fluorescence images presented in this study were representative of at least three independent experiments. Scale bar = 25micron.
Figure 4.
p62 aggregates sequester LC3-I.
Vectors expressing mRFP-p62, GFP-LC3, or GFP-LC3A120 were transfected into MEFatg5−/− cells as indicated (A and B). Images were taken at 48 h time point. The same sets of plasmids were transfected into MEFwt cells as indicated for 72 h. Images were recorded at 20 h and 72 h post transfection (C–F). Scale bar = 25micro. Arrows point to mRFP-p62 only punctae. The inserts were enlarged to show the puncta in detail.
Figure 5.
p62 aggregates sequester Atg7 in a LC3-dependent manner.
GFP-Atg7 (Aa), GFP-Atg7/Flag-LC3 (Ab), or GFP-Atg7C572A/Flag-LC3 (Ac) vectors were co-transfected with mRFP-p62 into MEFwt cells for 48 h. GFP-Atg7 punctae were shown; mRFP-p62 and Flag-LC3 with GFP-Atg7 vectors (B) or with GFP-Atg7C572A(C) were co-transfected into MEFwt cells for 48 h; mRFP-p62 and Flag-LC3A120 with GFP-Atg7 vectors (D) or with GFP-Atg7C572A (E) were co-transfected into MEFwt cells for 48 h. Flag-LC3, GFP-Atg7, or GFP-Atg7C572A expression vectors were co-transfected into HEK293 cells as indicated for 48 h. Total cell lysates were used for IP by the Flag M2 antibody. Immunoblotting was conducted using the antibodies as indicated (F and G). Expressing vectors of GFP-Atg12 and mRFP-p62 were co-transfected into MEFwt cells for 48 h (H). All images were recorded at 48 h post-transfection. Scale bar = 25 micron. The inserts were enlarged to show the puncta in detail.
Figure 6.
p62 aggregates do not sequester Atg3 and LC3-Atg3.
GFP-Atg3 and mRFP-p62 (A) or with Flag-LC3 vectors (B) were co-transfected into MEFwt cells for 48 h. GFP-Atg3C264S, mRFP-p62 and Flag-LC3 vectors were co-transfected into MEFwt cells for 48 h (C). Flag-LC3, GFP-Atg3, GFP-Atg3C264S, or GFP-Atg3C264A expression vectors were co-transfected into HEK293 cells for 48 h. Immunoprecipitation was conducted using total cell lysates. Immunoblotting was conducted as indicated. Images were recorded at 48 h post-transfection. Scale bar = 25 micron.
Figure 7.
Mutual exclusion of Atg4 or Atg3 with p62 in LC3 complex.
HEK293 cells were co-transfected with mRFP-p62/GFP-Atg3/Flag-LC3 or mRFP-p62/GFP-Atg4B/Flag-LC3 for 48 h. Total cell lysates were used to conduct IP using correspondent antibodies. Immunoblottings were then applied using antibodies as indicated (A and B). HEK293 cells were transfected with GFP-Atg4B or GFP-Atg4BC74A alone or with mRFP-p62 for 48 h (C). HEK293 cells were transfected with GFP-Atg3 alone or with mRFP-p62 for 48 h (D). Total cell lysates were used to detect LC3 species as indicated. Relative band intensity of protein of interest was divided by correspondent band intensity of β-actin loading control. Fold of increase or decrease was then determined by setting the control sample as 1.
Figure 8.
p62 preferentially binds LC3-II under autophagic stimulation.
HEK293-GFP-LC3 cells were cultured in nutrient-rich media (Aa) or treated with CPP for 2 h (Ab) to induce large GFP-LC3 positive vesicles. HEK293-GFP-LC3A120 cells were used as a control (Ac); (B) HEK293-GFP-LC3 cells were transfected with mRFP-p62 for 24 h, and then treated with CPP for 2 h to induce large GFP-LC3/mRFP-p62 positive vesicles. (C) HEK293-GFP-LC3 cells were transfected with mRFP-p62-LRS-mut.2 for 24 h, and then treated with CPP for 2 h. HEK293-GFP-LC3 cell line was cultured in regular media (D) or treated with CPP for 2 h (E) before fixed and immunostained for the endogenous LC3 and p62. (F) HEK293-GFP-LC3 cells were cultured in regular media or treated with CPP for 2 h. Total cell lysates were used for immunoprecipitation of endogenous p62. Western blot analysis was conducted to detect p62 bound GFP-LC3 species. Arrow points to GFP-LC3 positive vesicle. Arrow head points to p62 aggregate. Scale bar = 25 micron.
Figure 9.
(A) p62 aggregates’ effect on cytosolic LC3 and Atg7 levels. A549 cells stably expressing GFP-LC3 were infected with Ad-mRFP-p62 at 20 MOI (D1) and 10 MOI (D2) for 48 h.
Cells were then cultured in nutrient-rich media or starved in EBSS for 2 h. Total cell lysates were centrifuged at 15,000 g to separate p62 aggregates. The supernatants were used for immunoblot assays as indicated. Image J was used to quantify the related band densities using β-actin as a loading control. Relative band intensity of protein of interest was divided by the correspondent band intensity of β-actin. Fold of increase or decrease was then determined by setting the control sample as 1. CM = complete media. S = starvation. (B) Summary of p62 interaction with LC3. As Atg4B occupies or blocks the binding site for p62, there is no interaction between p62 and LC3 (a). Atg7 binds LC3 but does not block the p62 interaction domain allowing the interaction of p62 and LC3 (b). As Atg3 occupies or blocks the binding site for p62, there is no interaction between p62 and LC3(c). Membrane bound LC3-II exposes the binding site for p62 after it is converted from the LC3-Atg3 and becomes the receptor for p62.