Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes

Abstract

Autophagy is responsible for nonspecific, bulk degradation of cytoplasmic components. Recent work has revealed also that there is specific, autophagic degradation of polyubiquitinated protein aggregates, whose buildup occurs during neurodegenerative disease. Here, we report that simple mono-ubiquitination of normally long-lived cytoplasmic substrates is sufficient to target these substrates for autophagic degradation in mammalian cells. That is, upon their ubiquitination, both small [i.e., red fluorescent protein (RFP)] and large (i.e., peroxisomes) substrates are efficiently targeted to autophagosomes and then degraded within lysosomes upon autophagosome-lysosome fusion. This targeting requires the ubiquitin-binding protein, p62, and is blocked by the Class III phosphatidylinositol 3-kinase (PI3K) inhibitor, 3-methyladenine (3-MA), or by depletion of the autophagy-related-12 (Atg12) protein homolog. Mammalian cells thus use a common pathway involving ubiquitin and p62 for targeting diverse types of substrates for autophagy.

Fig. 1.

Ubiquitin targets cytosolic RFP to autophagosomes for degradation. (A and B) COS-7 cells transiently cotransfected with GFP-LC3 and UB-RFP (A), or GFP-LC3 and RFP (B). Cells were imaged 24 h after transfection. Arrowheads in (A) indicate obvious examples of colocalized GFP-LC3 and UB-RFP. (C and D) COS-7 cells transiently cotransfected with either GFP-LC3 and UB-RFP (C) or GFP-LC3 and RFP (D), and treated with 0.25 mM leupeptin for 20 h before imaging. (E) COS-7 cells transiently coexpressing GFP-LC3 and UB-RFP, and treated with 0.25 mM leupeptin and 10 mM 3-MA for 20 h before imaging. (F) Fluorescence Protease Protection assay of COS-7 cells coexpressing GFP-LC3 and UB-RFP. 24 h after cotransfection and leupeptin treatment, cells were washed and then treated with 0.6% [vol/vol] digitonin (D) for 10 min and than with 0.005% [wt/vol] trypsin (T), followed by imaging. Arrowheads indicate obvious examples of colocalized GFP-LC3 and UB-RFP. (Scale bars, 10 μm.)

Fig. 2.

Monomeric ubiquitin is sufficient to target RFP for degradation by autophagy. (A) Immunoblot of cell lysate from COS-7 cells expressing RFP, UB-RFP, or UBko-RFP. 25 μg of total protein was resolved by SDS/PAGE and immunoblot using rabbit anti-RFP antibodies and donkey anti-rabbit IgG conjugated to horseradish peroxidase. Asterisks indicate the position of three higher molecular weight species of polyubiquitinated UBko-RFP. The single arrowhead indicates the position of mono-ubiquitinated RFP, whereas the double and triple arrowheads indicate the position of RFP alone and a cross reacting band, respectively. Molecular masses (in kDa) are indicated on the left side of the blot. (B–E) COS-7 cells cotransfected with LAMP1-GFP and either UB-RFP (B and C), UB-RFP (D), or RFP (E). Leupeptin (0.25 mM) was added 20 h before imaging. Cells shown in (C) were treated also with 10 mM 3-MA. (Scale bars, 10 μm.) (F) Quantification of the percentage of cells with five or more punctate RFP signals that colocalized with GFP-LC3 in cells coexpressing GFP-LC3 and various RFP constructs as indicated. Cells were also incubated with either leupeptin alone (white bar), or with leupeptin and 3-MA (dark gray bars). Shown are the averages ± standard deviations from three independent experiments with each experiment including at least 50 cells scored.

Fig. 3.

p62 is required for sequestering UB-RFP into punctate structures. (A and B) HeLa cells transfected with either siRNA pools directed against p62 (A) or control siRNA (B). Twenty-four hours later, cells were transfected again with the respective siRNA and also with a plasmid encoding UB-RFP. Four hours after the second transfection, leupeptin (0.25 mM) was added to the cells, and 20 h after that, cells were fixed and stained with anti-p62 and Alexa 543-goat anti-rabbit antibodies. White lines in micrographs illustrating endogenous p62 immunostaining frame the outline of cells coexpressing UB-RFP. Arrowheads in (B) indicate obvious examples of colocalized UB-RFP and endogenous p62. (Scale bars, 10 μm.) (C) Quantification of the percentage of cells with 5 or more punctate structures containing either RFP or UB-RFP and GFP-LC3 in cells transfected with also control siRNA or p62 siRNA and treated with leupeptin as in (A and B). Shown are the averages ± standard deviations from three independent experiments with each experiment including at least 50 cells scored.

Fig. 4.

Ubiquitination of a PMP results in a decrease in the number of peroxisomes within a cell. (A) Schematic illustration of the predicted topological orientation of PMP34-GFP-UBko, UB-GFP-SKL, UBko-PEX3-GFP, and PEX3-GFP-UBko. (B–E) COS-7 cells transiently expressing either PMP34-GFP (A), empty vector (mock) (B), PMP34-GFP-UB (C), or PMP34-GFP-UBko (D) were fixed and stained with anti-catalase and Alexa 543-goat anti-rabbit antibodies 48 h after transfection. Note that two PMP34-GFP-UBko-transformed cells can be seen in (D), both of which display a reduced number of peroxisomes relative to cells transformed with the empty vector (B) or PMP34-GFP (A). (F) Immunoblot of cell lysate from COS-7 cells expressing PMP34-GFP, PMP34-GFP-UB or mock treated. Cells were lysed, and 25 μg of total protein was subjected to SDS/PAGE and then immunoblotted with antibodies against PMP70 or the cytosolic protein glyceraldehydes phosphate dehydrogenase (GAPDH) serving as a protein loading control. (G) Percentage of total number of transfected cells with less than 50 peroxisomes per cell expressing various proteins as indicated 48 h after transfection. Shown are the averages ± standard deviations from three independent samples with each experiment including at least 100 cells scored.

Fig. 5.

The autophagic pathway mediates a decrease in ubiquitinated peroxisomes. (A and B) COS-7 cells coexpressing CFP-LC3 and either PMP34-Venus (A) or PMP34-Venus-UBko (B). Arrowheads in (B) indicate obvious examples of colocalized PMP34-Venus-UBko and CFP-LC3. (C–E) COS-7 cells coexpressing LAMP1-Cherry and either PMP34-GFP-UBko (C and D) or PMP34-GFP (E). Cells were also treated with leupeptin (0.25 mM) and chloroquine (0.1 mM) 8 h after transfection. The higher magnified images shown in (D) are a maximum intensity projection of a z series of the magnified area of the single slice image of the cell outlined in (C). (F) Percentage of the total number of transfected cells with less than 50 peroxisomes per cell expressing either PMP34-GFP or PMP34-GFP-UB, and with or without 3-MA, after 24-h or 48-h post-transfection. Shown are averages ± standard deviation from three independent samples with each experiment including at least 100 cells scored. (Scale bars, 10 μm.)

Fig. 6.

Silencing of Atg12 expression prevents ubiquitin-mediated peroxisome degradation. (A) Immunoblot of cell lysate from HeLa cells before Atg12 siRNA treatment (day 0), or 1 and 2 days after Atg12 siRNA treatment as indicated. Cells were lysed and 25 μg of total protein was subjected to SDS/PAGE and then immunoblotted with antibodies against either Atg12 or GAPDH serving as a loading control. Depletion in Atg12 expression was indicated by the decrease in the Atg12-Atg5 protein conjugate. (B–C) HeLa cells were transfected with either siRNA pool directed against Atg12 (B), or a nontargeting control siRNA (C). Twenty-four hours after the initial transfection, cell were transfected again with the appropriate siRNA along with a plasmids encoding PMP34-GFP-UBko. Forty-eight hours after the second transfection cells were fixed and stained with anti-Atg12 and Alexa 543-goat anti-rabbit antibodies. (D) Percentage of the total number of cells with less than 50 peroxisomes per cell expressing either PMP34-GFP or PMP34-GFP-UBko and treated with either control siRNA or Atg12 siRNA. Shown are the averages ± standard deviations from three independent samples with each experiment including at least 100 cells scored. (Scale bars, 10 μm.)

Fig. 7.

p62 is required for the degradation of peroxisomes. (A and B) HeLa cells transiently expressing either PMP34-GFP-UBko (A) or PMP34-GFP (B) were fixed and stained with anti-p62 and Alexa 543-goat anti-rabbit antibodies 48 h after transfection. Arrowheads in (A) indicate obvious examples of colocalized PMP34-GFP-UBko and endogenous p62. The area of the cell outlined in the merge image in (B) is shown at higher magnification in the inset; note the lack of any obvious colocalizations of expressed PMP34-GFP and endogenous p62 in this cell. (C–F) HeLa cells transfected with either control siRNA or a siRNA pool for p62 (p62 siRNA) as indicated. Twenty-four hours after the initial transfection, cells were retransfected with siRNA and also with a plasmids encoding either PMP34-GFP-UBko (C and D) or PMP34-GFP (E and F). Cells were fixed and stained as above either 48 h after first transfection treatment. All images shown are maximum projections of z-series. (G) Percentage of the total number of transfected cells with less than 50 peroxisomes expressing either PMP34-GFP or PMP34-GFP-UBko and treated with either control siRNA or p62 siRNA at 72-h post-transfection. Shown are the averages ± standard deviations from three independent samples with each experiment including at least 75 cells scored. (H) The mean fluorescence intensity of the immunolabeled endogenous catalase from at least 25 cells treated with either control siRNA or a siRNA pool for p62. Also shown are the standard deviations of the mean values (P < 0.01). (Scale bars, 10 μm.)