Ubiquilin functions in autophagy and is degraded by chaperone-mediated autophagy

Abstract

Autophagy is the process by which organelles and portions of the cytoplasm are degraded in lysosomes. Several different forms of autophagy are known that are distinguishable chiefly by the mode in which cargo is delivered to the lysosome for degradation. Ubiquilin was recently reported to regulate macroautophagy, the form of autophagy in which cytosolic cargo is packaged in a double-membrane structure or autophagosome that fuses with lysosomes for degradation. We confirm here using different morphological and biochemical procedures that ubiquilin is present in autophagosomes in HeLa cells and in brain and liver tissue of mouse. Coimmunoprecipitation studies indicated that ubiquilin binds the autophagosome marker LC3 in a complex and that reduction of ubiquilin expression reduces autophagosome formation, which correlates with a reduction in maturation of LC3-I to the LC3-II form of the protein. We found that ubiquilin is degraded during both macroautophagy and during chaperone-mediated autophagy (CMA), the latter of which involves the active transport of proteins into lysosomes. We discuss the implication of this degradation in mediating cross-talk between macroautophagy and CMA. Finally, we demonstrate that ubiquilin protects cells against starvation-induced cell death propagated by overexpression of mutant Alzheimer's disease PS2N141I protein and green fluorescent protein (GFP)-huntingtin exon-1 fusion protein containing 74 polyglutamines.

Figure 1.

Ubiquilin colocalizes with LC3 in autophagosomes. (A) Immunofluorescence microscopy of HeLa cells stained for endogenous ubiquilin (a) and LC3 proteins (b) and the result of merging of the two images (c). Arrows in this and subsequent panels show examples of colocalization of ubiquilin and LC3 in characteristic autophagosome-like structures. Fluorescent images of a HeLa cell transfected with mRFP- and GFP-tagged ubiquilin-1 (d) and LC3 (e) proteins and the resulting merged image (f). Fluorescent images of HeLa cells transfected with mRFP- and GFP-tagged LC3 (g) and ubiquilin-1 (h) proteins and the resulting merged image (i). (B) Immunofluorescence microscopy of HeLa cells transfected with double-tagged mCherry-GFP-LC3 reporter and stained for ubiquilin. The left-hand panels show the images of the primary fluorescence of GFP (a), mCherry (c) and ubiquilin staining (e) and the right-hand images are the result produced after merging two of the three primary images. The white arrow shows the colocalization of ubiquilin with GFP and mCherry fluorescence. The arrowhead shows colocalization of ubiquilin with predominantly mCherry and faint GFP fluorescence. The yellow arrow indicates rare ubiquilin-positive puncta in which GPF or mCherry fluorescence was not detected. Bar, 5 µm.

Figure 2.

Ubiquilin, LC3 and polyubiquitinated proteins bind together in a complex. (A) Equal portions of a HeLa cell lysates were used to immunoprecipitate proteins with either an anti-ubiquilin antibody or its pre-immune serum and the resulting precipitates were probed with antibodies to detect different proteins, as indicated. (B) Cultures of HeLa cells were transfected with cDNAs encoding GFP alone, or GFP-LC3, or GFP-LC3 and ubiquilin-1 (+ indicates lanes in which the cDNAs were transfected). GFP proteins were immunoprecipitated from lysates of the cultures and the resulting precipitates were examined for coimmunoprecipitation of ubiquilin by immunoblotting (bottom panel). Also shown are blots of the total lysates probed with different antibodies, as indicated. (C) Duplicate samples of K48 and K63 in vitro assembled ubiquitin chains (− and + indicate lanes in which the chains were omitted or added, respectively) were immunoblotted with antibodies that specifically recognize K48 or K63 chains, illustrating their specificity. (D) HeLa cells grown on coverslips were transfected with cDNA encoding mCherry-GFP-LC3 and after 24 h the cells were fixed and stained with antibodies to specifically detect either ubiquilin (a–d), or K48-ubiquitin chains (e–h), or K63-ubiquitin chains (i–j). GFP (a, e and i), mCherry (b, f and j) and Cy5.5 (c, g and k) fluorescence images were captured and used to examine whether ubiquilin, K48 or K63 chains colocalized with the mCherry-GFP-LC3 reporter. The right-hand panels are the images produced after merging the three images shown to the left of each of them. Bar, 5 µm.

Figure 3.

Knockdown of ubiquilin inhibits autophagosome formation and maturation of LC3 protein. (A–C) HeLa cells were either mock transfected or transfected with siRNAs to specifically knockdown ubiquilin-1 and -2 proteins and 24 h later they were both re-transfected with cDNA encoding mCherry-GFP-LC3. Twenty hours later, the cells were fixed and the percent of GFP transfected cells that displayed five or more GFP puncta was quantified. (A) Representative images of cells showing knockdown of ubiquilin reduces autophagosome formation as measured using the mCherry-GFP-LC3 reporter. Bar, 10 µm. (B) Graphs of the quantification of cells with autophagosomes described in (A) obtained from three independent experiments. The graphs in this and subsequent figures show the mean and the error bars show the standard deviation. (C) Immunoblots showing effective knockdown of ubiquilin proteins in the cultures after 44 h of knockdown (+ and − indicate the cells that were transfected with siRNAs to knockdown ubiquilin-1 and -2 proteins, or which were mock-transfected, respectively). (D) Cultures of HeLa cells were transfected with siRNAs to knockdown ubiquilin-1 and -2 proteins or with negative control siRNAs and lysates were collected 0, 24, 48 and 72 h later. The lysates containing equal amounts of protein were immunoblotted with antibodies to detect ubiquilin, actin and LC3 proteins. Also shown are graphs of the quantification of the ubiquilin and LC3-I and -II bands. Similar results were seen in two separate experiments.

Figure 4.

Ubiquilin is degraded during macroautophagy. (A) HeLa cell cultures were treated with puromycin or puromycin and bafilomycin A1 for the time periods as indicated. At the intervals shown, the cells were fixed and stained for ubiquilin. The percent of cells displaying five or more ubiquilin puncta was quantified after capturing images under the microscope with a ×40 objective lens. (B) Immunoblots of equal amounts of protein lysate made from HeLa cell cultures treated for 0–7 h with puromycin and probed for ubiquilin, LC3 or actin. (C) Same as in (B) except that additional lysates from cultures that were treated for 2, 4, and 7 h with both puromycin and bafilomycin A1 were analyzed. Also shown are graphs of the quantification of the ubiquilin [lower two bands corresponding to ubiquilin-1 and -2 proteins (from three separate experiments), and LC3-II bands (from two separate experiments)]. (D) Immunoblots of equal amounts of protein lysate prepared from HeLa cell cultures treated for 2 or 7 h with different combinations of puromycin and 3MA. Actin was used to monitor protein loading. Also shown are graphs of the quantification of the ubiquilin and LC3-II bands obtained from three separate experiments.

Figure 5.

Ubiquilin is enriched in purified autophagosomes and lysosomes active for CMA. Subcellular fractions from mouse liver were isolated as described under material and methods and 100 µg protein of each fraction were subjected to immunoblot analysis for the indicated proteins. Hom, homogenate; Cyt, cytosol; AV, autophagosomes; Lys C, lysosomes; CMA−, lysosomes with low activity for CMA; CMA+, lysosomes active for CMA.

Figure 6.

Ubiquilin is a substrate of CMA. (A) GST-ubiquilin-1 (2 µg) was incubated with freshly isolated intact lysosomes (30 µg) treated (+) or not (−) with protease inhibitors. Where indicated, a non-CMA substrate Ovalbumin (Ova) (lanes 6–7) or a CMA substrate GAPDH (lanes 8–9) were added to the incubation media. i: input (0.5 µg). (Bottom) Quantification of binding, uptake and association of GST-ubiquilin-1 to lysosomes. Values are expressed as percentage of the protein added into the reaction and are mean + SE of the densitometric analysis of the four separate experiments. (B) Competition of CMA uptake of GST-ubiquilin-1 by increasing concentrations of GAPDH. GST-ubiquilin-1 (2 µg) was incubated with lysosomes either alone or in the presence of increasing concentrations (as indicated) of GAPDH in an experiment similar to that shown in (A). The graph shows the quantification of binding and uptake of GST-Ubiquilin by lysosomes in the presence of GAPDH. Values were obtained from the densitometric analysis of three different experiments. Inset shows a representative immunoblot. (C) Blockage of ubiquilin CMA with antibodies against LAMP-2A and hsc70. GST-ubiquilin-1 (2 µg) was incubated with lysosomes as in (A) in the absence or presence of neutralizing antibodies against LAMP-2A or hsc70. Quantification of association of GST-ubiquilin-1 to lysosomes is shown. Values are mean + SE and were obtained from the densitometric analysis of three different experiments. Inset shows a representative immunoblot.

Figure 7.

Ubiquilin overexpression reduces starvation-induced cell death caused by overexpression of presenilin-2 proteins. (A and B) Cultures of HeLa cells were transfected with a total of 6 µg of DNA, 2 µg of which corresponded to an expression plasmid encoding either GFP alone, or wild-type PS2-GFP, or PS2N141I-GFP protein and the remainder of which contained either 0, 2 or 4 µg of cDNA encoding ubiquilin-1 protein or with empty vector DNA. Twenty hours after transfection, the cultures were washed and incubated in starvation medium for either 0 or 4 h after which time cell death of GFP expressing cells was quantified. (A) Immunoblots of lysates from the transfected cultures blotted for expression of the GFP-tagged proteins, ubiquilin proteins and actin. (B) Quantification of cell death of GFP expressing cells in the cultures obtained in three independent experiments.

Figure 8.

Knockdown of ubiquilin expression enhances starvation-induced cell death in HeLa cells stably expressing GFP-Htt74Q protein. (A–C) Cultures of HeLa cells stably expressing GFP-Htt74Q protein were either mock transfected or transfected with siRNAs to specifically knockdown ubiquilin-1 and -2 proteins. Twenty-four hours after transfection, the medium was replaced with starvation medium and cell death assays were performed at 0 and 4 h after starvation. (A) Phase-contrast microscopy images of cells just before transfection and 24 h post-transfection after culturing for 0 and 4 h in starvation medium. Bar, 100 µm. (B) Hoechst staining of cells at 0 and 4 h of starvation. Bar, 100 µm. (C) Quantification of cell death seen in the cultures after 24 h post-transfection. (D) Immunoblots showing knockdown of ubiquilin in lysates from the cultures.

Figure 9.

Ubiquilin immunoreactivity is associated with plaques in the brain of PS1/APP mice. (A–F) Mouse brain sections from a 12-month-old PS1/APP mouse (B–F) and an age-matched non-transgenic control (A) stained with antibodies against ubiquilin by immunohostochemistry. Examinations of plaques at higher magnifications reveal neurites demonstrating a clear, punctate labeling pattern (C–F, arrows and C and D, inset). Bars A–F: 20 µm; C inset and D inset: 10 µm.

Figure 10.

Immunogold localization of ubiquilin in dystrophic neurites of brain from a 12-month-old PS/APP mouse using a pre-embedding silver enhancement technique. Ubiquilin immunoreactivity is highly specific to AVs within dystrophic neurites (A–C, arrows) and is absent in other organelles (C, arrowheads). Bar: 500 nm.