Autophagy mediates SUMO-induced degradation of a polyglutamine protein ataxin-3

Abstract

Previously, we reported that small ubiquitin-like modifier-1 (SUMO-1) promotes the degradation of a polyglutamine (polyQ) protein ataxin-3 and proposed that proteasomes mediate the proteolysis. Here, we present evidence that autophagy is also responsible for SUMO-induced degradation of this polyQ protein. The autophagy inhibitor 3-MA increased the steady-state level of ataxin-3 and stabilized SUMO-modified ataxin-3 more prominently than the proteasome inhibitor MG132. Interestingly, SUMO-1 overexpression enhanced the co-localization of ataxin-3 and autophagy marker LC3 without increasing LC3 puncta formation suggesting that SUMO-1 is involved in the substrate recruitment rather than the induction of autophagy. To assess the importance of a putative SUMO-interacting motif (SIM) in ataxin-3 for SUMO-induced degradation, we constructed a SIM mutant of ataxin-3. Substitution of putative SIM (V165G) facilitated the degradation of polyQ-expanded ataxin-3, which is more resistant to SUMO-induced degradation than the normal ataxin-3. These results together indicate that SUMO-1 promotes the degradation of ataxin-3 via autophagy and the putative SIM of ataxin-3 plays a role in this process.

Keywords: Ataxin-3; SUMO; SUMO-interacting motif; autophagy.

Figure 1.

Effects of MG132 or 3-MA treatment on ataxin-3 degradation. To assess the involvement of different proteolytic pathways on ataxin-3 degradation, BOSC cells expressing HA-tagged ataxin-3 (26Q or 73Q) and FLAG-tagged SUMO-1 were incubated with 5 µM MG132 (a proteasome inhibitor) (A) or 5 mM 3-MA (an autophagy inhibitor) (B). After 24 h, cells were collected, lysed and the cell lysate was subjected to immunoblot analysis using anti-HA antibody (ataxin-3) and anti-β-actin antibody. Arrows indicate the SUMO-modified form of ataxin-3.

Figure 2.

SUMO-1 overexpression does not increase ubiquitylation of ataxin-3. To test if SUMO-1 overexpression leads to the increased ubiquitylation of ataxin-3, HA-tagged ataxin-3 (26Q or 73Q), myc-tagged ubiquitin (Ub) and FLAG-tagged SUMO-1 were transfected into BOSC cells. When necessary, cells were incubated with MG132 (5 µM) to block the degradation of ataxin-3. The cell lysate was prepared and subjected to immunoprecipitation with anti-HA agarose beads followed by immunoblot with anti-HA antibody to ensure that ataxin-3 proteins were properly isolated (upper panel) or with anti-myc antibody to detect poly-ubiquitylated ataxin-3 (bottom panel).

Figure 3.

SUMO-1 enhances the co-localization of ataxin-3 and LC3. After 24 h of transfection of HA-tagged ataxin-3 (26Q or 73Q) with FLAG-tagged SUMO-1, BOSC cells were fixed and permeabilized. To determine the localization of ataxin-3 and LC3 (autophagy marker), cells were incubated with anti-HA antibody followed by FITC-anti-IgG antibody (for ataxin-3) and anti-LC3 antibody followed by Cy3-anti-IgG antibody (for LC3). DAPI was used for nuclear staining. Arrowheads indicate co-localization of ataxin-3 and LC3 (upper panel). For quantification of signal co-localization, images were analyzed by ImageJ (Coloc 2 plug-in) for co-localization correlation (Pearson correlation coefficient r). Bar graphs represent r values of three independent images selected from each group (mean ± SD) (lower panel).

Figure 4.

Effects of SIM mutation on the degradation of ataxin-3. To determine the role of SIM in ataxin-3 degradation, the effects of SUMO-1 overexpression on the normal (WT) and SIM mutant (V165G) of ataxin-3 (26Q and 73Q) were compared. (A) BOSC cells coexpressing HA-tagged ataxin-3 (1 µg) with different amount of FLAG-tagged SUMO-1 (0, 1, 2, 3 µg) were collected and the cell lysate was prepared. (B) After 24 h of transfection with ataxin-3 (normal and SIM mutant) and SUMO-1 (each 1 µg), cells were treated with 20 µg/ml of cycloheximide to block the synthesis of proteins. From this point (t = 0), cells were collected every 8 h (up to 24 h) and then processed for the immunoblot analysis. Immunoblot analysis of ataxin-3 with anti-HA antibody was performed as described in Figure 1. Graphs in the lower panels are the results of image quantification from three independent experiments. Bars = SD