Ubiquilin4 is an adaptor protein that recruits Ubiquilin1 to the autophagy machinery

Abstract

Ubiquilins (Ubqlns)-a family of ubiquitin-binding proteins-are involved in several protein degradation pathways and have been implicated in various neurodegenerative diseases. Ubqln1 regulates autophagosome maturation during autophagy-mediated degradation. We now show that Ubqln4 mediates the interaction between Ubqln1 and the autophagy machinery by recruiting Ubqln1 to LC3. This targeting of Ubqln1 to autophagosomes requires the Ubqln4 UBL domain and the Ubqln1 UBA domain. This study identifies a new role for Ubqln4, expanding the role for Ubqlns in protein degradation.

Figure 1

Ubqln1 interacts with Ubqln4. (A) Inducible GFP-S-Ubqln1 293 cell line was treated with 1 μg/ml of doxycycline for 24 h. GFP-S-Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis. Red sequences indicate Ubqln4 peptides identified by mass spectrometry. (B) 293 cells were co-transfected with GFP-S or GFP-S-Ubqln1 and HA-Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti-GFP and anti-HA antibodies. Lower molecular weight GFP-tag-containing bands that appear in the GFP-S-Ubqln1 lane might represent degradation products. (C) Endogenous Ubqln1 in 293 cells was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG. The immunoprecipitates were analysed by western blotting using anti-Ubqln1 and anti-Ubqln4 antibodies. (D) Confocal images of Hela cells transfected with GFP-S-Ubqln1 and RFP-Ubqln4. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross-section. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; Ubqln, Ubiquilin; WCL, whole-cell lysate.

Figure 2

Ubqln4 interacts with LC3. Confocal images of (A) Hela cells transfected with GFP-LC3 and RFP-Ubqln4 and starved for 1 h or (B) Hela cells starved for 1 h and stained for endogenous LC3 and Ubqln4. The squares show colocalization. The insets show the enlargement of the indicated areas. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross-section. (C) 293 cells were transfected with GFP-S, GFP-S-Ubqln1 or GFP-S-Ubqln4 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti-GFP and anti-LC3 antibodies. (D) Recombinant His-Ubqln1, His-Ubqln4, His-S5a or His-p62 was mixed with LC3 covalently coupled to agarose beads. The beads were washed, eluted and detected by western blot using anti-His antibody. The extent of Ubqln1, Ubqln4, s5a or p62 co-immunoprecipitation with LC3 was measured. Bars for Ubqln1, Ubqln4 and s5a represents the average and s.e.m. of four independent experiments. *P-value=0.0094. (E) 293 cells were transfected with GFP-S-Ubqln4 WT, GFP-S-Ubqln4 ΔSTI1 12, GFP-S-Ubqln4 Δ between STI1 or GFP-S-Ubqln4 ΔSTI1 34 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti-GFP and anti-LC3 antibodies. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule-associated protein light chain 3; Ubqln, Ubiquilin; WCL, whole-cell lysate; WT, wild-type.

Figure 3

Ubqln1 punctate distribution is dependent on Ubqln4. (A) Confocal images of Hela cells transfected with GFP-S-Ubqln1 and also transfected with either control or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 1 h. (B) Quantification of the number of GFP-S-Ubqln1 WT puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P-value=0.011, **P-value=0.0005 and ***P-value=0.0001. (C) Depletion of Ubqln4 with siRNA in (A) was verified by western blot using anti-Ubqln1, anti-Ubqln4 and anti-tubulin antibodies. (D) Hela cells transfected with GFP-S-Ubqln1 and also transfected with either control or Ubqln4 siRNA were starved for 1 h. GFP was immunoprecipitated and analysed by western blot using anti-GFP and anti-LC3 antibodies. (E) Confocal images of GFP-S-Ubqln1 WT in Hela cells transfected with RFP, RFP-Ubqln4 or RFP-Ubqln4 ΔUBL. (F) Quantification of the number of GFP-S-Ubqln1 WT puncta per cell in (E). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P-value=0.0001 and **P-value=0.0003. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule-associated protein light chain 3; UBL, ubiquitin-like; Ubqln, Ubiquilin; WCL, whole-cell lysate; WT, wild-type.

Figure 4

Ubqln1 interacts with Ubqln4 through its UBA domain. (A) 293 cells were co-transfected with GFP-S-Ubqln1, GFP-S-Ubqln1 ΔUBL (deletion of UBL domain) or GFP-S-Ubqln1 ΔUBA (deletion of UBA domain) and HA-Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti-GFP and anti-HA antibodies. (B) The extent of Ubqln4 co-immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln1, Ubqln1ΔUBL and Ubqln1ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P-value=0.0041. (C) 293 cells were co-transfected with GFP-S or GFP-S-Ubqln1 and HA-Ubqln4, HA-Ubqln4 ΔUBL or HA-Ubqln4 ΔUBA. GFP was immunoprecipitated and analysed by western blot using anti-GFP and anti-HA antibodies. (D) The extent of Ubqln4 co-immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4, Ubqln4ΔUBL and Ubqln4ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P-value=0.0003 and **P-value=0.0001. (E) 293 cells were co-transfected with GFP-S-Ubqln1 and HA-Ubqln4, HA-Ubqln4 ΔUBA or HA-Ubqln4 ΔUBA I55A (I55 in the UBL domain of Ubqln4 was mutated to A). GFP was immunoprecipitated and analysed by western blot using anti-GFP and anti-HA antibodies. (F) The extent of Ubqln4 co-immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4, Ubqln4ΔUBA and Ubqln4ΔUBA I55A. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average of two independent experiments. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; UBA, ubiquitin-associated; UBL, ubiquitin-like; Ubqln, Ubiquilin; WCL, whole-cell lysate; WT, wild-type.

Figure 5

Depletion of Ubqln4 inhibits autophagosome–lysosome fusion. (A) Confocal images of Hela cells transfected with mCherry-GFP-LC3 and also transfected with control, Ubqln1 or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 8 h. (B) Quantification of the number of mCherry/GFP double-positive and mCherry single-positive puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. The number of mCherry single-positive puncta of Ubqln1 and Ubqln4 siRNA was compared with that of control siRNA. *P-value=0.0475 and **P-value=0.0373. GFP, green fluorescent protein; LC3, microtubule-associated protein light chain 3; Ubqln, Ubiquilin.