Figure 6. RTN3 LIR motifs are required for ER tubules fragmentation.

(A) A549 cell lysates were added to beads with immobilized GST fusion LC3-like modifiers: GST, GST-LC3A, GST-LC3B, GST-GABARAP-L1, GST-GABARAP-L2), followed by WB using an antibody against endogenous RTN3. (B) Domain architecture of RTN3L and alignment of the LIR motifs. Blue: reticulon homology domain (RHD), red: LC3-interacting region (LIR). (C) RTN3L lacking all six LIR domains (∆6) fails to bind to GST fusion LC3-like modifiers when over-expressed in HEK-293T cells. (D) Immunofluorescence of HA and LC3B in U2OS TRex FLAG-HA-RTN3L and FLAG-HA-RTN3L∆6LIRs after 24 hr treatment with 1 µg/ml of doxycycline and starved for 6 hr with EBSS plus Bafilomycin A1, 200 ng/ml. RTN3L was monitored using an anti HA antibody, while autophagy induction was visualized using anti-LC3B antibody. Scale bars: 10 µm. (E) Quantification of cells presenting at least one ER tubule fragment after 6 hr starvation with EBSS plus Bafilomycin A1, 200 ng/ml. Number of cells >500 for each condition. Data are representative of three independent biological experiments. *p<0.01. Error bars represent s.d. (F) Immunofluorescence of HA and LAMP1 in U2OS TRex FLAG-HA-RTN3L or FLAG-HA-RTN3L∆6LIRs cells induced 24 hr with 1 µg/ml of doxycycline and subsequently starved for 6 hr with EBSS plus Bafilomycin A1, 200 ng/ml. RTN3L was monitored using an anti-HA antibody, while lysosomes are visualized using anti-LAMP1 antibody. Scale bars: 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.25555.025

Figure 6—figure supplement 1. RTN3L directly binds to the LC3s/GABARAPs modifiers.

(A,B) HEK293T cells were transfected with the HA tagged RTN3L and RTN2L (A) and RTN3S (B). Cell lysates were added to beads with immobilized GST-fusion LC3-like modifiers (GST, GST-LC3A, GST-LC3B, GST-LC3C, GST-GABARAP, GST-GABARAP-L1, GST-GABARAP-L2, GST-Ub, GST-4XUb), followed by WB using an antibody against HA tag. (C) A549 cell lysates were added to beads with immobilized GST-fusion LC3-like modifiers or the LC3 like modifiers lacking the unique N-Terminus, followed by WB using an antibody against endogenous RTN3. (D) A549 cell lysates were added to beads with GST-LC3B or GST-LC3BF52A-V53A mutant and followed by WB using an antibody against endogenous RTN3. (E) HEK293T were transfected with the FLAG tagged RTN3L with mutation in one, five or six LIR domains mutated. Cell lysates were added to beads with immobilized GST-fusion LC3-like modifiers, followed by WB using an antibody against FLAG tag.

DOI: http://dx.doi.org/10.7554/eLife.25555.026

Figure 6—figure supplement 2. LIR motifs are required to deliver RTN3L to lysosomes.

(A,B) Immunofluorescence of HA, GABARAP-L1 (A) and LC3B (B) and LAMP1 in U2OS TRex FLAG-HA-RTN3L and FLAG-HA-RTN3L∆6LIRs cells after 24-hr treatment with 1 µg/ml of doxycycline. Cells were kept in standard growing condition (DMEM with 10% FBS) or starved with EBSS for 6 hr. RTN3L was monitored using anti HA antibody, while autophagy induction is monitored using GABARAP-L1 (A) and (B) lysosomes were visualized using anti-LAMP1 antibody. Bafilomycin A1 was added at a final concentration of 200 ng/ml. Scale bars: 10 µm. (C) HeLa TRex mCherry-EGFP-RTN3 or mCherry-EGFP-RTN3∆6LIRs were treated with 1 µg/ml doxycycline for 24 hr and starved with EBSS for 6 hr in the absence of Bafilomycin A1. Scale bars: 10 µm. (D) Endogenous labeling of CALNEXIN and REEP5 in U2OS TRex cells and in U2OS TRex cells after RTN3L∆6LIRs over-expression. Scale bars: 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.25555.027

Figure 6—figure supplement 3. RTN3L over-expression does not affect autophagy flux.

(A) Co-immuno-precipitation of endogenous GABARAP with RTN3L and RTN3L∆6LIRs in U2OS TRex cells. (B,C) Western blot analysis of LC3B lipidation and p62 degradation in U2OS TRex FLAG-HA-RTN3L and FLAG-HA-RTN3L∆6LIRs cells after Torin1, 250 nM (B) or EBSS (C) treatment for the indicated time.

DOI: http://dx.doi.org/10.7554/eLife.25555.028