The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis
- Howard Hughes Medical Institute, University of California, Berkeley, United States
Figures
Figure 1 with 1 supplement
In vitro reconstitution of endogenous LC3 lipidation.
(A) The distribution of LC3-I and LC3-II between the cytosol (C) and membrane (M) fractions from indicated cells. Cytosol and membranes from indicated cells were separated and evaluated by …
Figure 1—figure supplement 1
Characterization of the in vitro-lipidated LC3.
(A) LC3-II distributes in the 16,000×g membrane pellet fraction. Reactions similar to those of Figure 1B were performed. After the indicated times, the post-reaction mixtures were centrifuged at …
Figure 2 with 2 supplements
The in vitro lipidation of LC3 is regulated by ATG5, starvation and PI3K.
(A) Starvation-promoted and ATG5-dependent lipidation of LC3. Indicated cells were either untreated or starved for 30 min. The in vitro lipidation reaction with the indicated combination of cytosols …
Figure 2—figure supplement 1
Starvation-promoted lipidation of LC3 by COS-7 or HEK293T cytosol.
COS-7, HEK293T and Atg5 KO MEF cells were either untreated or starved for 60 min. The in vitro lipidation reaction with indicated combination of cytosols and membranes was performed followed by …
Figure 2—figure supplement 2
Purification and verification of GST-FYVEs.
(A) Purification of GST-fusion PI3P binding FYVE domains and mutants (C/S). (B) PIP Strip blot with 10 µg/ml GST-FYVE. (C) PIP Strip blot with 10 µg/ml GST-FYVE (C/S).
Figure 3 with 1 supplement
Recapitulation of the major regulatory pathways for autophagy by in vitro lipidation of T7-LC3.
(A) Starvation-induced lipidation of T7-LC3. HEK293T and Atg5 KO MEF cells were either untreated or starved for 90 min. The in vitro lipidation reaction was performed by incubating T7-LC3 with …
Figure 3—figure supplement 1
Purification of the T7-tagged LC3 and characterization of the lipidation.
(A) Purification of HisT7-LC3 and the G/A mutant. (B) Lipidation of HisT7-LC3. The in vitro lipidation reaction was performed by incubating HisT7-LC3 or G/A mutant with indicated cytosols and Atg5 …
Figure 4
Membrane fractionation scheme.
Briefly, Atg5 KO MEFs were homogenized and the lysates were subjected to differential centrifugations with indicated g forces. The ability of each fraction to trigger T7-LC3 lipidation was examined. …
Figure 5
Separation of the total membrane by differential centrifugations.
(A–D) A differential centrifugation experiment was performed as depicted in Figure 4. The total PC of each fraction was measured and presented as a percentage of the total membrane (C) and adjusted …
Figure 6
Separation of the 25k pellet fraction by sucrose gradient ultracentrifugation.
(A–D) A sucrose step gradient ultracentrifugation to further separate the 25k pellet fraction was performed as depicted in Figure 4. The total PCs of each fraction were measured and presented as a …
Figure 7 with 2 supplements
Separation of the L fraction by OptiPrep gradient ultracentrifugation.
(A–B) An OptiPrep gradient ultracentrifugation was used to resolve membranes in the L fraction, as depicted in Figure 4. 10 fractions were collected. The total PCs of each fraction were measured and …
Figure 7—figure supplement 1
The ERGIC membrane promotes LC3 lipidation without altering cytosolic factors.
(A) The major autophagy factors are cytosolic. Immunoblot of the fractions from Figure 7 and the cytosol (Cyt) used for the in vitro lipidation assay was performed with indicated antibodies. (B) The …
Figure 7—figure supplement 2
The lipidation activity of the ERGIC-enriched fractions are regulated by starvation and PI3K.
(A) Lipidation of T7-LC3 from the ERGIC-enriched fractions is enhanced by starvation. An in vitro lipidation reaction similar to that in Figure 7 was performed with HEK293T cytosols from starved …
Figure 8 with 1 supplement
ERGIC directly triggers in vitro LC3 lipidation.
(A) Immunodepletion of ERGIC membrane from L fraction reduces in vitro lipidation activity. The L fraction was prepared as shown in Figures 4 and 5. An immunodepletion experiment with indicated …
Figure 8—figure supplement 1
Mitochondrial-associated endoplasmic reticulum membranes (MAM) are not active to trigger in vitro LC3 lipidation.
(A) Indicated membrane fractions were prepared as described by Wieckowski et al. (Wieckowski et al., 2009) and in vitro lipidation was performed as shown in Figures 4–7. T, total membrane; S, …
Figure 9 with 1 supplement
ERGIC is required for in vitro LC3 lipidation.
(A and B) In vivo depletion of ERGIC abolishes the in vitro lipidation of LC3. Atg5 KO MEFs were treated without or with 10 µg/ml Brefeldin A (BFA) for 30 min and then incubated with the indicated …
Figure 9—figure supplement 1
Immunofluorescence showing the effect of indicated drugs on ERGIC and Golgi.
Atg5 KO MEFs were treated with indicated drugs or drug combinations as depicted in Figure 9A,B. Cells were fixed for immunofluorescence with the indicated antibodies. Bar, 10 µm.
Figure 10 with 2 supplements
ERGIC is required for starvation-induced LC3 puncta formation.
(A) Drugs that disrupt ERGIC inhibit LC3 puncta formation. MEFs were transfected with plasmids encoding Myc-LC3. After transfection (24 hr), the cells were either non-starved (NT) or starved (ST) in …
Figure 10—figure supplement 1
Drugs that disrupt ERGIC inhibit starvation-induced ATG16 puncta formation.
(A) MEFs were transfected with plasmids encoding ATG16-Myc. After transfection (24 hr), the cells were either non-starved (NT) or starved (ST) in the absence or presence of the indicated drugs …
Figure 10—figure supplement 2
Effects of SAR1A variants on ERGIC.
MEFs were transfected with plasmids encoding the indicated SAR1A-DsRed variants or control DsRed. After transfection (24 hr), immunofluorescence with indicated antibodies was performed. Bar, 10 µm.
Figure 11
ERGIC is required for the starvation-induced localization of ATG14 and DFCP1 to puncta.
(A) H89 inhibits ATG14 and DFCP1 puncta formation. MEF cells were transfected with plasmids encoding EGFP-tagged ATG14 or DFCP1. After transfection (24 hr), cells were starved in the absence or …
Figure 12 with 2 supplements
ERGIC is required for membrane recruitment of ATG14 and DFCP1.
(A) Disruption of ERGIC inhibits membrane recruitment of ATG14 and DFCP1. Atg5 KO MEFs were either untreated or treated with H89. Membranes were collected and incubated with cytosol of HEK293T cells …
Figure 12—figure supplement 1
Establishment of the in vitro membrane recruitment assay.
(A) Membrane dependence for the flotation of ATG14 and DFCP1. Cytosol from HEK293T cells expressing ATG14-HA and EGFP-DFCP1 was incubated with or without membrane. A buoyant density gradient …
Figure 12—figure supplement 2
Atg14L and DFCP1 puncta colocalize with ERGIC.
(A and B) MEF cells were transfected with plasmids encoding ATG14-EGFP (A) or EGFP-DFCP1 (B). 24 hr after transfection, the cells were starved for 20 min. Cells were then fixed and visualized by …
