Figure 6. Cis-interacting ATG9A and LC3B... | Rockefeller University Press

Figure 6.

$Cis-interacting ATG9A and LC3B are co-resident on autophagosomes and putative autophagosome precursor membranes accumulating in ATG2 DKO cells. (A) Schematic of the three types of IPs performed to look for ATG9A associating with GFP-LC3B in membrane fractions collected as in Fig. 3. (B) Immunoblot of GFP IPs performed on membranes in the AP fraction collected from WT cells or the bulk membrane fraction collected from ATG2 DKO cells. WO (untreated samples pulling down Whole Organelles); SMA (samples incubated with 12:1 SMA:protein (∼2.5%); TX (samples treated with 1% Triton X-100). Input protein for each IP: 50 µg. Loaded input protein: 3 µg (6%). Loaded IPs: 10% of the total collected beads. (C) Densitometric quantification of ATG9A, GFP-LC3B, LC3B-II, Calnexin, and TOM20 in each type of IP displayed as average ± SD. The intensity of the bands in B were normalized to the input, and statistical significance was assessed by two-way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. Both cell lines, n = 3 biological replicates, except for WT Triton X-100 IPs where n = 2 biological replicates. Quantifications between cell lines are shown in Fig. S3. (D) Immunoblot of reverse IP confirming ATG9A can pull down LC3B-II. The AP fraction from WT cells expressing FLAG-ATG9A was solubilized with either 12:1 SMA:protein (∼2.5%) or 1% Triton X-100. Input protein for IPs: 100 µg. Loaded input protein: 10 µg (10%). Loaded IPs: 25% of the total collected beads. (E) Immunoblot of reverse IP demonstrating that ATG9A cannot pull down LC3B-II in autophagy deficient cells. Bulk membrane fraction from FIP200 KO cells was solubilized with either 12:1 SMA:protein (∼2.5%) or 1% Triton X-100. Input protein for IPs: 100 µg. Loaded input protein: 10 µg (10%). Loaded IPs: 25% of the total collected beads. (F) Left: schematic outlining how protein denaturation can be used to distinguish proteins interacting in trans (in separate SMALPs) vs in cis (within the same SMALP). ATG9A and GFP-LC3B are in the same SMALP only if they continue to co-purify after denaturation with guanidine hydrochloride (GdnHCl). Middle: immunoblot of denaturation experiment conducted with the AP fraction from WT cells expressing GFP-LC3B. Shown are sequential IPs, first with GFP-LC3B, then the sample was treated with 6 M GdnHCl before diluting over 25X and performing a second IP against ATG9A. Input protein for GFP IPs: 50 µg. Input for ATG9A IPs: 98% of the material collected from the GFP IPs. Loaded input protein: 5 µg (10%). Loaded GFP IPs: 1% of the total collected from the beads. Loaded ATG9A IPs: 50% of the total collected from the beads after GdnHCl treatment. Right: densitometric quantification of GFP-LC3B recovered in the final IP after GdnHCl treatment displayed as average ± SD. Two biological replicates are shown with 300 μl of anti-GFP beads (black) and one biological replicate with only 100 μl of anti-GFP beads (blue).$

Cis-interacting ATG9A and LC3B are co-resident on autophagosomes and putative autophagosome precursor membranes accumulating in ATG2 DKO cells. (A) Schematic of the three types of IPs performed to look for ATG9A associating with GFP-LC3B in membrane fractions collected as in Fig. 3. (B) Immunoblot of GFP IPs performed on membranes in the AP fraction collected from WT cells or the bulk membrane fraction collected from ATG2 DKO cells. WO (untreated samples pulling down Whole Organelles); SMA (samples incubated with 12:1 SMA:protein (∼2.5%); TX (samples treated with 1% Triton X-100). Input protein for each IP: 50 µg. Loaded input protein: 3 µg (6%). Loaded IPs: 10% of the total collected beads. (C) Densitometric quantification of ATG9A, GFP-LC3B, LC3B-II, Calnexin, and TOM20 in each type of IP displayed as average ± SD. The intensity of the bands in B were normalized to the input, and statistical significance was assessed by two-way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. Both cell lines, n = 3 biological replicates, except for WT Triton X-100 IPs where n = 2 biological replicates. Quantifications between cell lines are shown in Fig. S3. (D) Immunoblot of reverse IP confirming ATG9A can pull down LC3B-II. The AP fraction from WT cells expressing FLAG-ATG9A was solubilized with either 12:1 SMA:protein (∼2.5%) or 1% Triton X-100. Input protein for IPs: 100 µg. Loaded input protein: 10 µg (10%). Loaded IPs: 25% of the total collected beads. (E) Immunoblot of reverse IP demonstrating that ATG9A cannot pull down LC3B-II in autophagy deficient cells. Bulk membrane fraction from FIP200 KO cells was solubilized with either 12:1 SMA:protein (∼2.5%) or 1% Triton X-100. Input protein for IPs: 100 µg. Loaded input protein: 10 µg (10%). Loaded IPs: 25% of the total collected beads. (F) Left: schematic outlining how protein denaturation can be used to distinguish proteins interacting in trans (in separate SMALPs) vs in cis (within the same SMALP). ATG9A and GFP-LC3B are in the same SMALP only if they continue to co-purify after denaturation with guanidine hydrochloride (GdnHCl). Middle: immunoblot of denaturation experiment conducted with the AP fraction from WT cells expressing GFP-LC3B. Shown are sequential IPs, first with GFP-LC3B, then the sample was treated with 6 M GdnHCl before diluting over 25X and performing a second IP against ATG9A. Input protein for GFP IPs: 50 µg. Input for ATG9A IPs: 98% of the material collected from the GFP IPs. Loaded input protein: 5 µg (10%). Loaded GFP IPs: 1% of the total collected from the beads. Loaded ATG9A IPs: 50% of the total collected from the beads after GdnHCl treatment. Right: densitometric quantification of GFP-LC3B recovered in the final IP after GdnHCl treatment displayed as average ± SD. Two biological replicates are shown with 300 μl of anti-GFP beads (black) and one biological replicate with only 100 μl of anti-GFP beads (blue).