ATG2A is proximal to ARFGAP1 positive membranes.

(A) Proximity labeling of HEK293 ATG2 DKO cells stably expressing APEX2-GFP-ATG2A was performed as previously described26. Three biological replicates were averaged and plotted as the log2 ratio between the full labeling reaction in complete media over cells that were not treated with hydrogen peroxide on the x axis, and -log10 of the p-value on the y axis. The red line demonstrates the p-value cutoff of 0.05. The proteomics revealed an enrichment of autophagy (labeled in green) and early secretory proteins (COPII/ER Exit Site proteins are labeled in orange, ARFGAP1 is labeled in red). (B) Strategy to identify phagophores by live cell imaging. Phagophores are defined as sites where both GFP-ATG2A and TagBFP-LC3B co-localize. (C-D) Live cell imaging of ATG2 DKO cells stably expressing GFP-ATG2A and TagBFP-LC3B and transfected with mRuby-SEC23A or RFP-SEC23IP/p125a or ARFGAP1-TagRFP. The cells were starved for 2-4 hours prior to imaging. The fraction of phagophores (white arrows) that colocalized with the third fluorescent protein (transfected gene) was quantified and plotted in (D). Statistical significance was assessed by one way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. ***, adjusted P value <0.001. ****, adjusted P value <0.0001. Data from three biological replicates were pooled for each condition. Maximum intensity projections of confocal images. (E) Global organelle profiling data from https://organelles.sf.czbiohub.org31. Whole organelle profiles were established by pulling down endogenously tagged proteins and performing quantitative proteomics. The enrichment value for each protein were calculated by taking the difference in median log2 LFQ values between the replicates and the null distribution.

Autophagic flux depends upon RAB1 but not ARFGAP1.

(A-B) In gel fluorescence image (top panel) and immunoblots demonstrating that siRNA KD of RAB1 inhibits autophagic flux. In gel fluorescence reveals HaloTag7s moiety bound to the TMR ligand. Indicated bands are full length (FL) HT-mGFP-LC3B protein and HaloTag7 after cleavage (HT) in the lysosome. The ratio between FL and HT demonstrates autophagic flux, quantified in (B). Statistical significance was assessed by one way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. ***, adjusted P value <0.001. ****, adjusted P value <0.0001. Data from three biological replicates were collected for each condition. (C-D) Immunoblot of lysates from three biological replicates demonstrating that siRNA KD of RAB1 results in an accumulation of LC3B-II. The band intensity of LC3B-II was normalized against the intensity of GAPDH for each lane. To normalize each replicate, each individual value was divided by value of the non-transfected sample. Statistical significance was assessed by one way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. ***, adjusted P value <0.001. ****, adjusted P value <0.0001. (E-F) Immunoblot showing CoIP between GFP-RAB1A and 3xFLAG-ATG2A compared to the same reaction with other GFP-tagged early secretory proteins. The cells were incubated with either complete media or EBSS for four hours prior to harvesting. For the CoIP, ∼3 mg of cell lysate was used per condition (slight deviations in total amount between replicates, but not between lanes). To quantify the results, the intensity of the 3xFLAG-ATG2A IP signal was ratioed against the input signal. To normalize each replicate, each individual value was divided by the average of the replicate. Statistical significance was assessed by two way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. ***, adjusted P value <0.001. ****, adjusted P value <0.0001. The media was not a significant source of variation (p = 0.3535).

ATG2A, LC3B, ARFGAP1, and RAB1A localize to tubules following disruption of the early secretory pathway.

(A-B) In gel fluorescence image (top panel) and immunoblots demonstrating that SAR1BH79G-mOrange2 (mO) overexpression negatively affects autophagic flux. The ratio between the FL and the free HT represents the autophagic flux in each condition (as in Fig. 2 A/B) and is plotted in (B). Statistical significance was assessed by two way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. ***, adjusted P value <0.001. ****, adjusted P value <0.0001. Data from four biological replicates were collected for each of the HT-mGFP conditions and five replicates were collected for each of the HT-mGFP-LC3B conditions. (C) Live cell imaging of ATG2 DKO cells stably expressing GFP-ATG2 that were transfected with SAR1BH79G-mOrange2 and starved for two hours. Note that GFP-ATG2A forms long tubules that do not colocalize with the SAR1BH79G-mOrange2 signal. Maximum intensity projection of a confocal image. Gray line was added to show cell outline. (D-E) Live cell imaging demonstrates the localization of ARFGAP1-TagRFP (D) and SNAP-RAB1A (E) on GFP-ATG2A/TagBFP-LC3B positive tubules following SAR1BH79G overexpression. Arrows denote thin GFP-ATG2A positive tubules whereas arrow heads denote thicker tubules that lack most GFP-ATG2A but contain high levels of LC3B, ARFGAP1, and RAB1A. Maximum intensity projections of confocal images. (F) CLEM-FIBSEM of cells prepared as in (C). GFP-ATG2A fluorescence corresponds to a thin tubular membrane that is connected to a thicker tubule. The small inset in the left image shows a single SEM slice. The turquoise arrow is pointing to the tubule which is segmented and displayed in turquoise in the larger image, whereas the yellow arrows are pointing at adjacent ER tubules which are segmented and displayed in the larger image in yellow. At many points, these ER structures come into very close apposition with the tubule which is denoted in red. The ER structures are removed in the right image for better visualization of the ATG2A positive tubule. Note that only the thin section has corresponding GFP-ATG2A fluorescence.

ATG2 deletion results in the accumulation of ARFGAP1 and RAB1A on autophagic membranes.

(A) Schematic of the membranes that accumulate at the ATG2 DKO compartment based off the TEM images in Olivas et al9. The core of the compartment contains small vesicles that stain for ATG9A, LC3B, and P62. The periphery of the compartment is comprised of more complicated cup shaped membranes that stain for WIPI2. The entire compartment is enwrapped by ER tubules. (B) Immunofluorescence microscopy demonstrates that ARFGAP1 colocalizes with P62 in ATG2 DKO cells. Inset 1 highlights a cell undergoing mitosis, in which the colocalization of these two markers persists as denoted by the arrows. Inset 2 shows several cells, a ring-like accumulation of ARFGAP1 around P62 is clearly visible, separate from the Golgi. Maximum intensity projection of a confocal image. (C) Immunofluorescence images demonstrating the distribution of early secretory membranes around the ATG2 DKO compartment which is marked by either P62 (mouse antibody) or LC3B (rabbit antibody). The images presented are single confocal slices of the center of the ATG2 DKO compartment. The gray lines show the cell periphery and the insets focus on the largest accumulation of P62 or LC3B in the cell. (D) Quantification of the images in (C) and in Sup Fig 4A. The enrichment factor is equal to the mean of the given protein at the periphery of the ATG2 DKO compartment compared to its mean throughout the cell. Statistical significance was assessed by one way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. ***, adjusted P value <0.001. ****, adjusted P value <0.0001. Data from three biological replicates were pooled together for each of the conditions. (E-F) Immunofluorescence images demonstrating the enrichment of overexpressed GFP-RAB1A (E) or SNAP-RAB1A (F) at the periphery of the ATG2 DKO compartment as determined by the presence of P62 (E) or WIPI2 (F). Presented as a single confocal slice. The gray lines show the periphery of the cell and the insets focus on ATG2 DKO compartments that are distal from the Golgi.

ATG2A and ARFGAP1 colocalize during starvation.

(A-C) Immunoblot showing the restoration of LC3B flux (B) and P62 clearance (C) in ATG2 DKO cells stably expressing APEX2-GFP-ATG2A. The restoration of a wild type phenotype indicates that the fusion protein is both localizing and functioning properly. The band intensity of LC3B (B) and P62 (C) were normalized against the intensity of GAPDH for each lane. To normalize each replicate, each individual value was divided by the average of the replicate. Statistical significance was assessed by two way ANOVA. Data from five biological replicates were collected for each condition, save for the WT + Baf and WT + Baf + EBSS, which each had four replicates. (D-F) Single slice confocal immunofluorescence images (D) and immunoblots (E-F) demonstrating that the addition of biotin phenol and hydrogen peroxide to ATG2 DKO cells stably expressing APEX2-GFP-ATG2A results in the biotinylation of proteins proximal to ATG2A. The omittance of either biotin phenol or hydrogen peroxide resulted in the loss of the anti-biotin signal, save for endogenously biotinylated proteins. The immunoblot in (F) demonstrates that the biotinylated proteins can be enriched on streptavidin beads. (G) Proximity labeling of HEK293 ATG2 DKO cells stably expressing APEX2-GFP-ATG2A was performed as described in Fig. 1A. (H-J) FLASH-PAINT analysis confirming the proximity of ATG2A to ARFGAP1. ATG2 DKO cells stably expressing GFP-ATG2A were labeled with all of the antibodies listed in (J), which in turn were preincubated with secondary antibodies with unique DNA sequences as depicted in (H). Each target (T1, T2… Tn) was visualized sequentially by adding an adaptor DNA strand that matched the target sequence (A1, A2… An) and an additional sequence to recruit the imager DNA strand (H). To remove the imager strand from each sequential target, a unique eraser strand was added that recognized the entirety of the adaptor sequence (e.g. A1) and part of the imager sequence, resulting in the total sequestration of the adaptor DNA strand. The subsequent adaptor sequence was then added to visualize the next target protein. Seven of the ten visualized proteins are depicted in (I), which highlights the proximity of ATG2A and ARFGAP1 at autophagic and early secretory membranes. The heatmap presented in (J) shows the median distance within 200 nm between all visualized proteins. (I); pink arrow heads = ERES. white arrow heads = exogenous beads used as a fiduciary marks in imaging. (I; insets), brackets indicate immediate proximity of ATG2 (green) and ARFGAP (maroon) as frequently observed in ERES.

ATG2A CoIPs weakly with ER Exit Site proteins.

(A-D) Immunoblot showing the weak CoIP between 3xFLAG-ATG2A and various ER Exit Site proteins. For the CoIP, ∼3 mg of cell lysate was used per condition (slight deviations in total amount between replicates, but not between lanes). Immunoblots quantified as in Fig. 2B. Statistical significance was assessed by one way ANOVA. *, adjusted P value <0.05. **, adjusted P value <0.01. ***, adjusted P value <0.001. ****, adjusted P value <0.0001.

SAR1BH79G overexpression hinders autophagy and leads to the formation of ATG2A positive membrane tubules.

(A-B) Live cell imaging shows that SAR1BH79G overexpression negatively correlates with LC3B puncta number. ATG2 DKO cells stably expressing GFP-ATG2A and BFP-LC3B were transfected with SAR1BH79G-mOrange2 and starved for 2 hours. The number of transfected cells with GFP-ATG2A tubules (white arrow in A) was counted against the total number of transfected cells, resulting in a formation rate of 1.5±0.8%. ROIs were drawn over each transfected cell, and the resulting SAR1BH79G intensity was plotted against the number LC3B puncta in (B). A linear trendline was added in black with a 95% confidence interval. (C) CLEM-FIBSEM of cells prepared as in (Fig. 3C). GFP-ATG2A fluorescence corresponds to a thin tubular membrane. A single slice SEM slice is depicted in (i). Note that the segmented tubule comes in and out of the plane of this slice, so the tubule that is highlighted in turquoise is not complete. The yellow arrows are pointing at adjacent ER structures which are segmented and displayed in (ii) in yellow. At many points, these ER structures come into very close apposition with the tubule which is denoted in red. The ER structures are removed in (iii) for better visualization of the tubule. The GFP-ATG2A fluorescence signal is depicted in (iv).

Most early secretory markers are not enriched at the periphery of the ATG2 DKO compartment.

(A-B) Immunofluorescence images demonstrating the distribution of endogenous (A) or overexpressed (B) early secretory membranes around the ATG2 DKO compartment which is marked by p62. The images presented are single confocal slices of the center of the ATG2 DKO compartment. The gray lines show the cell periphery, and the insets focus on the largest accumulation of p62 in the cell.

Antibody Targets and Sequences for FLASH PAINT

Imager Sequence

Adaptor Sequences

Eraser sequences

Order of sequential antibody labeling for FLASH-PAINT

Mass Spectrometry Data

Key Resource Table