Figure S1. Isolation membranes are associated with the ER in absence of VMP1,related to Figure 1.(A) GFP was knocked into the C-terminus of VMP1 in HeLa cells by CRISPR/Cas9to create VMP1-GFP. The ER is indicated with DsRed-KDEL. Scale bars: 5 μm;insert, 2 μm.(B and C) In fluorescence protease protection (FPP) assays, after digitonin treatment,HeLa cells transfected with GFP-VMP1 (B) or VMP1-GFP (C) were treated with proteinase K (pK). Time-lapse images show that both the N-terminal andC-terminal GFP are digested. When overexpressed, VMP1-GFP forms somepunctate structures that are resistant to pK digestion. Scale bars: 5 μm.(D) Schematic illustration of the VMP1 topology based on the FPP assay. VMP1 ispredicted to contain six transmembrane domains.(E) Levels of
p62 and LC3-II are dramatically elevated in Vmp1 KO MEF cells in animmunoblotting assay. VMP1 is absent in KO cells. Quantifications of theLC3-II/I ratio and LC3-II levels (normalized by Actin levels) are also shown. Theconversion from LC3-I to LC3-II can be used to determine at which stepautophagosome formation is impaired in autophagy-deficient cells. Levels ofother autophagy proteins, including WIPI2, FIP200, ATG9, VPS34 and Beclin1,remain unchanged in Vmp1 KO MEF cells. *, unspecific band.(F) Mutation in VMP1 generated by CRISPR/Cas9 in COS7 cells.(G) Immunoblotting results showing that levels of
p62 and LC3-II are increased in VMP1 KO COS7 cells. VMP1 is effectively depleted in VMP1 KO cells.(H) Immunoblotting results showing that levels of
p62 and LC3-II are increased inshVMP1 HeLa cells compared with WT cells. VMP1 is effectively depleted inshVMP1 cells.(I) The hierarchical recruitment of autophagy proteins to the autophagosome formation site upon autophagy induction. It is important to note that the order in which ATG proteins are recruited does not reflect the order in which they function in autophagosome formation. The same ATG protein may function at multiple steps and may also affect the function of ATG proteins upstream in the order of recruitment.(J) Size of LC3-positive puncta from WT (n=156 puncta from 20 cells) and Vmp1 KO(n=148 puncta from 20 cells) MEF cells are quantified and presented as mean ± SD.(K and L) Compared to WT MEF cells (K), many LC3 puncta are formed andassociate with GFP-Sec61beta in Vmp1 KO MEF cells under basal conditions (L).Scale bars: 5 μm; inserts, 2 μm.(M and N) Compared to WT MEF cells (M), many LC3 puncta are formed andcolocalize with GFP-DFCP1 in Vmp1 KO MEF cells under basal conditions (N).Scale bars: 5 μm; inserts, 2 μm.(O-Q) WT (O) or VMP1 KO (P) COS7 cells cotransfected with GFP-FIP200 andRFP-LC3 were starved and subjected to live-cell imaging. In WT cells, an LC3punctum (arrows) emerges from a GFP-FIP200-positive site (arrowheads) and then the two puncta separate (O), while in VMP1 KO cells, an LC3 punctum stably associates with GFP-FIP200-positive structures (arrows) (P).Quantification data (WT, n=15 puncta from 3 cells; KO, n=16 puncta from 3 cells)are presented as mean ± SD (Q). Scale bars: 1 μm.(R-T) LC3 puncta are partially associated with the ER, labeled by CANX-GFP, in WTMEF cells 1 h after starvation (S), but completely localize on the ER in Vmp1 KOMEF cells (T). Quantification results are shown as mean ± SD (n=216 punctafrom 14 cells for S, n=94 puncta from 12 cells for T) in (R). Scale bars: 5 um;inserts, 2 um.(U and V) After 4 h starvation, LC3 puncta largely colocalize with LAMP1-labeledlysosomes in control (ctrl) MEF cells (U), but are separable from LAMP1structures in Vmp1 KO MEF cells (V). Scale bars: 5 μm; inserts, 2 um.(W-Y) LC3 puncta partially associate with the ERES, labeled by Sar1a-GFP, in WTMEF cells 1 h after starvation (W), but mostly associate with Sar1a-GFP in Vmp1KO MEF cells (X). Quantification results are shown as mean ± SD (n=188 punctafrom 10 cells for W, n=144 puncta from 10 cells for X) in (Y). Scale bars: 5 μm;inserts, 2 μm.(Z) Schematic steps for isolation of microsomes from WT and Vmp1 KO MEFs. PNS,postnuclear supernatant; S8K, supernatant after 8,000 g centrifugation.(A1 and B1) Immuno-EM of WT (A1) and Vmp1 KO (B1) MEFs with anti-LC3antibody (black arrowheads). The right panels show enlargements of the indicatedareas in the left panels. Black arrows indicate the ER-IM contact sites. Red 4arrows indicate the outer membrane of the IM. Red arrowheads indicate the innermembrane of the IM. Scale bars: 200 nm, left panels; 40 nm, right panels.