Supplemental Information. Pacer Mediates the Function of Class III PI3K. and HOPS Complexes in Autophagosome. Maturation by Engaging Stx17

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1 Molecular Cell, Volume 65 Supplemental Information Pacer Mediates the Function of Class III PI3K and HOPS Complexes in Autophagosome Maturation by Engaging Stx17 Xiawei Cheng, Xiuling Ma, Xianming Ding, Lin Li, Xiao Jiang, Zhirong Shen, She Chen, Wei Liu, Weihua Gong, and Qiming Sun

2 Supplemental figures and legends Figure S1. Related to Figure 1.

3 Figure S1. Sequence alignment of Pacer from different species. Amino acids in high similarity were highlighted in blue and grey.

4 Figure S2. Related to Figure 1 and 2

5 Figure S2. Pacer subcellular localization analysis. (A) U2OS cells transfected with Pacer-GFP were fixed and stained with anti-eea1 (label early endosome) antibody. For colocalization analysis with DFCP1, U2OS cells were cotransfected with Pacer-GFP and mcherry-dfcp1. Scale bars, 10 µm. (B) Recombinant protein GST-Pacer (1-200aa) purified from E. Coli was used as the immunogen for making polyclonal antibody in Rabbit. Anti-Serum was affinity-purified using GST-Pacer (1-200aa)-conjugated agarose beads. (C) Detection of endogenous Pacer. 293T cells with Pacer knockdown (KD) or knockout (KO) were analyzed for Pacer levels using Western blot. (D) Detection of exogenous Pacer, Pacer-GFP (wt), Pacer-GFP (1-600aa) and Pacer-GFP (1-560) were expressed in 293T cells and analyzed by Western blot. (E) Detection of p62 level in Pacer WT, OE and KO 293T cells. 293T cells were treated with BafilomycinA (BafA1), and the whole cell lysates were prepared and analyzed for p62 level by Western blot. (F) Autophagosome maturation assay in Hela cells. mcherry-gfp-lc3 was expressed in Hela (vector) or Hela (Pacer OE) cells, and analyzed by confocal microscopy analysis. (G) The colocalization of LAMP1 and LC3 was analyzed in Pacer WT or KD U2OS cells by staining endogenous LAMP1 and LC3, and subsequently quantified. Data are shown as mean ± SD, **p < 0.01.

6 Figure S3. Related to Figure 3.

7 Figure S3. Pacer is a component of PI3KC3 complex through direct interaction with UVRAG. (A) Pacer interaction with UVRAG-PI3KC3 subcomponents. Flag-tagged Vps34, UVRAG, Beclin1, Beclin2, hnrbf2, Atg14 or Rubicon were co-expressed with HA-Pacer individually, and analyzed by anti-flag IP, followed by anti-ha Western blot. (B, C) Dissect the Pacer-interacting domain of Beclin1. HA-Pacer was coexpressed with Flag-tagged full-length Beclin1 or mutants. IP was performed using anti-flag antibody-conjugated beads. IP samples were analyzed by Western blot. (D, E) Dissect the Pacer-interaction domain of UVRAG. HA-Pacer was coexpressed with Flag-tagged full-length UVRAG or mutants in 293T cells. IP was performed using anti-flag antibody-conjugated beads. IP samples were analyzed by Western blot. (F) Structural prediction of aa of Pacer. (G) Analysis of recombinant proteins for GST-UVRAG-His6, Flag-Rubicon, Flag-Pacer or Flag-PacerΔ40 purified from insect cells by SDS-PAGE and Coomassie Blue staining. (H) In vitro GST pulldown assay. GST or GST-UVRAG-His6 was allowed to bind to beads first, incubated with Flag-Rubicon, Flag-Pacer or Flag-PacerΔ40, washed for three times, analyzed by anti-flag Western blot. (I, J) In vitro competition assay. GST-UVRAG-His6 was allowed to bind to beads first, which was followed by three washes and incubation with Flag-Rubicon, after another three washes, varying concentration of Flag-Pacer or Flag-PacerΔ40 were added into the reaction. After final three washes, proteins bound to the beads were analyzed by anti-flag Western blot. (K) Pacer destabilizes Rubicon. Rubicon levels were analyzed in 293T cells overexpressing Pacer-WT or Pacer-5A in the presence of Mg132 or Bafilomycin A1.

8 Figure S4. Related to Figure 4.

9 Figure S4. Pacer regulates PI3P biogenesis and targets UVRAG to autophagosomes. (A) Pacer knockdown decreases the activity of PI3KC3 in vivo. GFP-FYVE2 was expressed at a low level in Pacer KD, OE and WT U2OS cells. Control cells were treated with 5mM 3-MA. GFP-FYVE2 puncta number indicating relative in vivo level of PtdIns(3)P were quantified. Scale bars, 10 µm. Data are shown as mean ± SD from 90 micrographs of three independent experiments, **p < (B) Pacer and LC3 colocalization analysis in UVRAG knockdown cells. Pacer-GFP was transfected into UVRAG knockdown or control U2OS cells, and then analyzed for colocalization with LC3 by confocal microscopy. Scale bars, 10 µm. Data are shown as mean ± SD from 40 micrographs of three independent experiments. (C) Measure the effect of PATS on UVRAG localization. U2OS cells were transfected with plasmids expressing either WT UVRAG or a chimeric protein with PATS fusion to UVRAG, stained with anti-lc3 antibody, analyzed by confocal microscopy; colocalization with LC3 was quantified in (D). Scale bars, 10 µm. Data are shown as mean ± SD from 60 micrographs of three independent experiments, **p < (E) Pacer targets UVRAG to autophagosomes. GFP, Pacer-GFP or PATS-GFP was coexpressed with HA-UVRAG in U2OS cells, stained with anti-lc3 and HA antibodies. Colocalization with LC3 was quantified in (F), data are shown as mean ± SD from 60 micrographs of three independent experiments, ***p < 0.001; the effect on LC3 puncta formation by Pacer and UVRAG coexpression was quantified in (G), Scale bars, 10 µm. Data are shown as mean ± SD from 60 micrographs of three independent experiments, *p < 0.05.

10 Figure S5. Related to Figure 5.

11 Figure S5. Pacer recruits PI3KC3 complex to autophagosomes. (A) PacerΔ40 and Pacer-5A failed to recruit Beclin1, UVRAG and Vps34 to autophagosomes. U2OS cells coexpressed with PacerΔ40-GFP or Pacer-5A-GFP and Beclin1-HA, UVRAG-HA or Vps34-HA were fixed and stained with anti-ha and LC3 antibodies, analyzed by confocal microscopy, scale bars, 10 µm. (B) Analysis of colocalization of GFP-FYVE2 and LC cell lines were transfected with GFP-FYVE2 at a low level. Cells were then fixed, stained for LC3, analyzed by confocal microscopy, and quantified for the colocalization of LC3 and GFP-FYVE2. Scale bars, 10 µm. Data are shown as mean ± SD from 60 micrographs of three independent experiments, **p < (C) PI3P biogenesis rescue assay. GFP-FYVE2 was expressed at a low level in Pacer WT, Pacer KO, and the rescued U2OS cells. GFP-FYVE2 puncta number indicating relative in vivo level of PtdIns(3)P was quantified. (D) Detection of LC3-II level in Pacer WT and Pacer KO cells rescued by Pacer WT or PacerΔ( ). (E) PI3P biogenesis assay. GFP-FYVE2 was expressed at a low level in U2OS cells stably overexpressing Pacer WT, Pacer-5A or Pacer ( ). GFP-FYVE2 puncta number indicating relative in vivo level of PtdIns(3)P was quantified. Scale bars, 10 µm. Data are shown as mean ± SD from 60 micrographs of three independent experiments, **p < 0.01, *p < (F). (G) mcherry-gfp-lc3 was expressed in U2OS cells stably overexpressing Pacer-5A or Pacer WT, respectively. LC3 was monitored by fluorescence microscope. GFP-negative mcherry-positive (GFP-mCherry+) puncta, which indicates autolysosome, were quantified and summarized and quantified. Scale bars, 10 µm. Data are shown as mean ± SD from 35 micrographs of three independent experiments, *p < 0.05, **p < 0.01.

12 Figure S6. Related to Figure 5.

13 Figure S6. Pacer relocates HOPS complex to autophagosomes. (A) Analysis of Pacer interaction with HOPS complex. Flag-tagged Pacer WT or PacerΔ was coexpressed with Vps16-HA or Vps18-HA in 293T cells, and analyzed by anti-flag IP, followed by anti-ha Western blot. (B) Analysis of Pacer-5A interaction with HOPS complex. Flag-tagged Pacer-WT or Pacer-5A was coexpressed with Vps41-HA or Vps39-HA in 293T cells, and analyzed by anti-flag IP, followed by anti-ha Western blot. (C) Analysis of colocalization of HOPS with LC3 in Pacer WT or Pacer KD U2OS cells. U2OS cells were transfected with plasmid expressing each individual subunit of HOPS complex, cells were fixed and stained by anti-ha and anti-lc3 antibody, colocalization was quantified. Scale bars, 10 µm. (D) Analysis of Pacer-5A targeting HOPS to autophagosomes. Pacer WT-GFP or Pacer-5A-GFP was cotransfected with Vps39-HA or Vps41-HA, cells were fixed and stained by anti-lc3 and anti-ha antibodies, colocalization was analyzed and quantified in (E). Scale bars, 10 µm. (F) Analysis of Rab7-interaction with Pacer, Rubicon or PLEKHM1. GFP-tagged Rab7 was coexpressed with Pacer-Flag, Rubicon-Flag or PLEKHM1-Flag in 293T cells, and analyzed by anti-flag IP, followed by anti-gfp Western blot.

14 Figure S7. Related to Figure 6.

15 Figure S7. Pacer colocalizes and interacts with autophagosomal Stx17. (A, B) Analysis of Pacer colocalization with UVRAG or Atg14. U2OS cells were coexpressed with Pacer-GFP and mcherry-atg14, or Pacer-GFP and mcherry-uvrag, and analyzed by confocal microscopy. The colocalization was quantified in (C). Scale bars, 10 µm. Data are shown as mean ± SD from 150 micrographs of three independent experiments, *p<0.05. (D, E) Quantify relative intensity of each individual cololcalized puncta of Pacer-GFP and mcherry-uvrag or Pacer-GFP and mcherry-atg14 by a Phosphorimager and normalized by calculating as the percentage of the intensity of merged puncta. (F) Colocalization analysis of Stx17, Pacer (Δ ) and LC3. U2OS cells coexpressed with Stx17-HA, Pacer (Δ )-GFP were fixed and stained with anti-ha and LC3 antibodies, analyzed by confocal microscopy. Scale bars, 10 µm. (G) Analysis of the colocalization of Stx17 and Pacer with Vps34, Beclin 1 or Beclin 2. Stx17-Myc and Pacer-GFP were co-transfected with Vps34-HA, Beclin 1-HA or Beclin 2-HA in U2OS cells. Cells were then fixes and stained, followed by confocal microscopy analysis. Scale bars, 10 µm. (H) Analysis of Pacer s effect on SNARE complex formation. Flag-Stx17 and HA-VAMP8 or Flag-Stx17 and HA-SNAP29 cotransfected with Myc-Pacer or Vector alone, anti-flag IPs were performed, and analyzed by Western-blot. (I) Analysis of Pacer-5A interaction with Stx17. Flag-Pacer WT or Flag-Pacer-5A was expressed in 293T, and anti-flag IPs were performed and analyzed by Western-blot. (J) The working model. Pacer and Rubicon form a molecular switch to regulate autophagy by engaging Stx17, PI3KC3 and HOPS.

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