PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex

Abstract

Endoplasmic reticulum (ER)–Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and disease. Here, we identify UVRAG, a beclin-1-binding autophagic factor, as a phosphatidylinositol-3-phosphate (PtdIns(3)P)-binding protein that depends on PtdIns(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1–beclin-1–PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi–ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis.

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Figure 1: UVRAG interacts with phosphoinositides.
Figure 2: UVRAG localizes at the ER.
Figure 3: UVRAG association with the ER is PtdIns(3)P-dependent.
Figure 4: UVRAG interacts with the ER tethering complex.
Figure 5: UVRAG interaction with the ER tether and PtdIns(3)P is required for COPI-dependent retrograde transport to the ER.
Figure 6: UVRAG and its interaction with RINT-1 and PtdIns(3)P are required for cis-Golgi maintenance.
Figure 7: UVRAG dissociates from the RINT-1 complex, and interacts with the beclin 1 complex during autophagy.
Figure 8: The role of beclin 1, RINT-1 and PtdIns(3)P in UVRAG-mediated Atg9 trafficking during autophagy.

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Acknowledgements

The authors wish to acknowledge V. Hsu, Z. Yue, J. Lippincott-Schwartz, T. Yoshimori, W. M. Yuan, S. Firestein and J. Laporte for providing reagents, D. Hauser and E. Barron for performing electron microscopy. We thank all the members of the Liang laboratory for helpful discussions. The authors wish to thank M. Torres for her editorial assistance. This work was supported by the American Cancer Society (RSG-11-121-01-CCG to C.L.), National Institutes of Health grants (R01 CA140964 and R21 CA161436 to C.L.), and core services performed through grant NIAID U19AI083025.

Author information

S.H., D.N. and B.M. performed most experiments of this study and analysed the data. S.H. contributed extensively to the revision work. J-H.L. performed protein–lipid binding assays and confocal microscopy. T.Z. conducted autophagy-related assays. I.G., S.D.P., Z.Z. and S.O. helped with data collection. N.B., B.L. and H.S.Y. conducted bioinformatics analysis. W-H.L., Y.T., H-G.W., V.D., R.P. and M.T. provided critical constructs and antibodies. A.M. and P.F. advised on gel filtration analysis. C.L designed research, analysed data and wrote the paper.

Correspondence to Chengyu Liang.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 UVRAG is a phosphoinositide-binding protein.

(a) Coomassie stained SDS gel showing purified UVRAG C2 (left panel) or full-length UVRAG proteins. Asterisks indicate degradation products of GST-UVRAG. The GST fusion was later cleaved with thrombin. (b) PI(3)P-binding property of UVRAG C2. Liposomes with serial dilutions of PI(3)P (19.5, 13, 6.5, and 3.25 pmol) were incubated with purified UVRAG C2 domain GST-fusion protein, and the GST-fusion proteins bound to PI(3)P were detected using an anti-GST antibody. The purified FYVE domain from Hrs was used as a control. (c) Distribution of the PI(4)P-binding FAPP1-PH and the endosome-associated PI(3)P-binding p40phox(PX) in MTMR3-expressing cells. HeLa cells co-transfected with MTMR3-mStrawberry and GFP-FAPP1-PH (1st row) or GFP-p40phox(PX) (2nd row) were processed for confocal microscopy analysis. Asterisks indicate MTMR3-transfected cells. Scale bars, 10 μm. (d) Wortmannin does not affect the PI(4)P pool of UVRAG. HeLa cells transfected with the PI(4)P probe GFP-FAPP1-PH were incubated in the absence (untreated, UT) or presence of 100 nM wortmannin for 1 hr, and processed for confocal microscopy analysis using anti-UVRAG antibody. Insets highlight the relative distribution of PI(4)P and UVRAG, which was not altered by wortmannin treatment. Scale bars, 10 μm.

Supplementary Figure 2 UVRAG interaction with the ER tethering complex.

(a) Schematic representation of RINT-1 (WT) and its deletion mutants and summary of their interactions with UVRAG. Interaction was determined by coimmunoprecipitation of Flag-RINT-1 with HA-UVRAG from 293T cell lysates. +, strong binding; ±, weak binding;–, no binding. CCD, coiled coil domain. Rad50-BD, Rad50-binding domain of RINT-1. (b) Interactions of the RINT-1 truncated mutants with UVRAG. At 48 hr post-transfection with Flag-UVRAG and GST-tagged RINT-1 mutants, 293T whole cell lysates (WCLs) were subjected to GST-pull down, followed by immunoblotting with anti-Flag. (c) RINT-1 deletion of the residues 65-220 abolishes UVRAG binding. At 48 h post-transfection with HA-UVRAG and Flag-RINT-1, or their mutants, 293T WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with anti-HA antibody. (d) ZW10 does not compete with UVRAG for RINT-1 interaction. The 293T cells were transfected with both Flag-UVRAG and GST-RINT-1 1−256 with increasing amounts of HA-ZW10. WCLs were used for GST-pulldown followed by immunoblotting with anti-Flag. (e) UVRAG does not compete with ZW10 for RINT-1 interaction. The 293T cells were transfected with both HA-ZW10 and GST-RINT-1 1−256 with increasing amounts of Flag-UVRAG. WCLs were used for GST-pulldown followed by immunoblotting with anti-HA. (f) Confocal microscopy analysis of the colocalization of endogenous UVRAG and RINT-1. Inset highlights UVRAG-RINT-1 colocalization. Scale bars, 10 μm. (gRINT-1 knockdown inhibits UVRAG interaction with ZW10 and NAG. 293T cells were transfected with control shRNA or RINT-1-specific shRNA for 72 hr, followed by IP with a UVRAG antibody and immunoblotting for UVRAG and the RINT-1–ZW10–NAG complex subunits. The right panel shows endogenous protein expression. Actin serves as a loading control. (h) Schematic representation of the UVRAG-RINT-1-ZW10 multiprotein complex that interacts with the COPI vesicle. CT, C-terminus.

Supplementary Figure 3 Effect of UVRAG on the function of the ER tethering complex.

(aUVRAG knockdown does not affect the ER localization of endogenous RINT-1 (top) and ZW10 (bottom). Control shRNA- or UVRAG shRNA-treated HeLa cells (green) were immunostained for endogenous PDI (red), RINT-1 or ZW10 (blue), followed by confocal microscopy. Scale bars, 10 μm. (b) Cytosolic dispersion of β′-COP in UVRAG-knockdown cells. HeLa cells were treated with control shRNA or UVRAG-specific shRNA expressing GFP as an expression marker for 72 hr and stained for β′-COP. Representative images of β′-COP (red) staining in these cells are shown (left panel). The percentage of shRNA-expressing cells (green) with β′-COP dispersion was quantified (right panel). 100 cells from randomly chosen fields (60x) were analyzed for each experiment. Means were calculated from the data collected from three independent experiments (n = 300). Scale bars, 10 μm. (c) Confocal microscopy analysis of the distribution of δ-COP in cells depleted of UVRAG, RINT-1, or ZW10. HeLa cells were transfected with the gene-specific shRNA as indicated, and then stained with anti- δ-COP (red). The percentage of shRNA-transfected cells with δ-COP dispersion was evaluated (right panel). Data represent mean ± SD; n = 200 cells obtained from three independent experiments. (d) Retrograde transport of ts045-VSVG-KDELR-YFP from Golgi to the ER. Representative confocal microscopy images of the Golgi-like (1st row; at 32°C) and reticular ER-like (2nd row; at 40°C) pattern of the retrograde cargo and its colocalization with GM130 and PDI, respectively. Scale bars, 10 μm. (e) Effects of autophagy-related proteins on COPI-dependent retrograde transport indicated by the Golgi-to-ER redistribution of VSVG-KDELR. HeLa cells transfected with controlshRNA, Beclin1 shRNA (left), or Atg16L1 shRNA (middle), or Atg7+/+ and Atg7−/− iMEF cells (right), were transfected with VSVG-KDELR-YFP. The ER pattern for the chimeric KDELR was quantified. Data represent mean ± SD; n = 120 cells obtained from three independent experiments. n.s., not significant. Endogenous protein expression was confirmed by immunoblotting (bottom).

Supplementary Figure 4 Effect of UVRAG knockdown on the ER and Golgi structure.

(a) Electron microscopy (EM) analysis of Golgi structure after RINT-1 suppression. HeLa cells were transfected with RINT-1-specific shRNA followed by EM analysis. Golgi fragmentation and scattering were detected. Scale bar, 100 nm. (bUVRAG knockdown leads to cis-Golgi dispersion in 293T and Cos7 cells. The 293T and Cos7 cells transfected with pGIPZ-UVRAG shRNA for 72 hr, were immunostained for cis-Golgi with anti-GM130 (red). Asterisks denote the UVRAG-depleted cells, and arrows indicate control (non-transfected) cells. Scale Bars, 10 μm. (c) Confocal microscopy analysis of the GM130 distribution upon UVRAG knockdown in U2OS cells. U2OS cells were treated with control- or UVRAG-specific shRNA followed by immunostaining of endogenous GM130 (red). Asterisks indicate cells with Golgi dispersion. Scale bars, 10 μm. (d) HeLa cells were transfected with pGIPZ-UVRAG-shRNA to deplete endogenous UVRAG for 72 hr, followed by confocal microscopy analysis using the indicated antibodies, including anti-p115 for cis-Golgi, anti-PDI for the ER, anti-TGN46 for the TGN, and anti-Sec31a for COPII. GFP signals highlight the shRNA-transfected cells and DNA was stained with DAPI (blue). For the distribution of GalNac-TII (bottom two rows), HeLa cells were transfected with pLKO.1-control shRNA or –UVRAG shRNA, together with the GFP-GalNac-TII plasmid. UVRAG knockdown was verified by confocal microscopy using anti-UVRAG antibody (red). Asterisks denote UVRAG-depleted cells, and arrows indicate control cells. Scale Bar, 10 μm. (e) Depletion of RINT-1 or ZW10 leads to cis-Golgi dispersion. HeLa cells were transfected with RINT-1-, or ZW10-specific shRNA expressing GFP as a marker and then stained for GM130 (red). Nuclei were stained with DAPI (blue). Asterisks denote the shRNA-transfected cells with Golgi dispersion. Arrows indicate control cells. Scale bars, 10 μm.

Supplementary Figure 5 UVRAG is required for maintenance of Golgi integrity.

(a-b) The effect of Brefeldin A (BFA) on the Golgi was not discernably affected upon UVRAG knockdown. HeLa cells were treated with scrambled shRNA or shRNA against UVRAG, and then transfected with the GalNacT2-GFP (a) or ManII-GFP (b) plasmids. BFA (0.5 μg/ml) was then added to cells for indicated time points. Cells were fixed, and examine for the distribution of GalNacT2-GFP (a) and ManII-GFP (b) using immunofluorescence microscopy. Data (in a and b) represent mean ± SD; n = 100 shRNA-transfected cells obtained from three independent experiments. Scale bar, 10 μm. Kinetics of BFA-induced redistribution of GalNacT2 (a; right panel) and ManII (b; right panel) from the Golgi to the ER are shown. (cBeclin1 deficiency has minimal effect on GM130 distribution. Beclin1 wild-type (beclin1+/+) and knockdown (beclin1-/-) mouse ES cells were stained using anti-GM130 for cis-Golgi integrity and DAPI for nuclei, followed by confocal microscopy analysis. Western blot shows the levels of Beclin1 in these cells with actin serving as a loading control (right). Scale bars, 10 μm. (d) The C-Vps complex is not required for cis-Golgi integrity. HeLa cells transfected with control-, or Vps16-, or Vps18-specific shRNA, were subjected to confocal microscopy analysis using anti-GM130 (red). Western blots show the levels of Vps16 and Vps18 in these cells with actin serving as a loading control (right). Scale bars, 10 μm.

Supplementary Figure 6 UVRAG switches from the RINT-1-containing ER tethering complex to the Beclin1-containing autophagy complex during autophagy.

(a) UVRAG dissociates from the ER tether and interacts with the Beclin1-Bif-1-PI(3)KC3 complex during SMER28-induced autophagy. The 293T cells were treated with SMER28 (50 μM, 2 h) and subjected to IP with anti-UVRAG (lane 3-4), followed by immunoblotting with the indicated antibodies. Input (10% of whole-cell-lysates; lane 1-2) shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data. (b) Differential colocalization of endogenous UVRAG with RINT-1 and Beclin1 upon starvation. HeLa cells were incubated either in complete medium (untreated, UT) or starvation condition (HBSS, 2 hr), and stained for endogenous UVRAG (green), RINT-1 (red), and Beclin1 (blue). Insets show high magnification of the selected areas. Confocal microscopic quantification of colocalization of UVRAG with RINT-1 and Beclin1 was shown (right panel). Data are mean ± SD, n = 60 cells obtained from three independent experiments; *, p<0.05; **, p<0.01. Scale bars, 10 μm. (c) Interactions of the RINT-1-binding defective mutant and K78A/R82A mutant of UVRAG with the ER tether and the Beclin1-complex upon rapamycin treatment. The 293T cells transfected with empty vector (vec; lane 2), Flag-UVRAG (WT; lane 3-4), Flag-UVRAG Δ270−442(lane 5-6), or Flag-UVRAG K78A/R82A (lane 7-8), were treated with rapamycin (50 nM, 2 hr). WCLs were used for IP with anti-Flag, followed by immunoblotting with the indicated antibody. The input (10% WCLs) shows endogenous protein expression (lane 1) and actin serves as a loading control. See Supplementary Fig. S9 for uncropped data. (d) Differential interaction of UVRAG with RINT-1-ZW10 and Beclin1-Bif-1 upon the Golgi-ER retrograde pulse induced by temperature shift. The 293T cells transfected with the retrograde cargo VSVG-KDELR were incubated at 32°C (permissive temperature; lane 1-2) overnight, then shifted to 40°C (non-permissive temperature; lane 3-4) to induce a retrograde traffic wave from Golgi to the ER. WCLs were used for IP with either IgG (lane 1 and 3) or anti-UVRAG (lane 2 and 4), followed by immunoblotting with the indicated antibodies. See Supplementary Fig. S9 for uncropped data. (e) Confocal microscopy analysis of the co-localization of endogenous Atg9 and GM130 (cis-Golgi) in HeLa cells. Insets highlight co-localization. Scale bars, 10 μm.

Supplementary Figure 7 Effect of UVRAG, Beclin1, RINT-1, and Atg16L1 on autophagy-induced Atg9 traffic.

(a) The autophagy-induced dispersal of Atg9 can be blocked by knockdown of UVRAG or Beclin1, but not RINT-1, or Atg16L1. Control- (1st row), UVRAG- (2nd row), Beclin1-(3rd row), RINT-1-(4th row), or Atg16L1-(5th row) knockdown HeLa cells were incubated in complete medium (untreated, UT), starvation medium (HBSS, 2 hr), or treated with rapamycin (100 nM, 2 hr) or SMER28 (50 μM, 2 hr). The distribution pattern of endogenous Atg9 (green) was analyzed by confocal microscopy (left). The percentage of cells with dispersed Atg9 foci was calculated (middle). For each sample, 500 cells were counted. Immunoblotting of UVRAG, Beclin1, RINT-1, and Atg16L1 are shown (right). Data represent mean ± SD; n = 500 shRNA-transfected cells obtained from three independent experiments. n.s. not significant; *** p<0.001. Scale bars, 10 μm. (b) Autophagy activity of the UVRAG mutants. The 293T cells expressing empty vector, Flag-tagged wild-type UVRAG or its mutants as indicated, were cultured in complete medium or treated with SMER28 (50 μM). The cell lysates were subjected to immunoblotting with LC3, p62, Flag, and actin antibodies. Densitometric quantification of the LC3-II/LC3-I ratios under the indicated conditions is shown at the bottom of the LC3 blot. Similar results were obtained from three independent experiments. See Supplementary Fig. S9 for uncropped data.

Supplementary Figure 8 Schematic representations of UVRAG functional domains and a hypothetical model of UVRAG-coordinated Golgi-ER retrograde transport and autophagy-related Atg9-trafficking.

(a) Domain organization of UVRAG. The PR, C2, CCD, and C-terminal regions are indicated. Amino acid positions are shown at the top. The binding sites for UVRAG interacting proteins are indicated with arrows. C-Vps, class C vacuole protein sorting (Vps) complex; Ku-DNA-PK, Ku70-Ku80-associated DNA dependent protein kinase complex. (b) UVRAG assembles different protein complexes under basal and autophagy-inducing conditions. Under basal condition, UVRAG, a PI(3)P effector at the ER, associates with the RINT-1–ZW10–NAG tethering complex for efficient COPI-vesicle transport to the ER; Induced Golgi-ER traffic pulse can increase this complex assembly. Upon autophagy induction, UVRAG dissociates from the ER tethering complex and associates with the Beclin1-containing autophagy complex, which is required for Atg9 translocation from the Golgi to the autophagosome biogenesis site. The partner change of UVRAG during autophagy highlights coordinated regulation of intracellular membrane trafficking events. See the Discussion for detail.

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He, S., Ni, D., Ma, B. et al. PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex. Nat Cell Biol 15, 1206–1219 (2013) doi:10.1038/ncb2848

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