SUPPLEMENTARY INFORMATION

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1 Figure S1 UVRAG amino acid sequence. Bold letters indicate the peptides identified from mass spectrometry analysis. Symbols indicate the prolines in the N-terminal proline-rich sequence (*), the C2 domain (underlined), and the central coiled-coil domain (double underlined). 1

Figure S2 UVRAG, Becin1 and Bcl-2 interaction. (a) Native gel. 0.2 nmol of the MBP-UVRAG CCD (lane 1), His 6 -Beclin1 CCD (lane 2), and preformed complex (lane 3) were separated through native gel.

2 Figure S2 UVRAG, Becin1 and Bcl-2 interaction. (a) Native gel. 0.2 nmol of the MBP-UVRAG CCD (lane 1), His 6 -Beclin1 CCD (lane 2), and preformed complex (lane 3) were separated through native gel. (b) SDS-PAGE. 0.2 nmol of MBP-UVRAG CCD-His 6 -Beclin1 CCD preformed complex (lane 3) was recovered by Mono Q column and separated through SDS-PAGE. 0.2 nmol of the MBP-UVRAG CCD (lane 1) and His 6 -Beclin1 CCD (lane 2) were included as controls. (c) vbcl-2 AAA mutant does not interact with PI3KC3. At 48 h posttransfection with GST, GST-vBcl-2, or GST-vBcl-2 AAA construct together with Flag-tagged PI3KC3 and V5-tagged Beclin1, WCL of 293T cells were used for GST pulldown followed by immunoblotting with anti-flag. The raw data for this experiment is presented in the Supplementary information, Fig S5. (d) Triple complex. 0.2 nmol of the MBP-UVRAG CCD, His 6 -Beclin1 CCD, Bcl- XL loop and TM were mixed for 30 min and subjected to amylose column purification. Purified proteins were separated through SDS-PAGE. 2

Figure S3 UVRAG-induced Autophagy. (a) Electron microscopic quantitation of autophagosome. Results shown represent the mean ±SEM number of autophagic vacuoles per cell. (b).

3 Figure S3 UVRAG-induced Autophagy. (a) Electron microscopic quantitation of autophagosome. Results shown represent the mean ±SEM number of autophagic vacuoles per cell. (b). Essential role of UVRAG in Beclin1- mediated autophagy. HeLa cells were transfected with control sirna or UVRAG-specific sirna together with the GFP-LC3 expression vector. At 24, 48 and 72 h posttransfection, GFP-LC3-positive cells with punctate staining were photographed (top panel) and counted (bottom panel). Autophagy was quantified as the percentage of GFP-positive cells with punctate staining. The scale bars represent 20µm. 3

4 Figure S4 No effect of UVRAG wt and mutants on the viability of HCT116 cells. 1x10 5 HCT116 cells carrying vector, UVRAG wt or its mutants were seeded into 6 well plates and cell viability (%) was determined by counting live cells after trypan blue staining. The results represent means ((SD) of three independent assays. 4

5 Figure S5 Original gel scan. 5

6 Figure S5 Original gel scan. 6

7 Figure S5 Original gel scan. 7

8 Supplemental Data Experimental Procedures Cell Culture HCT116, NIH3T3, HeLa, MCF7 and 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mm L-glutamine, 1% penicillin-streptomycin (Gibco-BRL). MCF7.control cells, MCF7.Beclin1 cells and MCF7.Beclin ECD cells were generously provided by Dr. B. Levine and maintained in DMEM supplemented with 10% Tet-system-approved FBS (Clontech), 300 µg/ml hygromycin and 1 µg/ml doxycycline, as described previously 18. Transient transfections were performed with FuGENE 6 (Roche), Lipofectamine 2000 (Invitrogen), or calcium phosphate (Clontech) following the manufacturer s instructions. HCT116 and NIH3T3 stable cell lines were established using a standard protocol of selection with 2 µg/ml of puromycin. Plasmid Construction All constructs for transient gene expression in mammalian cells were derived from pcdna5/frt/to (Invitrogen). A DNA fragment corresponding to the coding sequence of the γhv-68 M11 gene was amplified from S11 cell genomic DNA by polymerase chain reaction (PCR) and subcloned into plasmid pcdna5/frt/to between restriction sites KpnI and NotI or pef-ires-puro between AflII and NotI for selection of stable transfectants. The GST-tagged vbcl-2 sequence was cloned into a pcdna5/frt/to derivative encoding an N-terminal GST epitope tag between BamHI and NotI sites. HA-tagged vbcl-2 and KSHV Bcl-2, the Flag-tagged and HA-tagged versions of both PI3K and UVRAG were cloned between to the KpnI and NotI 1

9 sites of pcdna5/frt/to encoding an N-terminal HA tag or Flag tag. V5-tagged Beclin1 was expressed from a modified pcdna5/frt/to encoding a C-terminal V5 tag; the Belcin1 sequence was inserted between the BamHI and XhoI sites. HA-tagged vbcl-2 AAA mutant was generated by PCR using oligonucleotide-directed mutagenesis to replace serine-85, glycine-86 and arginine-87 with alanine; the PCR product was subcloned into pcdna5/frt/to. Truncated mutants of PI3KC3, UVRAG and Beclin1 were made by subcloning PCR products of cdna fragments containing each separate domain of the associated gene into pcnda5/frt/to. The UVRAG CCD mutant, lacking the coiled-coil domain (aa ), was constructed by twostep PCR directed mutagenesis. All constructs were sequenced using an ABI PRISM 377 automatic DNA sequencer to verify 100% agreement with the original sequence. The HA-tagged Bcl-2 family expression vectors were kindly provided by Dr. M.J. Hardwick (John Hopkins University). pegfp-lc3 and p40px-egfp plasmids were kindly provided by Drs. T. Yoshimori (Osaka University Graduate School of Dentistry) and S. Field (University of California, San Diego), respectively. In vivo GST Pulldown, Protein Purification and Mass Spectrometry To identify vbcl-2-binding proteins, 293T cells expressing GST or GST-vBcl-2 fusion proteins were harvested and lysed with NP40 buffer (50 mm HEPES, ph 7.4, 150 mm NaCl, 1 mm EDTA, 1% (v/v) NP40) supplemented with a complete protease inhibitor cocktail (Roche). Post-centrifuged supernatants were pre-cleared with protein A/G beads at 4 0 C for 2 h. Precleared lysates were mixed with a 50% slurry of glutathione-conjugated Sepharose beads (Amersham Biosciences), and the binding reaction was incubated for 4 h at 4 0 C. Precipitates were washed extensively with lysis buffer. Proteins bound to glutathione beads were eluted and 2

10 separated on a Nupage 4-12% Bis-Tris gradient gel (Invitrogen). After silver staining (Invitrogen), specific protein bands were excised and analyzed by ion-trap mass spectrometry at the Harvard Taplin Biological Mass Spectrometry facility, and amino acid sequences were determined by tandem mass spectrometry and database searches. Immunoblot Analysis and Immunoprecipitation Assay For immunoblotting, polypeptides were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane (Bio-Rad). Immunodetection was achieved with anti-v5 (1:5000) (Invitrogen), anti-flag (1:5000) (Sigma), anti-ha (1:5000), anti- GST (1:2000) (Santa Cruz Biotech), anti-tubulin (1:1000) (Santa Cruz Biotech), anti-beclin1 (1:500) (BD Bioscience), anti-pi3kc3 (1:250) (Abgent). Rabbit anti-uvrag was diluted 1:500 for immunoblotting. The proteins were visualized by a chemiluminescence reagent (Pierce) and detected by a Fuji Phosphor Imager. For immunoprecipitation, cells were harvested and then lysed in NP40 buffer supplemented with a complete protease inhibitor cocktail (Roche). After pre-clearing with protein A/G agarose beads for 1 h at 4 0 C, whole-cell lysates were used for immunoprecipitation with the indicated antibodies. Generally, 1-4 µg of commercial antibody was added to 1 ml of cell lysate, which was incubated at 4 0 C for 8 to 12 h. After addition of protein A/G agarose beads, the incubation was continued for 1 h. Immunoprecipitates were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 5 min Protein Stability 3

11 At 24 h posttransfection with Beclin1 with UVRAG, 293T cells were treated with 20 µg/ml cycloheximide for 8 h. At each time point, cells were collected and subjected to immunoblotting with anti-flag or anti-tubulin. Production of recombinant Beclin1 CCD and UVRAG CCD. The constructs were generated based on a standard polymerase chain reaction protocol. Beclin1 CCD (residues ) and UVRAG CCD (residues ) were expressed in the bacterial strain BL21 (DE3) (Novagen) individually using the vectors pproex Hta (Invitrogen) and a modified pmal-c2 (New England Biolabs), respectively, or co-expressed using a vector construct containing Trc or Tac promoter at the upstream of each gene segment. Beclin1 CCD and UVRAG CCD contained a His 6 tag and a His 6 -fused MBP tag, respectively, at the N- terminus of each protein. Two proteins were individually purified using Ni-NTA columns (Novagen), and the complex of both proteins produced by coexpression was purified using amylose resin (New England Biolabs) and further purified using a Mono Q column (Amersham Pharmacia) after cleaving the tags with TEV protease. Circular dichroism spectroscopy. Data were collected on a JASCO J-810. CD spectrum was recorded with the preformed Beclin1 CCD and UVRAG CCD complex (2.5 M) in 20 mm phosphate buffer (ph 7.5) at 25 o C over the range of nm in a nitrogen atmosphere. The spectrum was the accumulation of three scans corrected by subtracting signals from the buffer control. Lysosomal volume and enzymatic assay 4

12 To measure the lysosomal volume, cells were dissociated with cell dissociation buffer (Sigma) and labeled with LysoTracker Green DND-26 (LTR; 50nM; Molecular Probes) for 45min 37 C, followed by flow cytometry analysis. Lysosomal acid phosphatase activity was assayed using 4- methylumbelliferyl phosphate (4-MUP) substrates. HCT116 cells were lysed with enzyme lysis buffer (50mM Tris-HCL ph 7.4, 1% Triton-X100, 300mM NaCl) supplemented with a complete protease inhibitor cocktail (Roche). The supernatants were incubated at 37 C for 60-90min with 1mM 4-MUP substrate in 0.1M sodium acetate buffer (Ph 4.5), the reaction was stopped by the addition of 0.5M glycine buffer ph 10.3, and the fluorescence was measured at excitation 355nm/ emission 460nm. Characterization of Cell Growth In Vitro HCT116 cells ( ) or NIH3T3 cells ( ) were plated into 6-well microtiter plates. Cell numbers were counted with a hemocytometer (Beckman Coulter) at each time point for 10 days. Cell counts were performed in triplicate. Soft Agar Colony Formation Assay To evaluate anchorage-independent colony formation, HCT116.vector, HCT116.UVRAG, HCT116.UVRAG CCD or HCT116.UVRAG CCD cells ( ) were suspended in complete medium containing 0.3% Nobel agar (Difco) supplemented with 2 µg/ml puromycin and plated in 6-well plates over a basal layer of complete medium containing 0.5% agar. Colonies were scored 10 days after plating and were photographed by phase-contrast microscopy. Images were captured with the QCapture software program. Clonogenicity was determined in repeated experiments. 5

13 Tumorigenicity in Nude Mice Nude mice were subcutaneously injected with HCT116.vector, HCT116.UVRAG or HCT116.UVRAG CCD cells. At 16 days postinoculation, mice were euthanized, and their tumors were weighed. 6

Supplementary Information: Materials and Methods. Immunoblot and immunoprecipitation. Cells were washed in phosphate buffered

Supplementary Information: Materials and Methods Immunoblot and immunoprecipitation. Cells were washed in phosphate buffered saline (PBS) and lysed in TNN lysis buffer (50mM Tris at ph 8.0, 120mM NaCl