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SUPPLEMENTARY INFORMATION Materials and Methods Cell Culture All Dictyostelium cells were grown in axenic HL5 medium. Knockout and Knock-in cell lines were selected with either blasticidin or hygromycin but were maintained in HL5 medium once the cell lines were established. Overexpressing cells lines were maintained in HL5 with 20 g/ml or 40 g/ml G418 according to the expression level. Gene Knockout, Gene Knock-in and Overexpression Constructs We generated a disgorgin knockout construct by inserting the blasticidin or hygromycin resistance cassette into the BamHI sites created at base 1196 of the disgorgin gene. The disgorgin knock-in construct were created by inserting a v5-tag gene at the end of the disgorgin gene upstream of the stop codon, TAA, followed by blasticidin resistance cassette and 3’ NTR. lvsD was disrupted at base 6599 with either hygromycin or Cre-loxP blasticidin to generate multiple gene disruptions (Faix et al., 2004). To knock out the lvsA gene, we used the recirculated REMI construct to disrupt the gene at base 9393 and selected with blasticidin. The disgorgin, dajumin, drainin, and lvsD genes were amplified by PCR from Ax2 genomic DNA, and cloned into the expression vector EXP-4(+) with either v5, the GFP gene, or the RFP gene to make N-v5-Disgorgin, N-GFP-Disgorgin, C-RFP-Dajumin, N-RFP-Drainin and N-GFP-LvsD constructs. All Rab genes were amplified by PCR from a Dictyostelium cDNA library and cloned into the N-RFP-EXP-4(+) vector. All mutations or deletions were generated using PCR. Successful mutagenesis was confirmed by sequencing. All knockouts and overexpression cell lines were generated in Dictyostelium Ax2 cells. drainin- cells and LvsAOE cells were from the De Lozanne lab. The genes of Rab14, Rab14ca, Rab14DN, Rab11A, Rab11Aca, Rab11ADN, 1 Rab11C, Rab8A, Rab8Aca, Rab8ADN, Drainin, and Disgorgin (1147-2151) were cloned into the pGEX-6p-1 vector for protein purification. Indirect Immunofluorescence Staining Inderect immunofluorescence staining was performed as described (Sesaki et al., 1997). Anti-v5 antibody (Abcam) is used at 1:200. Anti-H+-ATPase (VatM) mAb N2 and anti-Calmodulin mAb 2D1 were nice gifts from the Clarke lab and used at 1:50 and 1:100 respectively. REMI Screening REMI suppressor/enhancer screening is a modification of the protocol used previously (Dynes et al., 1994). Briefly, log-phase disgorgin- cells with hygromycin resistance were electroporated with DpnII and the REMI vector containing the blasticidin resistance cassette. The transformants were selected in 10 g/ml blasticidin and plated on SM agar with Klebsiella aerogenes. Colonies were picked onto 96-well plates and visualized under phase-contrast microscopy for different sizes of vacuoles. The genomic DNA of candidate cells was purified, cut with a restriction enzyme, re-circulated, and transformed into SURE Electroporation-Competent cells (Stratagene) to allow the sequencing of the gene. Detection of the Disgorgin Complex and Mass Spectrometry Vegetative cells were washed with Na/K phosphate buffer and resuspended at a density of 4 x 107 cells/ml in Na/K phosphate buffer. Cells were lysed with 2X lysis buffer (1% NP-40, 300 mM NaCl, 40 mM MOPS, pH 7.0, 20% glycerol, 2 mM Na3VO4). We performed the purification of the Disgorgin complex and mass spectrometry as described previously (Lee et al., 2005). 2 Figure S1. Southern and northern blot of disgorgin- cells and cell fractionation assay. (A) Southern blot analysis was performed as described previously (Aubry et al., 2003). We generated a disgorgin knockout construct by inserting the blasticidin resistance cassette into the BamHI sites created at base 1196 of the disgorgin gene. The genomic DNA was digested with EcoRV/EcoRI or EcoRI/XhoI and probed with 32 P-labeled 1135-1617 fragment of the disgorgin gene. The expected size is 2.5kb in wild-type Ax2 cells and 3.8kb from disgorgin mutant with EcoRV/EcoRI digestion and 7.4kb in wild-type Ax2 cells and 1.3kb from disgorgin mutant with EcoRI/XhoI digestion. (B) Northern blot analysis was performed as described previously (Aubry et al., 2003). RNA blot was probed with 32 P-labeled 1135-1617 fragment of the disgorgin gene. The expression of ckII shows the loading. (C) Cell fractionation assay. 2X107 vegetative cells were harvested, washed twice and resuspend in 300l iso-tonic buffer. Cells were mechanically lysed by filtration through 3m Nucleopore Track Etch membrane (Whatman). The lysate were spun at 13,000 rpm for 10 minutes. Supernatant and pellet were collected and subjected to SDS-PAGE. The membrane and cytosolic fractions were probed with anit-v5 for the endogenous Disgorgin in DisgorginKI cells, anti-GFP for GFP-Disgorgin overexpressing cells, anti-VatM for V-ATPase as membrane associated fraction and anti-ERK1 for cytosolic fraction P: Pellet; S: Supernatant. (D) Localization of GFP-Disgorgin with markers for endosomes or lysosomes. disgorgin- cells expressing GFP-Disgorgin treated either with TRITC-Dextran for 2 hours to label endosomes, or with LysoTracker red DND-99 to mark lysosomes. Arrows indicate the vacuoles which GFP-Disgorgin localizes to. Figure S2. Quantitation Data. (A) Quantitation of the percentage of cells with different vacuolar sizes. The percentage of cells with vacuole size >=8 m, >=6 m, >=4 m, or <=2.5 m for Ax2, disgorgin-, GFP-Disgorgin/disgorgin-, GFP-DisgorginR515A/disgorgin-, GFP-DisgorginQ551A/disgorgin-, GFP-DisgorginR515A /Ax2, GFP-DisgorginQ551A/Ax2, drainin-, drainin-/disgorgin-, GFP-Drainin/disgorgin-, GFP-Disgorgin/drainin-, 3 LvsAOE/disgorgin-, and lvsD-/disgorgin- strains. Vacuole size was measured by the diameter of the round vacuoles or the average of the length of the oval ones. At least 100 cells from each cell line were measured. (B) LvsAOE cells exhibit more CV bladders than wild-type cells. Quantitation of the percentage of cells with different number of CV bladders as visualizing by RFP-Dajumin in wild-type Ax2 cells and LvsAOE cells. At least 100 cells from each cell line were measured. Figure S3. Amino acid sequence alignment of the 70 amino acid upstream of TBC domain of Disgorgin (DDB0218275, D. discoideum); Drainin (AAD00520, D. discoideum); TRE2 (P35125, H. sapiens); RN-tre (Q92738, H. sapiens); Epi64 (AAK35048, Homo sapiens); Evi5-like (Q96CN4, H. sapiens). Figure S4. Mass spectrometry analysis of the Disgorgin complex and the co-immunoprecipitation of Disgorgin and two interacting proteins. (A) Mass spectrometry analysis of the Disgorgin complex. The Disgorgin-containing complex was immunoprecipitated and analyzed by mass spectrometry to determine the Disgorgin-interacting proteins. Wild-type Ax2 cells were used as a control for the Disgorgin/disgorgin- and DisgorginR515A/disgorgin- cells. Proteins that are uniquely identified in the Disgorgin/disgorgin- and/or DisgorginR515A/disgorgin- cells were listed in the table. Both spectra count (upper number in column 1-3) and total chromatogram intensity (lower number in column 1-3) were listed for each protein in each sample. The total number of identified distinct peptides (last column) was also listed for each protein. The most abundant peptides are from the SKP1 orthologues FpaA and FpaB, components of the SCF ubiquitination complex. We do not know if Disgorgin is a substrate for an SCF complex or may function as an adaptor to associate a potential interaction with the SCF complex, targeting it for ubiquitination. We identified ubiquitin in the complex, but we did not observe any ubiquitination of immunoprecipitated, tagged Disgorgin in wild-type cells 4 (using an anti-myc antibody to detect expressed myc-ubiquitin or an anti-ubiquitin antibody), possibly because the ubiquitinated product is degraded very rapidly (data not shown). Previous studies showed that proteosome inhibitors do not function in vivo to block proteosome activity. The vacuolar H+-ATPase A subunit, VatA, which also localizes to contractile vacuoles, was identified in the complex. We do not know if Disgorgin directly interacts with the vacuolar H+-ATPase A subunit or if the interaction is indirect. Although many of the proteins identified here are components of lysosomes, we did not detect any lysosomal defect in disgorgin- cells (data not shown). (B) Interaction of Disgorgin and FpaA/FpaB in an F-box-dependent manner. The cell lysates from cells co-expressing T7-FpaA or T7-FpaB and v5-Disgorgin or v5-DisgorginFbox were immunoprecipitated with anti-v5 antibody and the Western blot was probed with an anti-T7 or anti-v5 antibody. Movie S1. A reconstruction of the 3D structure of RFP-Dajumin/Ax2 or RFP-Dajumin/disgorgin- in isotonic buffer or hypotonic buffer as indicated. Movie S2. The initial CV discharge in disgorgin- cells in water. Frames were taken every 3 seconds. Movie S3. disgorgin- cells in water after the initial discharge of the enlarged vacuoles. Frames were taken every 3 seconds. Movie S4. RFP-Rab8A and GFP-Disgorgin translocate to CV bladders contemporaneously in wild-type Ax2 cells in water. Frames were taken every 3 seconds. Movie S5. RFP-Dajumin and GFP-LvsA in wild-type Ax2 cells in water. Frames were taken every 3 seconds. 5