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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,
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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).
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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
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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 300l iso-tonic buffer. Cells were mechanically lysed by filtration through 3m
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-,
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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
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(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-DisgorginFbox 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.
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