Electronic Supplementary Material
The AAA ATPase Vps4 plays important roles in Candida albicans
hyphal formation and is inhibited by DBeQ
Yahui Zhang, Wanjie Li, Mi Chu, Hengye Chen, Haoyuan Yu,
Chaoguang Fang, Ningze Sun, Qiming Wang, Tian Luo, Kaiju Luo,
Xueping She, Mengqian Zhang and Dong Yang
METHODS
Hyphal induction
For pH-induced hyphal formation assays, 10 l of overnight culture
was transferred into 1ml GMM with 50 mM Tris-HCl (pH 7) and
incubated for 2.5 h at 37°C. For serum-induced hyphal formation assay,
10 l of overnight culture was transferred into 1 ml GMM with 20% calf
serum and incubated for 2.5 h at 37°C.
VPS4 gene cloning, deletion and reintegration
To construct Vps4 expression plasmid, VPS4 (orf19.4339) was
amplified from the C. albicans genomic DNA (primers listed in Table 2
as VPS4BamHIf and VPS4SalIr). It was then cloned into the BamHI and
SalI sites of pGEX-4T-2 (GE Healthcare). Then pGEX-4T-2-VPS4 was
used to transform E. coli strain BL21(DE3) to generate ZYL1 strain. The
C. albicans genomic DNA was extracted with MasterPure™ Yeast DNA
Purification Kit (Epicentre) according to the manufacturer’s instruction.
The vps4 mutant was constructed by sequentially deleting the 2
copies of VPS4 gene from BWP17 [1] (strains YL1, YLZ1 and YLZ2). A
gene deletion cassette was constructed by flanking a marker gene, ARG4
or HIS1, with AB and CD (primers listed in Table 2) DNA fragments
(~400 bp each) corresponding to the 5’ and 3’ untranslated regions (UTR)
of the target gene, respectively. Transformants were selected using
2
appropriate dropout GMM and correct deletion was confirmed by
genomic DNA PCR (data not show).
Generation of epitope tagged proteins
For N-terminal tagging of VPS4, the DNA fragments for PMET3 and
GFP were inserted into CIp10, respectively, between the KpnI-XhoI and
XhoI-ClaI restriction sites. Then the VPS4CDS (primers listed in Table 2
as V4f and V4r) was cloned between the ClaI and EcoRV sites (a PstI
site were added) to generate pMET3pt-GFP-VPS4. To create the
VPS4E235Q mutant, the QuickChange multi-site-directed mutagenesis kit
(Agilent Technologies) was employed to create the site-directed
mutagenesis (primers listed in Table 2 as v4E235Qf and v4E235Qr) on
pMETpt-GFP-VPS4.
For N-terminal tagging of C. albicans Cps1 (orf19.2686), its CDS
was cloned from SC5314 genomic DNA (primers listed in Table 2 as
CPSClaIF
and
CPSPstIR),
and
replaced
the
VPS4
CDS
in
pMET3pt-GFP-VPS4 between the ClaI and PstI sites to generate
pMET3pt-GFP-CPS1.
All the plasmids containing the above-mentioned constructs were
linearized at the unique SalI or NsiI site within the MET3 promoter
(PMET3), transformed into BWP17 or vps4Δ strains (YLZ1) and then
integrated at the MET3 genomic locus (strains YLZ3, YLZ4, YLZ5 and
3
YLZ6). Transformants were selected on uridine-dropout GMM plates and
confirmed with genomic DNA PCR (data not show).
Vps4 expression and purification
ZYL1 cells were grown in LB media to the mid-log phase and
expression was induced by 0.5mM IPTG at 37℃ for 4h. Cells were
harvested, resuspended in 50 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA,
1mM DTT, 0.2 mM PMSF (pH 8.0) and lysed by sonication. Lysate was
applied to Glutathione Sepharose 4B (GE healthcare) and eluted with
on-column cleavage by TEV protease. Cleaved Vps4 was further purified
by size-exclusion chromatography in 50 mM Tris, 300 mM NaCl, 1 mM
EDTA, 1 mM DTT (pH 8.0) using a Sephacryl S-300 HR size exclusion
column. To study the assembly of Vps4 in the presence of ATP, it was
first incubated with 1mM ATP for 2 h before being separated by
size-exclusion chromatography in 50 mM Tris, 300 mM NaCl, 1 mM
EDTA, 1 mM DTT, 1 mM ATP (pH 8.0).
Cloning, expression and purification of p97
p97 was amplified from the mouse cDNA (primers listed in Table 2
as VcpBamHIF and VcpSalI) and then cloned into the BamHI and SalI
sites of pGEX-4T-2. Expression of p97 in E.coli strain BL21(DE3) was
induced by 0.5mM IPTG at 37℃ for 4h. The protein was purified
according to the same procedure for Vps4.
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DBeQ inhibition assay
To study the in vitro inhibition of ATPase activity, DBeQ (Sigma)
was dissolved in DMSO and then diluted to various concentrations from
0.45 mM to 14.5 mM in the same solvent. The reaction mixture contains
50 mM Tris (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 20 M ATP, 0.5 l
of DBeQ and 25 nM Vps4 or p97 in a final volume of 50 l. The control
reaction contains all the components except that 0.5 l of DMSO was
used in place of DBeQ. The mixture (without ATP) was incubated on ice
for 20 min. After being restored to room temperature, reactions were
initiated by addition of ATP.
Reaction was performed at room
temperature for 30 min and then 50 l of BIOMOL GREEN reagent was
added. After 15 min of incubation, 10 l of 0.2 M sodium citrate was
added. After 5 min of incubation, absorbance at 630 nm was measured in
a Bio-Rad iMark680microplate reader.
For analyzing the effect on hyphal formation, 10l of overnight
culture was transferred into 1ml GMM at pH 7 or with 20% calf serum.
DBeQ was then added to 30M. After incubation for 2.5 h at 37°C, cells
were observed under the microscope and classified according to the
number of hyphae.
Structural modeling and molecular docking
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The structural file of DBeQ was downloaded from Zinc
(http://zinc.docking.org) [2] and the PDB file of the D2 domain of p97
(p97D2) was obtained from the protein data bank (http://www.rcsb.org
with PDB ID 3CF0). The C. albicans Vps4 structure was modeled in
Swiss Model (http://swissmodel.expasy.org). Structural alignment was
performed in Pymol. Docking was performed on the ATP binding pocket
of p97D2 using GOLD [3]. A genetic algorithm with default parameters
was used and a total of 100,000 operations were performed. Gold scores
were calculated to evaluate docking results and that with the highest score
was selected for visualization in Pymol.
CPS experiment
To study CPS sorting, 10 l of overnight culture of YLZ5 or YLZ6
was inoculated into 1 ml GMM and shaken at 200rpm, 30°C for 6 h. The
distribution of CPS was then analyzed by the fluorescence microscope.
Vacuole Staining
For FM4-64 staining, cells were incubated on ice in GMM containing 16
mM FM4-64 (Life Technologies) for 20 min. Cells were then pelleted,
washed and resuspended in warm GMM lacking FM4-64. Cells were
incubated at 30°C for 60 min (shaken at 200 rpm) and analyzed by the
fluorescence microscope.
Cell growth studies
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C. albicans cells were grown overnight in YPD medium to
exponential phase (about 107 cells/ml). Then cells were diluted to 106
cells/ml, 105 cells/ml, 104 cells/ml with the method of hemacytometer
counting. Every 4 l of diluent was spotted on the YPD solid medium.
Then the plate was cultivated in 30℃ incubator for 24 hr. To study the
effect of DBeQ, 0.1% of DMSO or 30 M of DBeQ was used.
SUPPLEMENTARY FIGURE LEGENDS
Figure S1 Z-stack images from vps4 C. albicans cells. Images were
collected at 0.325 M intervals using the 488 nm wavelength. GFP-CPS
is in green color. Class E compartments are apparent in every cell.
Figure S2 Quantification of the formation of class E compartments. The
y axis shows the fraction of cells with class E compartments calculated
from Z-stack images. All vps4 cells show class E compartments, while
none of the wild type cells (even with DBeQ) have this defect.
Figure S3 Effect on cell growth by VPS4 deletion and DBeQ. 10-fold
serial dilutions of overnight cultures were spotted to YPD plates and
incubated at 30 ℃for 24h. Strains from the top to bottom: Wild-type,
vps4, Wild-type with DMSO, Wild-type with DBeQ.
Figure S4 DBeQ does not cause the formation of class E compartments.
After the treatment of wild type cells with DMSO or DBeQ, the
distribution of GFP-CPS was recorded and there were no differences.
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REFERENCES
1. Enloe B, Diamond A, Mitchell AP. A single-transformation gene
function test in diploid Candida albicans. J. Bacteriol. 2000; 182:
5730–5736.
2. Irwin JJ, Sterling T, Mysinger MM, Bolstad ES, Coleman RG. ZINC:
a free tool to discover chemistry for biology. J. Chem. Inf. Model
2012; 52:1757-1768
3. Jones G, Willett P, Glen RC. Molecular recognition of receptor site
using a genetic algorithm with a description of desolvation. J. Mol.
Biol. 1995; 245:43-53
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