Manuscript
Manuscript. Figure ___. Membrane transport characteristics of Xenopus laevis oocytes transformed with clone#3992. mRNA encoding orf3992 was transcribed in vitro from plasmid pEP(clone#3992) and transformed into Xenopus laevis oocytes. (A) Sugar transport into oocytes in the presence and absence of 10 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Extracellular medium supplemented with 1 mM sugar was left with oocytes for 1 hour and then intracellular concentration of sugar was measured. (B) Measurement of beemolulose transport kinetics into oocytes in the presence of different sugars.
Figure ____. Modifications to orf3992 within A. candicus. (A) Schematic showing CRISPR/Cas9 with guide RNA (gRNA) that targets orf3992 co-transformed into A. candicus. (B) Effect of CRISPR/Cas9 disruption of orf3992 gene on A. candicus growth characteristics. Sugars were added to minimal growth medium and A. candicus was grown for 12 hours in minimal medium plus sugar. (C) Schematic showing transformation of expression plasmid encoding orf3992 into A. candicus. (D) Effect of orf3992 overexpression on A. candicus growth in minimal medium plus sugars.
Figure ____. Screening Austral candicus cDNA library. (A) cDNA library preparation diagram. mRNA was extracted from A. candicus, converted to cDNA via reverse-transcription, followed by cloning into an expression plasmid with promoter constitutively active within S. cerevisiae. Following transformation of plasmid library into S. cerevisiae, 109 clones were plated onto minimal medium with all amino acids added lacking any sugar but beemolulose. Resulting colonies were picked and grown in 96-well liquid format with the same medium as used for plating colonies. Clone #3992 was one of 20 colonies that showed growth in the 96-well plate, had the most robust growth, and was chosen for further study. (B) Growth of yeast strains on different sugars. Growth was in liquid minimal medium supplemented with sugar(s) indicated. In the right panel, equimolar combinations of beemolulose plus indicated sugar were supplemented into minimal growth medium.
Figure ____. Identification and cellular localisation of ORF3992 protein. (A) Sequencing of clone #3992 reveals an open reading frame (ORF3992) of 700 amino acids long with hydropathy plotting potentially showing transmembrane domains. (B) Adding FLAG-tag to C-terminus of ORF3992 followed by transformation into yeast strain W303. (C) SDS-PAGE analysis of cytoplasm, membrane, and nucleus fractions from (-) yeast cells alone and (+) yeast cells carrying pEP(clone #3992). (D) Western blot of cytoplasm, membrane, and nucleus fractions from (-) yeast cells alone and (+) yeast cells carrying pEP(clone #3992). Probed with a rabbit anti-FLAG tag primary antibody, followed by anti-rabbit secondary antibody, and visualised with enhanced chemiluminescence (ECL) reagent.
B. Sugar
0
1750
3500 Tr
an sp
or t R
at e
(p m
ol /m
in /o
oc yt
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A ra
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X yl
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ul os
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La ct
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F ru
ct os
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R ha
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M an
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S uc
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G lu
co se
CCCP (10 µM)
Control A.
Substrate 1 Substrate 2 Km (mM) Vmax (pmols/min/oocyte)
Beemolulose – 20 ± 1 3499 ± 380 Beemolulose Glucose 22 ± 2 3298 ± 350 Beemolulose Mannose 21 ± 1 3507 ± 373 Beemolulose Rhamnose 102 ± 5 3445 ± 401
Beemolulose Transport Characteristics
B.
Sugar
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1750
3500
T
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p
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r
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R
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S
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c
r
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s
e
G
l
u
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e
CCCP (10 µM)
Control
A.
Substrate 1Substrate 2Km (mM)Vmax (pmols/min/oocyte)
Beemolulose- 20 ± 1 3499 ± 380
BeemoluloseGlucose22 ± 2 3298 ± 350
BeemoluloseMannose 21 ± 13507 ± 373
BeemoluloseRhamnose102 ± 5 3445 ± 401
Beemolulose Transport Characteristics
A.
La ct
os e
R ha
m no
se
B ee
m ol
ul os
e
X yl
os e
M an
no se
F ru
ct os
e
S uc
ro se
G lu
co se
N on
e
Sugar added to minimal medium
0
Δorf3992 Wild-type
100
150
G ro
w th
(% )
C.
B.
pEP(clone #3992)
La ct
os e
R ha
m no
se
B ee
m ol
ul os
e
X yl
os e
M an
no se
F ru
ct os
e
S uc
ro se
G lu
co se
N on
e
Sugar added to minimal medium
0
pEP(clone#3992) Wild-type
100
200
G ro
w th
(% )
Cas9+ gRNA
orf3992
D.
A.
L
a
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t
o
s
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R
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N
o
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Sugar added to minimal medium
0
Δorf3992
Wild-type
100
150
G
r
o
w
t
h
(
%
)
C.
B.
pEP(clone #3992)
L
a
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t
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G
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N
o
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Sugar added to minimal medium
0
pEP(clone#3992)
Wild-type
100
200
G
r
o
w
t
h
(
%
)
Cas9+
gRNA
orf3992
D.
A ra
bi no
se
X yl
os e
La ct
os e
R ha
m no
se
A ra
bi no
se
X yl
os e
+ Beemolulose
B ee
m ol
ul os
e
La ct
os e
F ru
ct os
e
R ha
m no
se
M an
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M an
no se
S uc
ro se
S uc
ro se
G lu
co se
G lu
co se
109 independent clones
Minimal medium + beemolulose
B.
A.
Sugar(s) added to minimal medium
0
Yeast + pEP(clone #3992)
Yeast
50
100
G ro
w th
(% )
mRNA
Saccharomyces cerevisae
Austral candicus
Expression Plasmid (pEP)
cDNA
Yeast + pEP(clone #3992)
Yeast
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10
9
independent clones
Minimal medium
+ beemolulose
B.
A.
Sugar(s) added to minimal medium
0
Yeast + pEP(clone #3992)
Yeast
50
100
G
r
o
w
t
h
(
%
)
mRNA
Saccharomyces
cerevisae
Austral candicus
Expression
Plasmid
(pEP)
cDNA
Yeast + pEP(clone #3992)
Yeast
C.
220
KDa
100 120
80
50
60
40
20
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MW M
ar ke
r
Cytoplasm Membrane Nucleus – + – + – + D.
220
KDa
100 120
80
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60
40
20
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MW M
ar ke
r
Cytoplasm Membrane Nucleus – + – + – +
Western Blot 1° antibody: anti-FLAG
SDS-PAGE
pEP(clone #3992)
B.
pEP(clone #3992)
FLAG-tag
W303(pEP(clone #3992))
A.
ORF3992 Position of Protein
H yd
ro pa
th y
+3
0
+2 +1
-1 -2 -3
1 508254
N- -C
C.
220
KDa
100
120
80
50
60
40
20
30
M
W
M
a
r
k
e
r
CytoplasmMembraneNucleus
–
+
–
+
–
+
D.
220
KDa
100
120
80
50
60
40
20
30
M
W
M
a
r
k
e
r
CytoplasmMembraneNucleus
–
+
–
+
–
+
Western Blot
1° antibody: anti-FLAG
SDS-PAGE
pEP(clone #3992)
B.
pEP(clone #3992)
FLAG-tag
W303(pEP(clone #3992))
A.
ORF3992
Position of Protein
H
y
d
r
o
p
a
t
h
y
+3
0
+2
+1
-1
-2
-3
1
508
254
N- -C
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