Article
Methods
No statistical methods were used to predetermine sample size. The
experiments were not randomized and investigators were not blinded
to allocation during experiments and outcome assessment.
Construct design and cloning
PfHT1 was cloned into the GAL-inducible vector pDDGFP2. The result-
ing construct consisted of: residues 1–5 from rat GLUT5 to facilitate
recombinant expression, PfHT1 residues 20–504 (out of 504) (Uni-
Prot accession number: O97467), followed by a tobacco etch virus
(TEV) cleavage site and a C-terminal GFP–His 8 tag. The vector was
transformed into the Saccharomycies cerevisiae strain FGY217 (MATα,
ura3–52, lys2Δ201 and pep4Δ)^33 as previously described^34. The result-
ing translated sequence following TEV digestion, with the non-PfHT1
residues from GLUT5 and residues of the TEV cleavage site underlined,
is: MEKEDSGFFSTSFKYVLSACIASFIFGYQVSVLNTIKNFIVVEFEWCKG
EKDRLNCSNNTIQSSFLLASVFIGAVLGCGFSGYLVQFGRRLSLLIIYNFFFLV
SILTSITHHFHTILFARLLSGFGIGLVTVSVPMYISEMTHKDKKGAYGVMHQL
FITFGIFVAVMLGLAMGEGPKADSTEPLTSFAKLWWRLMFLFPSVISLIGIL
ALVVFFKEETPYFLFEKGRIEESKNILKKIYETDNVDEPLNAIKEAVEQNESA
KKNSLSLLSALKIPSYRYVIILGCLLSGLQQFTGINVLVSNSNELYKEFLDSH
LITILSVVMTAVNFLMTFPAIYIVEKLGRKTLLLWGCVGVLVAYLPTAIANEI
NRNSNFVKILSIVATFVMIISFAVSYGPVLWIYLHEMFPSEIKDSAASLASLV
NWVCAIIVVFPSDIIIKKSPSILFIVFSVMSILTFFFIFFFIKETKGGEIGTSPYIT
MEERQKHMTKSVVENLYFQ.
Large-scale production and purification
For large-scale production, 24 l of S. cerevisiae FGY217 cells were grown
in –URA medium containing 0.1% (v/v) glucose at 30 °C in 2-l shaking
flasks. Protein production was induced at an optical density at 600 nm
(OD 600 ) of 0.6 by the addition of galactose to a final concentration of
2% (w/v). After 24 h incubation at 30 °C, the cells were collected, resus-
pended in buffer containing 50 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.6 M
sorbitol and lysed by mechanical disruption as previously described^34.
Membranes were isolated by ultracentrifugation at 4 °C and 195,000g
for 2 h, homogenized in 20 mM Tris-HCl pH 7.5, 0.3 M sucrose, 0.1 mM
CaCl 2 , flash-frozen in liquid nitrogen and stored at −80 °C. The PfHT1-
containing membranes were solubilized for 2 h at 4 °C in equilibration
buffer, consisting of 1× PBS, 150 mM NaCl, 10% (v/v) glycerol and 1% (w/v)
n-dodecyl-β-d-maltopyranoside (DDM; Glycon). Non-solubilized mem-
branes were removed by ultracentrifugation at 195,000g for 45 min,
and the cleared supernatant was incubated with 15 ml of Ni2+-nitrilotri-
acetate affinity resin (Ni-NTA; Qiagen) for 2 h at 4 °C in the presence of
40 mM imidazole under mild agitation. The resin was transferred to a
30-ml Eco-column (Bio-Rad) and washed with 300 ml of equilibration
buffer containing 0.1% (w/v) n-undecyl-β-d-maltopyranoside (UDM;
Anatrace) and 50 mM imidazole. The immobilized protein was eluted
in 30 ml of equilibration buffer containing 0.1% (w/v) UDM and 250 mM
imidazole. The eluate was incubated with equimolar TEV protease at
4 °C overnight to cleave the GFP–His 8 tag during dialysis performed
against 3 l of dialysis buffer, consisting of 20 mM Tris-HCl pH 7.5, 150
mM NaCl and 0.08% (w/v) UDM. The dialysed and digested sample
was loaded onto a 5-ml HisTrap column (GE Healthcare) equilibrated
with dialysis buffer, and the PfHT1-containing flow-through was col-
lected and concentrated. The concentrated solution was applied onto a
PD-10 desalting column (Sephadex G-25, GE) pre-equilibrated in dialysis
buffer, and the initial 1.6 ml of the flow-through collected, concentrated
to 6–8 mg ml−1 and used for crystallization experiments. For proteoli-
posome-based transport assays, the on-column immobilized PfHT1
was washed, eluted and dialysed in equilibration buffer and dialysis
buffer containing DDM 0.1% (w/v) and 0.03% (w/v), respectively, and
concentrated to 2 mg ml−1.
PfHT1 mutants were generated by overlap PCR, cloned into the
pDDGFP 2 vector, and overexpressed in 6-l cultures as previously
described for the wild type. The PfHT1 mutants were purified as
described for the wild type, but without GFP–His 8 tag removal by TEV
protease cleavage. The purified PfHT1–GFP fusions were concentrated
to 2 mg ml−1 and judged to be monodisperse by size-exclusion chroma-
tography using an Enrich 650 10 × 300 column in buffer containing 20
mM Tris-HCl pH 7.5, 150 mM NaCl and 0.03% (w/v) DDM.Transport activity of PfHT1 reconstituted into liposomes
Total bovine brain lipid extracts (Sigma Aldrich) and cholesteryl-
hemisuccinate (CHS) (Sigma-Aldrich) powder were mixed in buffer
containing 10 mM Tris-HCl pH 7.5 and 2 mM MgSO 4 to a final concentra-
tion of 30 and 6 mg ml−1, respectively. The lipid mixture was subjected
to multiple rounds of freeze–thaw cycles by flash-freezing in liquid
nitrogen and thawing at room temperature interspersed with sonica-
tion. Lipid mixture was further spun down at 16,000g for 15 min and
the supernatant containing small unilamellar vesicles was collected.
To make proteoliposomes, 10 μg of purified PfHT1 was added to 500
μl of unilamellar vesicles, flash-frozen and thawed at room tempera-
ture. Large unilamellar proteoliposomes were prepared by extrusion
(LiposoFast, Avestin; membrane pore size, 400 nm). Transport assays
for PfHT1 mutants were carried as GFP fusions and compared with
PfHT1 wild type prepared in the same manner.
For the transport time-course experiments, 15 μl of prepared prote-
oliposomes were diluted into 45 μl of external buffer consisting of 10
mM Tris-HCl 7.5, 2 mM MgSO 4 and either: [^14 C]d-glucose (30 μM) (Ameri-
can Radiolabelled Chemicals and Moravek Biochemicals), [^3 H]d-xylose
(0.3 μM) (American Radiolabelled Chemicals), [^14 C]d-mannose (30 μM)
(Moravek Biochemicals) or [^14 C]d-galactose (30 μM) (American Radiola-
belled Chemicals), [^14 C]d-fructose (6.0 μM), (Moravek Biochemicals),
[^3 H]d-glucosamine (0.3 μM) (Perkin Elmer). The reaction was stopped
by the addition of 1 ml of 10 mM Tris-MgSO 4 buffer and followed by
rapid filtering through a 0.22-μm filter (Millipore). The on-filter col-
lected proteoliposomes were washed with 6 ml of buffer containing
10 mM Tris-HCl 7.5 and 2 mM MgSO 4 , transferred to scintillation vials
and emulsified in 5 ml of Ultima Gold scintillation liquid (Perkin Elmer)
before scintillation counting (TRI-CARB 4810TR 110 V; Perkin Elmer).
The proteoliposomes for [^14 C]d-glucose competitive-uptake assays
were prepared as described for the time-course experiments. [^14 C]d-
glucose competitive uptake was measured at 30 s in external buffer
containing unlabelled sugars at a final concentration of 50 mM.
For kinetic analysis, the KM and Vmax values for d-glucose and d-fruc-
tose transport for PfHT1 and mutants were determined by measuring
the initial transport velocities for d-glucose at 20 s and d-fructose at 60
s for increasing concentrations of these sugars in buffer containing sto-
chiometric amounts of [^14 C]d-glucose or [^14 C]d-fructose. The recorded
radioactivity from empty liposomes was subtracted from the recorded
decay counts of the transported sugars and fitted to Michaelis–Menten
kinetics using nonlinear regression by GraphPad Prism 7.0. KM and Vmax
values for d-mannose, d-galactose and d-glucosamine for PfHT1 were
determined in the same way, with initial transport velocities recorded
at 20 s for d-mannose and 60 s for d-galactose and d-glucosamine.
Time course and kinetics of PfHT1 and PfHT1–GFP were measured to
be comparable. To calculate kcat, the amount of transported sugar was
therefore normalized by the fraction of reconstituted PfHT1–GFP fusion
incorporated into liposomes, which could be calculated by fluores-
cence-detection size-exclusion chromatography (FSEC)^35 analysis of
3% DDM (w/v)-solubilized proteoliposomes. PfHT1–GFP orientation
into liposomes was estimated by incubating 150 μl of proteoliposomes
with and without TEV protease at a ratio of 1:3 (w/w) overnight at 4 °C
and the fraction of cleaved GFP estimated by in-gel fluorescence^34 with
a ratio of about 60:40 (outside: inside) calculated.
For inhibition assays, 15 μl of PfHT1 wild type- or Trp412Ala mutant-
containing proteoliposomes were diluted into 43 μl of buffer consisting
of 10 mM Tris-HCl 7.5, 2 mM Tris-MgSO 4 that had been pre-incubated
for 1 h in either 4% (v/v) DMSO or 4% (v/v) DMSO with 115 μl of the tested