Analysis of transport activities in
yeast
To express GeSUTs in yeast, 2 distinct GeSUT cDNA
sequences (1611 bp for GeSUT4 and 1560 bp for GeSUT3) were
amplified using Phusion polymerase (New England Biolabs, USA) using the
same gene-specific primers used for RT-PCR analysis. The cDNA was first
cloned into the pDONR221-f1 vector and subsequently transferred to the
pDRf1-GW vector using LR Gateway technology (Grefen et al. 2010).
The yeast strains YSL2-1 or EBY4000 were transformed with the resulting
constructs (pDRf1- GW-GeSUT4 or pDRf1-GW-GeSUT3) using the LiAc method
(Chen et al. 2015). The cDNA of the Arabidopsis H+/sucrose
cotransporter AtSUC2, was also amplified and cloned into pDRf1-GW
using the same strategy. Transformants were selected and spotted on
synthetic deficient media supplemented with or without various
concentrations of sugars as described previously (Ho et al.2019). Sequences of primers are provided in Table S1.
To analyze transport activities with radiotracers, yeast cultures were
prepared as described previously (Ho et al. 2019). For a
time-dependent uptake assay, uptake buffer
medium containing 2 mM sucrose and 1.5 µCi [14C] sucrose ml-1, in 50
mM sodium phosphate (pH 5) was prepared. For a concentration-dependent
uptake assay, uptake media with increasing sucrose concentrations (0.02,
0.2, 1, 2, 5, 10 and 20 mM sucrose in 50 mM sodium phosphate) and the
same molar ratios of µCi [14C] sucrose were prepared. For
competition assays, uptake buffer media (2 mM sucrose with 2 µCi
[14C] sucrose ml-1) supplemented with 20 mM of various unlabeled
sugars (glucose, galactose, fructose, mannose, sucrose, or maltose) were
prepared. For analysis under different pH conditions, uptake buffer
media containing 2 mM sucrose with 2 µCi [14C] sucrose ml-1 were
prepared at different pH values using 50 mM sodium phosphate buffer. For
CCCP treatment, yeast cells were pre-incubated with medium containing 10
µM CCCP for 10 min before the uptake analysis. All uptake assays and
import of radiotracer were analyzed as described previously (Ho et
al. 2019).