Yeast strains and cultivation.
Yeast strains are listed in supplementary Table S1
online. Strains were routinely grown at 30°C in either SD medium (6.8 g l−1
yeast nitrogen base, complete supplement or drop-out mix (from Qbiogene, Solon, OH, USA or Formedium, Hunstanton, UK), 20 g l−1
glucose) or YPD medium (10 g l−1
yeast extract, 20 g l−1
peptone and 20 g l−1
glucose), with the optional addition of 200 mg l−1
G418 (PAA Laboratories GmbH, Pasching, Austria) or 100 mg l−1
clonNat (Werner BioAgents, Jena, Germany). The strains EY57 and EY920 were maintained on YPGal medium (YPD medium containing 20 g l−1
galactose as a carbon source; Wykoff & O'Shea, 2001
). Low-phosphate YPD medium was prepared as described previously (Kaneko et al, 1982
; Werner et al, 2005
Manipulation of yeast strains.
Yeast genomic DNA was isolated by using the ‘smash-and-grab' protocol (Rose et al, 1990
). Plasmids were transformed by the method of Gietz et al (1992)
. Selection markers were exchanged and additional genes were deleted, truncated or tagged as described by Janke et al (2004)
. All strains were verified by PCR. Pho90- and Pho87-N-terminal truncations were obtained by homologous recombination directly in the genome of S. cerevisiae
(without the addition of new coding sequence except for new start or stop codons, ). At the same time, we exchanged the native promoter with the constitutive transcriptional enhancer factor (TEF) promoter (from the TEF1
Quantification of poly P and Ptot contents. Ptot
and poly P were determined and quantified as described previously (Werner et al, 2005
; Freimoser et al, 2006
). With the help of a phosphate standard, Ptot
and poly P levels were calculated by using 97 g mol−1
) and 80 g mol−1
(molecular weight of the HPO3
residues in poly P), respectively.
Phosphate-uptake measurements. Yeast cells were grown overnight in YPD (OD600≈4–8), inoculated in fresh medium (OD600≈0.25), harvested (OD600≈0.6–1) and washed twice (phosphate-free SD medium, 4% glucose with centrifugation at 1,500g, 4°C, 5 min). Cells were resuspended in the phosphate-free medium (OD600≈20) and kept at 4 °C. Before the uptake, experimental cells were shaken at 24 °C for 5 min. A 10 × phosphate solution (250 μM–10 mM with 3.8–4.5 × 106 counts per minute (CPM), or 20 and 50 mM with 8 × 106 CPM) was added to 0.9 volumes of the cell suspension; uptake was stopped by filtering and washing with 5 ml of a 500 mM phosphate buffer. Radioactivity was quantified by scintillation counting, the amount of phosphate that was taken up was calculated, and Vmax and Km were determined by non-linear curve fitting (Prism 5, GraphPad Software, La Jolla, CA, USA).
Phosphate efflux assay.
Cells were precultured overnight in YPD, transferred to fresh medium (OD600
≈1) and harvested after 4 h. After two washing steps (phosphate-free SD medium, 2% glucose), cells were resuspended in the same medium (OD600
≈20). The phosphate released into the medium was quantified after 2 h by the malachite green assay as described previously (Werner et al, 2005
31P nuclear magnetic resonance spectroscopy.
Yeast strains were grown overnight in 100 ml YPD medium and were harvested at OD600
P nuclear magnetic resonance spectroscopy was carried out as described previously (Pinson et al, 2004
), and the measurements for cytosolic orthophosphate and poly P are given as millimolar of orthophosphate residues.
The bait and prey constructs were generated with the vectors pNCW and pNubGx and used according to Iyer et al (2005)
. We used the lacZ
reporter and the two auxotrophic markers HIS3
to assess the interaction.
Northern blot analysis.
Transcript levels of SPL2
were determined by northern blot analysis as described previously (Pinson et al, 2004
Western blot analysis.
Proteins from cells expressing GFP–PHO90
were extracted, separated by SDS–PAGE gel electrophoresis and blotted as described previously (Horak & Wolf, 2001
). The blots were hybridized with GFP (1:4,000, Living colors A.v. Monoclonal Antibody ( JL-8), Clontech, Mountain View, CA, USA) and Hxk antibodies (Rockland Immunochemicals Inc., Gilbertsville, PA, USA), scanned (FluorChem SP, Alpha Innotech Corp., San Leandro, CA, USA), and protein levels were quantified by peak area integration of three independent blots.
Localization of GFP-tagged Pho90 and Pho90Δ375N was carried out as described previously by Hürlimann et al (2007)