Materials and Reagents

p-aminobenzoic acid (Sigma-Aldrich, catalog number: P5669 )
d-Biotin (Sigma-Aldrich, catalog number: B4501 )
Calcium-D-pantothenate (Sigma-Aldrich, catalog number: 21210 )
Riboflavine (Sigma-Aldrich, catalog number: R4500 )
Pyridoxine hydrochloride (Sigma-Aldrich, catalog number: P9755 )
KCl
MgSO_4^.7H₂O
KH₂PO₄
Na₂B₄O₇^.10H₂O
CuSO₄^.5H₂O
FeO₄P^.4H₂O
MnSO_4^.H₂O
Na₂MoO₄^.2H₂O
ZnSO₄^.7H₂O
NaOH
Tween 80 (Sigma-Aldrich, catalog number: P1754 )
Radiolabelled substrate
e.g. [8-³H]-xanthine, 22.8 Ci/mmol (Moravek Biochemicals, catalog number: MT537 )
[2,8-³H]-hypoxanthine, 27.7 Ci/mmol (Moravek Biochemicals, catalog number: MT700 )
[5-³H]-uracil, 23 Ci/mmol (Moravek Biochemicals, catalog number: MT610 )
Non radiolabelled substrate
e.g. Xanthine (Sigma-Aldrich, catalog number: X7375 )
Hypoxanthine (Sigma-Aldrich, catalog number: H9377 )
Uracil (Sigma-Aldrich, catalog number: U0750 )
Toluol (AppliChem GmbH, catalog number: A3393 )
Triton X-100
2,5-Diphenyloxazole (PPO) (Sigma-Aldrich, catalog number: D4630 )
1,4-bis (5-phenyloxazol-2-yl) benzene (POPOP) (Sigma-Aldrich, catalog number: P3754 )
Complete Media (CM) (see Recipes)
Minimal Media (MM) (see Recipes)
Scintillation Fluid (see Recipes)

Equipment

Petri dishes
Neubauer counting-chamber slide
Spatula
Orbital shaking incubator
Incubator at 37 °C
Nylon net filter 60 μm (Merck KGaA, catalog number: NY60 )
50 ml Falcon tubes
1.5 ml centifuge tubes
Centrifuge
Vortex
Scintillation vials
Scintillation counter
Heat block
Magnetic stirrer
Magnetic strirr bar
pH meter
Pasteur pipette

Software

GraphPad Prism software (Amillis et al., 2004)

Procedure

Inoculate a petri dish of CM with the strain of interest and let it reach full growth at 37 °C for 96 h.
Using a spatula transfer one quarter of the fully grown colony (~4 cm) in a 50 ml Falcon tube containing 2 ml of 0.01% v/v Tween 80 in water. This amount usually corresponds to 10⁸ conidiospores. The accurate amount of conidiospores can be estimated using a Neubauer counting-chamber slide or by measuring viable conidiospores after standard serial dilutions and plating on CM.
Vortex well the sample for separating the conidiospores.
Inoculate a 100 ml flask containing 25 ml MM supplemented with appropriate carbon (C) (e.g. Glucose 1% w/v) and nitrogen (N) (e.g. NaNO₃ 10 mM) sources and necessary vitamins (e.g. D-biotin 0.02 μg/ml) with the conidiospores filtered through a Nylon net filter 60 μΜ. (All necessary supplements and concentrations for A. nidulans strains can be found at www.fgsc.net.)
Incubate for 3-5 h at 37 °C, shaking with 140 rpm, for the germinating conidiospores to reach a stage just before germ tube emergence. The time and temperature of incubation can change depending on the expression profile of the transporter of interest and the auxotrophic requirements of the strain. All transporters studied up to date in our lab (for example the purine/pyrimidine transporters UapA, UapC, AzgA, FcyB, FurD) reach maximum expression before germ tube emergence, driven by an unknown developmental control, irrespectively of the presence or absence of their substrates or other physiological conditions (Amillis et al., 2004; Vlanti and Diallinas, 2008; Amillis et al., 2007). In mycelia, transporter expression is very much dependent on physiological conditions (e.g. induction by substrates or/and N or C catabolite repression).
While conidiospores germinate, prepare the stock solution of radiolabeled (usually ³H or ¹⁴C) substrate of interest in water or MM so that for each assay 25 μl of the stock will be used.
Collect the conidiospores by centrifuging the culture in a 50 ml Falcon tube for 5 min, at 3,000 x g, room temperature.
Discard the supernatant and resuspend the pellet in 5 ml standard MM.
Distribute the spores in 75 μl aliquots in eppendorf tubes and use as many as needed. Conidiospore suspensions can be kept at 4 °C for at least 24 h without loss of transport activity.
Incubate conidiospore aliquots at 37 °C in a heat block for 5 min before addition of radiolabeled substrate.
Radiolabeled substrate is added for different periods of time. Most transporters show linearly increased activities for at least 1 min. For measuring initial uptake rates, which are necessary for determining K_m and apparent V_max values, the proper time of incubation must be defined for each transporter through a time-course experiment. For steady state substrate accumulation a period of 5 min is used. Usual time points are 10, 20, 30, 60 and 120 sec. For each time point, measurements are performed in triplicate. The temperature used for the incubation with the radiolabeled substrate depends on the transporter being studied at each experiment and the experiment being held, temperature for most experiments is 37 °C. The transport reaction is stopped by adding an equal volume (100 μl) of cold unlabeled substrate at 100-1,000 fold excess concentration, related to radiolabelled substrate, and direct transfer of the assay/eppendorf tube in an ice bucket.
Centrifuge the samples at 11,000 x g for 3 min at 4 °C.
Remove the supernatant through aspiration under vacuum using a Pasteur pipette. It is important to remove all the supernatant without losing any cells.
Wash the pellet of cells once with 1 ml ice cold MM and centrifuge at 11,000 x g for 3 min. Remove the supernatant as before.
Resuspend the pellet in 1 ml of scintillation fluid and put the eppendorf tubes into scintillation vials. Use a scintillation counter to measure substrate accumulation in the cells.
Analysis of transport measurements is performed using GraphPad Prism software. Radioactive counts should be converted to substrate concentration/min/conidiospores, based on the concentration and specific activity of the stock of radioactive substrate used.
For K_m determination of a transporter, different substrate concentrations should be used, for a fixed incubation time, previously determined to reflect initial uptake rates. This is usually 1 min. The range of concentrations used is determined at first empirically. In the final experiment, at least three concentration points below and above the apparent K_m value should be used. For each concentration point measurements are performed in triplicate.
The stock solutions are prepared using a mixture of fixed radiolabeled substrate and increasing concentrations of non-radiolabeled substrate, so that for each assay 25 μl of the stock will be used.
Terminate transport assays and perform measurements as described above.
For K_i determination of a transporter, the method is identical to the one for K_m determination, but stock solutions are prepared using a mixture of fixed radiolabeled substrate and increasing concentrations of non-radiolabeled putative inhibitors. For each concentration point measurements are performed in triplicate.
K_m and V_max determination is carried out using typical Lineweaver-Burk plot analysis that is based on the Michaelis-Menten equation for enzyme kinetics V = V_max[S]/(K_m + [S]), where V is the reaction velocity (the reaction rate), K_m is the Michaelis–Menten constant, V_max is the maximum reaction velocity, and [S] is the substrate concentration. The Lineweaver-Burk plot depicts the linear expression of the previous equation which is transformed to the following: 1/V = (K_m/V_max).(1/[S]) + 1/V_max. The data obtained by this experiment correspond to the apparent velocity of the transporter for each substrate concentration. Ki measurements are determined by estimating IC₅₀ values (inhibitor concentration for obtaining 50% inhibition) of given substrate/inhibitor, using the formula K_i = IC₅₀/1 + [S]/K_m, where [S] is the fixed concentration of radiolabeled substrate used. Another way to analyse the data is by using the GraphPad Prism Software through a non linear regression curve fit and sigmoidal dose response analysis. The IC₅₀ value corresponds to the K_m/i of the transporter. Quality factors for the analysis result are: R² which should be > 0.99 and the Hill co-efficient which should be approximately -1 for a transporter with one binding site.
The method described can be modified and adapted for most filamentous fungi that produce asexual spores, e.g. A. fumigatus. (Goudela et al., 2008)

Recipes

Complete Media (1 L)
Vitamin solution from 100x stock solution* 10 ml
Salt solution from 50x stock solution** 20 ml
Glucose 10 g
Peptone 2 g
Yeast Extract 1 g
Cas-amino- acids 1 g
Agar 10 g
Add water to 1 L final volume
Adjust the pH to 6.8 using NaOH
Autoclave for 20 min

*Vitamin stock solution
p-aminobenzoic acid 20 mg
d-biotin 1 mg
Calcium-D-pantothenate 50 mg
Riboflavin 50 mg
Pyridoxine 50 mg
Add water to 1 L final volume

**Salt stock solution
KCl 26 g
MgSO_4^.7H₂O 26 g
KH₂PO₄ 76 g
Trace elements 20x stock solution*** 50 ml
Add water to 1 L final volume

***Trace elements stock solution
Na₂B₄O₇^.10H₂O 40 mg
CuSO₄^.5H₂O 400 mg
FeO₄P^.4H₂O 714 mg
MnSO_4^.H₂O 728 mg
Na₂MoO₄^.2H₂O 800 mg
ZnSO₄^.7H₂O 8 mg
Add water to 1 L final volume
Minimal Media
Salt solution from 50x stock solution* 20 ml
Add water until 1 L final volume
Adjust the pH to 6.8 using NaOH
Autoclave for 20 min
Scintillation Fluid (1 L)
Toluol 666 ml
PPO 2.66 g
POPOP 0.0066 g
2 h stirring in RT
Add Triton-X 100 333 ml
Overnight stirring

Acknowledgments

This protocol was adapted from the following publications: Diallinas et al. (1995); Koukaki et al. (2005); Meintanis et al. (2000); Tazebay et al. (1997). E.K. works in the laboratory of G.D, and is co-financed by the European Union (European Social Fund-ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: Thales, Investing in knowledge society through the European Social Fund.

References

Amillis, S., Cecchetto, G., Sophianopoulou, V., Koukaki, M., Scazzocchio, C. and Diallinas, G. (2004). Transcription of purine transporter genes is activated during the isotropic growth phase of Aspergillus nidulans conidia. Mol Microbiol 52(1): 205-216.
Amillis, S., Hamari, Z., Roumelioti, K., Scazzocchio, C. and Diallinas, G. (2007). Regulation of expression and kinetic modeling of substrate interactions of a uracil transporter in Aspergillus nidulans. Mol Membr Biol 24(3): 206-214.
Diallinas, G., Gorfinkiel, L., Arst, H. N., Jr., Cecchetto, G. and Scazzocchio, C. (1995). Genetic and molecular characterization of a gene encoding a wide specificity purine permease of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes. J Biol Chem 270(15): 8610-8622.
Goudela, S., Reichard, U., Amillis, S. and Diallinas, G. (2008). Characterization and kinetics of the major purine transporters in Aspergillus fumigatus. Fungal Genet Biol 45(4): 459-472.
GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA. www.graphpad.com.
Koukaki, M., Vlanti, A., Goudela, S., Pantazopoulou, A., Gioule, H., Tournaviti, S. and Diallinas, G. (2005). The nucleobase-ascorbate transporter (NAT) signature motif in UapA defines the function of the purine translocation pathway. J Mol Biol 350(3): 499-513.
Meintanis, C., Karagouni, A. D. and Diallinas, G. (2000). Amino acid residues N450 and Q449 are critical for the uptake capacity and specificity of UapA, a prototype of a nucleobase-ascorbate transporter family. Mol Membr Biol 17(1): 47-57.
Tazebay, U. H., Sophianopoulou, V., Scazzocchio, C. and Diallinas, G. (1997). The gene encoding the major proline transporter of Aspergillus nidulans is upregulated during conidiospore germination and in response to proline induction and amino acid starvation. Mol Microbiol 24(1): 105-117.
Vlanti, A. and Diallinas, G. (2008). The Aspergillus nidulans FcyB cytosine-purine scavenger is highly expressed during germination and in reproductive compartments and is downregulated by endocytosis. Mol Microbiol 68(4): 959-977.