The transport substrates of TpNTT3

Import measurements with radioactively labelled nucleotides on intact E. coli cells expressing the recombinant protein were conducted to experimentally clarify the biochemical features of TpNTT3. Initial transport measurements with [α³²P]ATP revealed that TpNTT3 mediates significant time-dependent uptake of ATP into E.coli (~ 300 pmol/mg protein in 30 min), whereas non-induced (control) cells showed no or only marginal accumulation of radioactivity (< 40 pmol/mg protein in 30 min) (Figure 9).

To investigate whether ATP is the only substrate of TpNTT3, we conducted [α³²P]ATP import measurements in presence of 10-fold excess of various non-labelled nucleotides or nucleotide derivates. [α³²P]ATP import via TpNTT3 was markedly reduced by a high number of added nucleotides (Table 4).

Figure 9: Time dependent uptake of [α³²P]ATP by TpNTT3. ATP import (50 µM) mediated by E.coli cells expressing the recombinant TpNTT3 (black rhombs) or by non-induced cells (grey squares). Data are the mean of at least three independent experiments, standard errors are given.

Table 4: Effects of various metabolites on [α³²P]-ATP uptake by TpNTT3. Uptake of [α³²P]ATP by recombinant TpNTT3 was measured at a substrate concentration of 50 µM, and non-labelled effectors were present in 10-fold excess. Import was stopped after 10 min. Rates of nucleotide uptake are net values (minus control: non-induced E. coli cells) given as percentage of the non-affected transport (set to 100%). Data are the mean of three independent experiments. Bold type is used to indicate significantly reduced import rates (< 50% residual activity).

Effector Import [%] SE [%] Effector Import [%] SE [%]

None 100.0 / UMP 59.5 +/- 3.7

ATP 7.4 +/- 1.1 ITP 4.4 +/- 1.2

GTP 7.1 +/- 2.0 IDP 23.4 +/- 1.6

UTP 64.0 +/- 2.0 IMP 6.8 +/- 1.2

CTP 93.3 +/- 1.2 dITP 37.1 +/- 0.3

dATP 16.8 +/- 5.6 dAMP 46.6 +/- 6.4

dGTP 13.4 +/- 2.1 dGMP 28.6 +/- 2.6

dCTP 81.1 +/- 6.2 dIMP 36.9 +/- 2.8

TTP 90.4 +/- 4.7 cAMP 3,5 12.2 +/- 2.1

ADP 13.2 +/- 1.4 cGMP 3,5 18.1 +/- 3.9

GDP 13.7 +/- 1.9 cIMP 3,5 11.3 +/- 1.6

AMP 4.8 +/- 2.5 cAMP 2,3 111.8 +/- 5.9

GMP 16.6 +/- 0.9 ADP-Glucose 80.0 +/- 3.1

dADP 67.2 +/- 5.4 UDP-Glucose 95.8 +/- 3.0

Adenosine 100.6 +/- 3.6 NAD 78.1 +/- 4.9

Guanosine 66.8 +/- 4.6 NADP 102.2 +/- 6.3

UDP 93.0 +/- 2.6

Generally, purine nucleotides, including inosine nucleotides, mono- and triphosphorylated (deoxy)nucleosides and cyclic mononucleotides, caused a significant decrease in [α³²P]ATP transport, whereas pyrimidine nucleotides or the representative deoxynucleoside diphosphate dADP showed no or comparably low effects (~ 60 % or more residual transport activity). Moreover, deoxymononucleotides and dITP were not as efficient (~ 30 to 47 % residual activity) as the majority of purine nucleotides (~ 20 % or lower residual activity). It is important to mention that only cyclic 3’,5’-AMP and cyclic 3’,5’-GMP (that act in cellular signalling and protein regulation) led to transport reduction, whereas presence of cyclic 2’,3’-AMP (product of mRNA degradation) did not affect [α³²P]ATP transport.

The observed transport reduction might be caused by inhibitory properties of the applied molecule or could be due to its competition during binding or translocation of [α³²P]ATP.

We performed import studies with selected radiolabelled purine nucleotides to investigate whether they represent substrates or inhibitors of TpNTT3 (Figure 10). All nucleotides tested were imported. The rates of ADP, AMP, dAMP, dATP and dGTP uptake (~ 100 to 140 pmol mg protein^-1) were in the range of the ATP import (~ 115 pmol mg protein^-1) and transport of GTP, GDP and cAMP was slightly higher (~ 200 pmol mg protein^-1). GMP, dGMP (~ 80 pmol mg protein^-1) and finally cGMP (~ 30 pmol mg protein^-1) were transported with lowest rates.

Therefore, we can conclude that TpNTT3 accepts an extraordinarily wide range of purine nucleotides as substrates. We did not investigate transport with radiolabelled inosine nucleotides because most are not commercially available. However, all other nucleotides investigated were identified as substrates and therefore it seems likely that also the structurally related inosine nucleotides are accepted by TpNTT3.

We also determined the maximal velocities (Vmax-values) and affinities (KM-values) of TpNTT3 for the different substrates (Table 5). The amount of functional, recombinant protein in the E.coli membrane influences the Vmax (the more protein, the higher the Vmax) whereas KM-values are independent of this factor. Therefore, KM- and Vmax-values of different substrates of one transporter can be compared. Comparisons of the KM-values of different heterologously expressed NTTs is also possible but not comparison of their Vmax -values. For determination of KM-values and Vmax-values we conducted import studies with rising concentrations of the labelled nucleotides, respectively. TpNTT3 exhibits comparatively high affinities for ATP, ADP, AMP, GTP, GDP and cAMP (from ~ 20 to 57 µM) whereas dGTP, GMP and dATP are transported with intermediary (from ~ 80 to 140 µM) and dGMP and dAMP with rather low affinity (200 and 330 µM). Most substrates are transported with a maximal velocities ranging from 1.2 to 2.0 nmol mg protein^-1 h^-1. However, dGMP, dGTP, cAMP and particularly dATP show higher Vmax-values (2.5 to 3.8 and 6.8 nmol mg protein^-1 h^-1). It should be noted that due to the low transport rate reliable determination of the transport parameters for cGMP was hampered.

The previous results suggest that TpNTT3 generally prefers a broad spectrum of purine nucleotides as substrates and among the transported purine nucleotides it seems to be not highly selective. Therefore, TpNTT3 immensely differs not only from the diatom isoforms NTT1 and NTT2 and PtNTT5, but also from all plastidial or bacterial NTT-type carriers described so far.

Figure 10: Import of radioactively labeled nucleotides via recombinant TpNTT3. E coli expressing the recombinant TpNTT3 and non-induced cells were incubated for 10 min in 50 µM of the given substrates. The displayed transport represents the net uptake via TpNTT3 and was calculated by subtraction of the corresponding value of the control. Data are the mean of at least three independent experiments, standard errors are given.

Table 5: KM and Vmax values of nucleotide transport by recombinant TpNTT3. Nucleotide uptake in the presence of rising concentrations of substrate (5 µM – 1000 µM) was allowed for time spans in the linear phase of corresponding transport at 50 µM. KM values are given in μM, Vmax values are given in nmol mg protein⁻¹ h⁻¹. Data are the mean of at least three independent experiments. Corresponding standard errors are given in brackets.

Substrate KM Vmax

ATP 19.6 (+/- 2.7) 1.96 (+/- 0.29)

ADP 36.9 (+/- 6.5) 1.40 (+/- 0.15)

AMP 28.4 (+/- 2.3) 1.22 (+/- 0.06)

dATP 142.7 (+/- 8.6) 6.78 (+/- 0.77) dAMP 332.5 (+/- 18.1) 1.89 (+/- 0.15) cAMP 56.8 (+/- 2.4) 3.34 (+/- 0.18)

GTP 22.4 (+/- 1.7) 1.77 (+/- 0.17)

GDP 20.7 (+/- 1.9) 1.62 (+/- 0.14)

GMP 108.0 (+/- 7.1) 1.68 (+/- 0.15) dGTP 82.7 (+/- 10.7) 3.79 (+/- 0.67) dGMP 208.8 (+/- 16.8) 2.51 (+/- 0.16)