Two Nucleotide Transport Proteins in Chlamydia trachomatis, One for Net Nucleoside Triphosphate Uptake and the Other for Transport of Energy

J. Tjaden; H. H. Winkler; C. Schwöppe; M. Van Der Laan; T. Möhlmann; H. E. Neuhaus

doi:10.1128/JB.181.4.1196-1202.1999

DOI: 10.1128/JB.181.4.1196-1202.1999

Article Figures & Data

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Fig. 1.
Alignment of predicted amino acid sequences of Npt1_Ct, Npt2_Ct, and RpTLC. Numbers indicate amino acid positions; dots mark artificial sequence gaps introduced to improve similarity between the proteins.
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Fig. 2.
Hydropathy analysis of the predicted amino acid sequences of Npt1_Ct, Npt2_Ct, and RpTLC. Hydropathy analysis was carried out as described by von Heijne (28). The values of H, which are essentially identical, have been offset to separate the plots for clarity. The predicted transmembrane domains in RpTLC are shown at the top.
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Fig. 3.
Time dependency of [α-³²P]ATP (●) and [α-³²P]ADP (○) uptake into intact E. colicells expressing npt1_Ct. IPTG-induced E. coli cells harboring plasmid pJT167 were incubated with 50 μM [α-³²P]ATP or [α-³²P]ADP for the indicated time periods. Control cells were not induced (−IPTG). Data are means of three independent experiments; standard deviations were less than 8% of the mean values.
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Fig. 4.
Exchange-mediated efflux of intracellular radioactivity. IPTG-induced E. coli cells expressingnpt1_Ct were preloaded by incubation with [α-³²P]ATP at 25 μM. After removal of external radioactivity, cells were resuspended in phosphate buffer with or without 1 mM AMP, ADP, or ATP.
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Fig. 5.
Time dependency of [α-³²P]ATP, [α-³²P]ADP, [α-³²P]GTP, [α-³²P]UTP, and [α-³²P]CTP uptake into intact E. coli cells expressingnpt2_Ct. (A) Time dependency of [α-³²P]ATP (0.8 mM; ●) or [α-³²P]ADP (1 mM; ○) uptake. (B) Time dependency of [α-³²P]GTP (0.05 mM), [α-³²P]UTP (0.5 mM), and [α-³²P]CTP (0.5 mM) uptake.

Tables

Figures

Table 1.

Effects of various NTPs or CCCP on transport activity of Npt1_Ct and Npt2_Ct^a

Effector	Rate of a NTP transport (nmol · mg of protein⁻¹ · h⁻¹)^b
	Npt1_Ct, ATP	Npt2_Ct
	Npt1_Ct, ATP	ATP	GTP	UTP	CTP
None	130.0	53.4	75.7	86.3	91.1
ATP			77.9	49.4	61.7
GTP	105.0	3.5		3.4	7.3
UTP	121.6	10.9	68.9		34.9
CTP	130.4	16.5	70.6	39.9
CCCP	91.2	7.1	10.6	19.8	20.9

↵a Substrate concentrations for labeled nucleotides: for Npt1_Ct, 50 μM (ATP); for Npt2_Ct, 1,000, 50, 400, and 400 μM (ATP, GTP, UTP, and CTP, respectively). The unlabeled NTPs were used at a 2.5-fold-higher concentration; CCCP was present at 1 mM.
↵b Mean of three independent experiments.

Table 2.

Effects of various nucleotides on transport activities of His₁₀-Npt1_Ctand His₁₀-Npt2_Ct

Effector	Rate of nucleotide transport (%)^a
Effector	ATP import His₁₀-Npt1_Ct	GTP import His₁₀-Npt2_Ct
None	100.0	100.0
dATP	96.5	100.7
dGTP	100.6	92.6
dCTP	106.4	97.0
dTTP	108.7	94.7
AMP	100.5	NM
GMP	NM	90.5
ADP	12.0	NM
GDP	NM	89.1
Guanosine tetraphosphate	NM	97.1

↵a ATP uptake was measured at a substrate concentration of 100 μM; GTP uptake was measured at a substrate concentration of 50 μM. The effectors were always present in a 2.5-fold-higher concentration than the substrates. ATP uptake by His₁₀-Npt1_Ct in the absence of effectors was 251.9 nmol · mg of protein⁻¹ · h⁻¹; GTP uptake by His₁₀-Npt2_Ct in the absence of effectors was 75.7 nmol · mg of protein⁻¹ · h⁻¹. NM, not measured. Data are means of three independent experiments.

Table 3.
K_m and V_maxvalues of Npt2_Ct for various nucleotides
Nucleotide Kinetic constant
K_m (μM) V_max(nmol · mg of protein⁻¹ · h⁻¹)
GTP 30.9 108.7
UTP 301.9 133.3
CTP 527.6 163.9
ATP 1,158.4 128.2
ADP 1,758.5 18.3