(Received for publication, May 30, 1995; and in revised form, July 18, 1995)
From the
PotA protein, one of the components of the
spermidine-preferential uptake system in Escherichia coli, was
purified to homogeneity, and some of its properties were examined. PotA
protein showed Mg- and SH-dependent ATPase activity.
The specific activity was approximately 400 nmol/min/mg of protein and
the K
value for ATP was 385
µM. The nature of the ATP binding site was explored by
identification of the amino acid residue photoaffinity-labeled with
8-azido-ATP. It was found that 8-azido-ATP was attached to cysteine 26.
In the spermidine transport-deficient mutant E. coli NH1596,
valine 135 of PotA protein, which is located between two consensus
amino acid sequences for nucleotide binding (50-57 and
168-173), was replaced by methionine (Kashiwagi, K., Miyamoto,
S., Nukui, E., Kobayashi, H., and Igarashi, K.(1993) J. Biol. Chem. 268, 19358-19363). This mutated PotA protein could be
labeled with 8-azido-ATP, but showed very low ATPase activity. To
identify which cysteine is involved in the function of potA protein,
cysteines 26, 54, and 276 were replaced by alanine, threonine, and
alanine, respectively. Among the three mutated PotA proteins, the
mutated PotA protein C54T only lost both ATPase and spermidine uptake
activities. The results taken together indicate that the adenine
portion of ATP interacts with a domain close to the
NH
-terminal end of PotA protein, and active centers of ATP
hydrolysis are located both within and between the two consensus amino
acid sequences for nucleotide binding. Association of PotA protein with
membranes was strengthened by the existence of channel forming PotB and
PotC proteins. ATPase of PotA protein was inhibited by spermidine,
suggesting that uptake inhibition by spermidine may function during
this process.
Polyamines (putrescine, spermidine, and spermine) are known to be necessary for cell growth(1, 2) . It is thus important to understand the mechanism by which the cellular polyamine is regulated. Polyamine transport is one of the important determination factors of polyamine content in cells. In Escherichia coli, polyamine uptake is energy-dependent, and the putrescine transport system is different from the spermidine (spermine) transport system(3, 4) . Furthermore, two transport systems for putrescine have been suggested in E. coli K12 grown in a low osmolarity medium(5) . We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79, and pPT71) in E. coli(6) . The system encoded by pPT104 was a spermidine-preferential uptake system and that encoded by pPT79 a putrescine-specific uptake system. Furthermore, these two systems were periplasmic systems (7) consisting of four kinds of proteins: pPT104 clone encoded PotA, PotB, PotC, and PotD proteins and pPT79 clone encoded PotF, PotG, PotH, and PotI proteins, judging from the deduced amino acid sequences of the nucleotide sequences of these clones(8, 9) . PotD and PotF proteins were periplasmic substrate binding proteins, and PotA and PotG proteins were membrane-associated proteins having the nucleotide-binding site. PotB and PotC proteins, and PotH and PotI proteins, were transmembrane proteins probably forming channels for spermidine and putrescine, respectively. In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (PotE protein) having 12 transmembrane segments (10) and was active in the excretion of putrescine from cells through putrescine-ornithine antiporter(11) . We also found that spermidine uptake by membrane vesicles was strongly dependent on PotD protein, and the uptake by intact cells was completely dependent on ATP through its binding to PotA protein(12) .
In this study, we tried to identify the functional domain in PotA protein using the purified and several mutated PotA proteins. We found that 8-azido-ATP was attached to cysteine 26, and replacement of cysteine 54 and valine 135 by threonine and methionine, respectively, led to the loss of ATPase and spermidine uptake activities. The results also indicate that PotA protein is associated with membranes through the interaction with PotB and PotC proteins.
To construct pKKpotA2BC, the 1.3-kb StyI fragment and the 5.1-kb StyI-DraIII fragment were prepared from pKKpotA and pKKpotABC, respectively. The 1.4-kb StyI-DraIII fragment prepared from pMWpotA2B was then ligated together with the 1.3-kb StyI and 5.1-kb StyI-DraIII fragments described above. Plasmid pKKpotA3BC was constructed by ligating the 0.8-kb SphI fragment of PCR product for the potA3 gene and the 7-kb SphI fragment of pKKpotABC. Plasmid pKKpotA4BC was constructed by ligating the 0.6-kb XbaI-DraIII fragment of pMWpotA4B and the 7.2-kb XbaI-DraIII fragment of pKKpotABC. Transformation of E. coli cells with various plasmids was carried out as described by Maniatis et al.(21) .
The strains and plasmids used in this study are listed in Table 1.
Figure 1:
Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis of proteins obtained after each purification step. Numbers on the left represent molecular mass in Da. Lane 1, 100,000 g supernatant; lane
2, precipitate with 50% saturation of
(NH
)
SO
; lane 3,
DEAE-Sephadex A-50 fraction; lane 4, Econo-Pac Q fraction
(1st); lane 5, Econo-Pac Q fraction
(second).
ATPase
activity of PotA protein was dependent on Mg, and 10
mM Mg
was necessary to obtain the maximal
activity (Fig. 2A). The ATPase activity was strongly
inhibited by spermidine, and the function of Mg
could
not be replaced by spermidine (Fig. 2B). Spermine, but
not putrescine, also inhibited the ATPase activity. Since spermidine
uptake was inhibited by the already accumulated spermidine, the
inhibition may be in operation during this process. The specific
activity was approximately 400 nmol/min/mg of protein (Table 2),
and the K
value for ATP was estimated to be 385
µM according to the Michaelis-Menten kinetics. Since
spermidine uptake was inhibited by N-ethylmaleimide (NEM) and p-chloromercuribenzoic acid(4) , effect of the
inhibitors on the ATPase activity was examined. As shown in Fig. 3, the ATPase activity of PotA protein was inhibited by NEM
and p-chloromercuribenzoic acid, and the inhibition was
restored by the addition of dithiothreitol.
Figure 2:
Effect of Mg (A) and spermidine (B) on ATPase activity of PotA
protein. Assays were performed under standard conditions except that
Mg
in the reaction mixture was changed and spermidine
was added to the reaction mixture as shown in the figure. Each value is
the average of three determinations. The standard deviation was within
± 10% for each data point.
Figure 3:
Effect of N-ethylmaleimide (A) and p-chloromercuribenzoic acid (B) on
ATPase activity of PotA protein. Assays were performed with () or
without (
) 2 mM dithiothreitol. Each value is the
average of three determinations. The standard deviation was within
± 10% for each data point.
Figure 4:
8-azido-[-
P]ATP
labeling of PotA1 (V135M) protein in inside-out membrane vesicles. Numbers on the left represent molecular mass in Da.
A. Coomassie Blue staining of protein; lane 1, the vesicles
prepared from E. coli JM105; lane 2, the vesicles
prepared from JM105/pKKpotA1BC. B,
8-azido-[
-
P]ATP labeling of proteins; lanes 1 and 2, labeling was performed using the
vesicles prepared from E. coli JM105 in the absence and
presence of 1 mM ATP, respectively; lanes 3 and 4, labeling was performed using the vesicles prepared from E. coli JM105/pKKpotA1BC in the absence and presence
of 1 mM ATP, respectively.
Figure 5: HPLC separation of tryptic peptides from 8-azido-ATP photoaffinity-labeled PotA protein. Amino acid sequences of peptides A and B were determined by automated Edman degradation. Cysteine in peptide A could not be determined, and it was inferred from the residual amino acid sequence of peptide A.
Figure 6: Structure and function of PotA protein. A, consensus amino acid sequences for nucleotide binding (sites A and B), photoaffinity-labeled amino acid with 8-azido-ATP (C26) and ATPase-deficient mutants (C54T and V135M) are shown in the figure. B, the secondary structure of PotA protein was analyzed according to the method of Chou and Fasman(32) .
Figure 7:
8-azido-[-
P]ATP
and N-[
C]ethylmaleimide labeling of
normal and mutated PotA proteins. Experiments were performed with
inside-out membrane vesicles prepared from E. coli JM105atp
carrying pKKpotABC,
pKKpotA2BC, pKKpotA3BC, and pKKpotA4BC. A, 8-azido-[
P]ATP labeling; B,
[
C]NEM labeling; C, Western blotting; D, Coomassie Blue staining. Numbers on the left represent molecular mass in Da. Arrows indicate the
position of PotA protein.
PotA2, PotA3, and PotA4 proteins were photoaffinity-labeled with 8-azido-ATP (Fig. 7A). In a foregoing paragraph, we identified cysteine 26 as photoaffinity-labeled amino acid. Thus, threonine 39 may be photoaffinity-labeled with 8-azido-ATP instead of cysteine 26 with regard to PotA2 (C26A) protein, as will be discussed later. The properties of PotA3 (C54T) protein were similar to those of PotA1 (V135M) protein. Although both proteins were photoaffinity-labeled with 8-azido-ATP, they showed very low ATPase activity.
Next, the binding of [C]NEM to
PotA2, PotA3, and PotA4 proteins was examined. As shown in Fig. 7B, [
C]NEM could bind to
the three mutated PotA proteins. When the amount of
[
C]NEM bound to the mutated PotA proteins was
calculated by measuring radioactivity and density of band from the data
of Fig. 7, B and D, it was approximately
60-70% of that to the normal PotA protein. The results suggest
that NEM can bind to all three cysteines (Cys-26, Cys-54, and Cys-276).
The secondary structure of PotA protein was analyzed by the method of
Chou and Fasman(32) . As shown in Fig. 6B, the
three cysteines are located at the turning region of the protein.
Figure 8: PotA protein in total and inner membrane proteins. PotA protein was analyzed by Western blotting. Total protein (A) and inner membrane protein from inside-out membrane vesicles (B) were prepared from E. coli JM105/pKKpotA (lane 1) and E. coli JM105/pKKpotABC (lane 2), respectively.
The spermidine-preferential uptake system belongs to periplasmic active transport systems (permeases), which consist of one periplasmic substrate-binding protein and three membrane-bound components(7) . One of the membrane components is a nucleotide-binding protein involved in energy supply. A model for the structure of the nucleotide-binding protein (HisP) in the histidine transport system was proposed by analogy to the adenylate kinase structure(33) . It was shown that site A (Gly-X-X-Gly-X-Gly-Lys) of consensus amino acid sequences for nucleotide binding is important for ATP hydrolysis. Furthermore, MalK protein, the nucleotide-binding protein in the maltose transport system, has been purified to homogeneity, and it was reported that the specific activity of MalK protein as ATPase was approximately 130 nmol/min/mg of protein(34) . The specific activity of PotA protein as ATPase was 396 nmol/min/mg of protein, higher than that of MalK protein, and the activity was inhibited by NEM. We found that cysteine 54, located in site A (GPSGCGKT), is involved in ATP hydrolysis. This supports the idea that site A is important for ATP hydrolysis(33) . We found that valine 135 is also located in the active center of ATPase in PotA protein. Replacement of proline 172 by threonine in site B (Pro-X-Val(Leu)-Leu-Leu-X-Asp-Glu) of the consensus amino acid sequences for nucleotide binding in HisP protein-stimulated ATPase activity(35, 36) . These results indicate that the active sites of ATP hydrolysis of the nucleotide-binding protein in periplasmic active transport systems are located both within and between the two consensus amino acid sequences for nucleotide binding.
In MalK protein, cysteine is located at the equivalent position of cysteine 54 of PotA protein(37) . When this cysteine was replaced by glycine, the transport activity did not change significantly(38) . This suggests that cysteine may not necessarily be essential, but still be important for the ATPase and transport activities. When ATPase activities of HisP and MalK were measured using proteoliposomes equivalent to rightside-out membrane vesicles, the existence of substrate and substrate-binding protein in proteoliposomes was essential for the ATPase activity(39, 40) . Structure of the nucleotide-binding protein in inside-out membrane vesicles or in free form may be different from that in proteoliposomes.
We also found that cysteine
26 was photoaffinity-labeled by 8-azido-ATP. Cysteine 26 is the 24th
amino acid from site A (GPSGCGKT) (Fig. 6A). It has
been reported that histidine 19 of HisP protein is
photoaffinity-labeled by 8-azido-ATP (28) and is the 21st amino
acid from site A. The results suggest that the adenine portion of ATP
interacts with a domain close to the NH-terminal end of the
nucleotide-binding protein in periplasmic active transport systems.
When cysteine 26 of PotA protein was replaced by alanine, the mutated
PotA protein was still photoaffinity-labeled with 8-azido-ATP.
Threonine 39 in the mutated PotA protein might be photoaffinity-labeled
by 8-azido-ATP instead of cysteine 26, since it has been reported that
serine 41 in HisP protein was also photoaffinity-labeled in addition to
histidine 19 in the presence of 5 mM Ca
and
10 mM Mg
(28) . Thus, the pocket for
ATP on the nucleotide-binding protein in periplasmic active transport
systems may be wide. The results taken together indicate that the
center of ATPase activity is located in the NH
-terminal
portion in PotA protein. In fact, the NH
-terminal peptide
consisting of 239 amino acids of PotA protein showed ATPase activity. (
)
It has been reported that the interaction of HisP protein with membranes was enhanced by HisQ and HisM membrane proteins(41) . We also found that the association of PotA protein with membranes was strengthened by the existence of PotB and PotC channel forming proteins. When the secondary structures of PotB and PotC proteins were compared, common amino acid sequences, LEAAR(K)DLGAS, were observed in the hydrophilic region(8) . These sequences may be involved in the interaction with PotA protein. It has been reported that a similar amino acid sequence has been found in a hydrophilic loop of channel forming membrane proteins in periplasmic active transport systems(42) .