Reconstitution in vitro of the catalytic portion (NtpA3-B3-D-G complex) of Enterococcus hirae V-type Na+-ATPase.
Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V(1); NtpA(3)-B(3)-D-G) and an integral membrane domain (V(0); NtpI-K(10)) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V(1) portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 microM for ADP or 3.1 microM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA(3)-B(3) heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA(3)-B(3) forming V(1) (NtpA(3)-B(3)-D-G) complex independent of nucleotides. The V(1) formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V(1) complex was as high as that of native V(1)-ATPase purified from the V(0)V(1) complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V(1) complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V(1)-ATPase complex.
Arai
Satoshi
S
Department of Biological Science and Technology, Tokyo University of Science, Noda-shi, Chiba 278-8510, Japan.
Yamato
Ichiro
I
Shiokawa
Asuka
A
Saijo
Shinya
S
Kakinuma
Yoshimi
Y
Ishizuka-Katsura
Yoshiko
Y
Toyama
Mitsutoshi
M
Terada
Takaho
T
Shirouzu
Mikako
M
Yokoyama
Shigeyuki
S
Iwata
So
S
Murata
Takeshi
T
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020134:Catalytic Domain; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry*; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D009711:Nucleotides; D021122:Protein Subunits; D000251:Adenosine Triphosphatases; C500332:V-type sodium ATPase, Enterococcus hirae