Cloning and sequencing of V-ATPase subunit d from mung bean and its function in passive proton transport.
We have previously shown that vacuolar H+-ATPase subcomplex V(o) from mung bean contains subunit d, however, its sequence and function were unknown. In the present study, we report the cloning and recombinant over expression of subunit d from mung bean in E. coli. To study the function of subunit d, two vacuolar H+-ATPase subcomplexes V(o) from mung bean were purified-one containing subunits a and c(c',c") and the other containing subunits a, c(c',c") and d. After reconstitution of the purified V(o) subcomplexes into liposomes, the proton translocation was studied. Our results show that the V(o) subcomplex in the absence of subunit d is a passive proton channel, while the V(o) subcomplex in the presence of the subunit d is not. Taken together, our data supports the conclusion that the subunit d of the plant vacuolar H(+)-ATPase from mung bean is positioned at the central stalk and involved in the proton translocation across the tonoplast membrane.
Ouyang
Zhuqing
Z
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101, Beijing, China.
Li
Zhuo
Z
Zhang
Xujia
X
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004058:Diffusion; D007887:Fabaceae / Q000201:enzymology*; D008969:Molecular Sequence Data; D021122:Protein Subunits; D011522:Protons; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D021122:Protein Subunits; D011522:Protons; D025262:Vacuolar Proton-Translocating ATPases