Quantification, organ-specific accumulation and intracellular localization of type II H(+)-pyrophosphatase in Arabidopsis thaliana.
Most plants have two types of H(+)-translocating inorganic pyrophosphatases (H(+)-PPases), I and II, which differ in primary sequence and K(+) dependence of enzyme function. Arabidopsis thaliana has three genes for H(+)-PPases: one for type I and two for type II. The type I H(+)-PPase requires K(+) for maximal enzyme activity and functions together with H(+)-ATPase in vacuolar membranes. The physiological role of the type II enzyme, which does not require K(+), is not clear. We focused on the type II enzymes (AtVHP2;1 and AtVHP2;2) of A. thaliana. Total amounts of AtVHP2s were quantified immunochemically using a specific antibody and determined to be 22 and 12 ng mg(-1) of total protein in the microsomal fractions of suspension-cultured cells and young roots, respectively, and the values are approximately 0.1 and 0.2%, respectively, of the vacuolar H(+)-PPase. In plants, AtVHP2s were detected immunochemically in all tissues except mature leaves, and were abundant in roots and flowers. The intracellular localization of AtVHP2s in suspension cells was determined by sucrose density gradient centrifugation and immunoblotting. Comparison with a number of marker proteins revealed localization in the Golgi apparatus and the trans-Golgi network. These results suggest that the type II H(+)-PPase functions as a proton pump in the Golgi and related vesicles in young tissues, although its content is very low compared with the type I enzyme.
Segami
Shoji
S
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Nakanishi
Yoichi
Y
Sato
Masa H
MH
Maeshima
Masayoshi
M
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D000939:Epitopes / Q000378:metabolism; D043564:Inorganic Pyrophosphatase / Q000378:metabolism*; D008969:Molecular Sequence Data; D021601:trans-Golgi Network / Q000378:metabolism*
D029681:Arabidopsis Proteins; D000939:Epitopes; D043564:Inorganic Pyrophosphatase