The V-ATPase from etiolated barley (Hordeum vulgare L.) shoots is activated by blue light and interacts with 14-3-3 proteins.
The vacuolar H(+)-ATPase (V-ATPase) is a key enzyme that controls the electrochemical proton potential across endomembranes. Although evidence suggests that V-ATPase is important for photo-morphogenesis, little is known about short-term regulation of V-ATPase upon initiation of the photo-morphogenetic programme by exposure of dark-grown plants to light. In this study, etiolated coleoptiles were given a short blue light treatment and V-ATPase characteristics were determined. The effectiveness of the light treatment was assessed by means of fusicoccin binding to the plasma membrane; this increased 5-fold. The short light treatment also induced a 2-fold to 3-fold increase in the hydrolytic activity of V-ATPase. Members of the 14-3-3 protein family are involved in both blue light perception and the subsequent activation of the P-type ATPase. We provide evidence that 14-3-3 proteins specifically interact with the catalytic A-subunit of the V-ATPase. First, the isolated V1-part of the V-ATPase co-purifies with 14-3-3 on a gel filtration column. Secondly, in an overlay experiment, 14-3-3 interacts with a 68 kDa band that was identified as the V1 A-subunit by mass spectrometry. Thirdly, in 14-3-3 affinity chromatography, both A- and B-subunits of the catalytic moiety of the V-ATPase were identified by matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI TOF/TOF MS) as 14-3-3-interacting proteins. It was shown that the A-subunit can be phosphorylated in vitro by a tonoplast-bound kinase, whose properties are affected by blue light. Taken together, the data show that besides the P- and F-type H(+)-ATPases, the V-type H(+)-ATPase also interacts with 14-3-3 proteins.
Klychnikov
O I
OI
The 14-3-3 Biology Group, Department of Structural Biology, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.
Li
K W
KW
Lill
H
H
de Boer
A H
AH
D048948:14-3-3 Proteins / Q000378:metabolism*; D003116:Color; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D001467:Hordeum / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects*; D008027:Light; D010940:Plant Proteins / Q000378:metabolism*; D018520:Plant Shoots / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects*; D011485:Protein Binding; D021122:Protein Subunits; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
D048948:14-3-3 Proteins; D010940:Plant Proteins; D021122:Protein Subunits; D025262:Vacuolar Proton-Translocating ATPases