JXB Advance Access originally published online on December 21, 2006
Journal of Experimental Botany 2007 58(5):1013-1023; doi:10.1093/jxb/erl261
RESEARCH PAPER |
The V-ATPase from etiolated barley (Hordeum vulgare L.) shoots is activated by blue light and interacts with 14-3-3 proteins
1The 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
2Department of Molecular and Cellular Neurobiology, Center of Neurogenomics and Cognitive Research, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
3Department of Structural Biology, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam, The Netherlands
* To whom correspondence should be addressed. E-mail: bert.de.boer{at}falw.vu.nl
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.
Key words: 14-3-3 interaction, blue light, phosphorylation, V-ATPase
Received 1 September 2006; Revised 23 October 2006 Accepted 11 November 2006