Research article

Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex

Christoph Kluge^1,2 , Thorsten Seidel¹ , Susanne Bolte² , Shanti S Sharma^1,3 , Miriam Hanitzsch¹ , Beatrice Satiat-Jeunemaitre² , Joachim Roß⁴ , Markus Sauer⁴ , Dortje Golldack¹ and Karl-Josef Dietz¹

¹Biochemistry and Physiology of Plants – W5, University of Bielefeld, Bielefeld, 33501, Germany

²CNRS, UPR 2355, Institut des Sciences du Végétale, Avenue de la terrasse, Gif Sur Yvette, 91198, France

³Department of Biosciences, H. P. University, Shimla, 171 005, India

⁴Applied Laser Physics and Laser Spectroscopy – D3, University of Bielefeld, Bielefeld, 33501, Germany

author email corresponding author email

BMC Cell Biology 2004, 5:29doi:10.1186/1471-2121-5-29

Published:	13 August 2004

Abstract

Background

Vacuolar H⁺-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed.

Results

To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells.

Conclusions

All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H⁺-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.