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

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

Dual role for ubiquitin in plant steroid hormone receptor endocytosis

Brassinosteroids are plant steroid hormones that control many aspects of plant growth and development, and are perceived at the cell surface by the plasma membrane-localized receptor kinase BRI1. Here we show that BRI1 is post-translationally modified by K63 polyubiquitin chains in vivo. Using both artificial ubiquitination of BRI1 and generation of an ubiquitination-defective BRI1 mutant form, we demonstrate that ubiquitination promotes BRI1 internalization from the cell surface and is essential for its recognition at the trans-Golgi network/early endosomes (TGN/EE) for vacuolar targeting. Finally, we demonstrate that the control of BRI1 protein dynamics by ubiquitination is an important control mechanism for brassinosteroid responses in plants. Altogether, our results identify ubiquitination and K63-linked polyubiquitin chain formation as a dual targeting signal for BRI1 internalization and sorting along the endocytic pathway, and highlight its role in hormonally controlled plant development

Inhibition of very long acyl chain sphingolipid synthesis modifies membrane dynamics during plant cytokinesis

Plant cytokinesis requires intense membrane trafficking and remodeling to form a specific membrane structure, the cell plate that will ultimately separate the daughter cells. The nature and the role of lipids involved in the formation of the cell plate remain unclear. Plant membranes are particularly rich in sphingolipids such as glucosylceramides with long (16 carbons) or very long (24 carbons) acyl chains. We reveal here that inhibition of the synthesis of sphingolipids with very long acyl chains induces defective cell plates with persistent vesicular structures and large gaps. Golgi-derived vesicles carrying material toward the cell plate display longer vesicle-vesicle contact time and their cargos accumulate at the cell plate, suggesting membrane fusion and/or recycling defects. In vitro fusion experiments between artificial vesicles show that glycosphingolipids with very long acyl chains stimulate lipid bilayer fusion. Therefore we propose that the very long acyl chains of sphingolipids are essential structural determinants for vesicle dynamics and membrane fusion during cytokinesis