RESUMO
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.
Assuntos
Caryophyllaceae/citologia , Proteínas de Plantas/análise , Frações Subcelulares/enzimologia , ATPases Vacuolares Próton-Translocadoras/análise , Sequência de Aminoácidos , Arabidopsis , Caryophyllaceae/enzimologia , Caryophyllaceae/genética , DNA Complementar/genética , Retículo Endoplasmático/enzimologia , Epitopos/análise , Transferência Ressonante de Energia de Fluorescência , Imuno-Histoquímica , Proteínas de Membrana/análise , Dados de Sequência Molecular , Cebolas/citologia , Folhas de Planta/citologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Raízes de Plantas/citologia , Raízes de Plantas/enzimologia , Reação em Cadeia da Polimerase , Isoformas de Proteínas/análise , Estrutura Terciária de Proteína , Subunidades Proteicas , Protoplastos , Proteínas Recombinantes de Fusão/análise , Saccharomyces cerevisiae , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/imunologia , Zea mays/citologia , Zea mays/enzimologiaRESUMO
From the ice plant, Mesembryanthemum crystallinum, McHKT1 was isolated encoding a protein 41-61% identical to other plant HKT1-like sequences previously described as potassium or sodium/potassium transporters. McHKT1 acts as a potassium transporter in yeast with specificity similar to that of wheat HKT1. In Xenopus oocytes it transports cations with a specificity Rb+ > Cs+ > [K+ = Na+ = Li+]. McHKT1 is exclusively localized to the plasma membrane. The isoform isolated is most highly expressed in leaves and is present in stems, flowers and seed pods but absent from the root where, according to immunological data, a second isoform exists which does not cross-hybridize with the leaf form in RNA blots at high stringency. McHKT1 transcript amounts increase during the first 6-10 h of stress and then decline to pre-stress levels with kinetics reminiscent of the initial influx of sodium into this halophyte. Immunocytological localization showed strong signals in the leaf vasculature and surrounding mesophyll cells but low-intensity signals are also detected in other cell types. In roots, McHKT is mainly confined to endodermis and stele. Possible functions of McHKT1 in ion homeostasis in the halophytic ice plant are discussed.