Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells
| Autor: | Miriam Hanitzsch, Karl-Josef Dietz, Christoph Kluge, Dortje Golldack, Joachim Ross, Markus Sauer, Thorsten Seidel |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Yellow fluorescent protein
Arabidopsis thaliana Protein subunit Recombinant Fusion Proteins Arabidopsis plant V-ATPase Bioengineering Applied Microbiology and Biotechnology colocalization Fluorescence microscope Fluorescence Resonance Energy Transfer health care economics and organizations Plant Proteins Mesembryanthemum biology Endoplasmic reticulum Protoplasts fungi Mesembryanthemum crystallinum Colocalization General Medicine biology.organism_classification Plants Genetically Modified VHA-a Protein Subunits Proton-Translocating ATPases Förster resonance energy transfer Ion homeostasis Biochemistry Vacuoles FRET VHA-c biology.protein Biophysics Biotechnology |
| Zdroj: | Journal of biotechnology. 112(1-2) |
| ISSN: | 0168-1656 |
| Popis: | The proton-translocating plant vacuolar H+-ATPase (VHA) is of prime importance for acidification of intracellular compartments and is essential for processes such as secondary activated transport, maintenance of ion homeostasis, and adaptation to environmental stress. Twelve genes have been identified that encode subunits of the functional V-ATPase complex. In this study, subunits c and a of the V-ATPase from the plant Mesembryanthemum crystallinum were fused to cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and were transiently coexpressed in protoplasts. Two-colour scanning confocal fluorescence microscopy demonstrates that the fusion proteins VHA-c-CFP and VHA-a-YFP are colocalized at the tonoplast, the plasmamembrane, and at endoplasmic membrane structures indicating expression in cytoplasmic vesicles. Furthermore, fluorescence resonance energy transfer (FRET) was used to visualize the interaction of VHA-c and VHA-a in vivo on the nanometer length scale. Excitation of CFP as donor fluorophore caused increased emission of YFP-fluorescence in protoplasts due to FRET. Our results give strong evidence for physical interaction of subunits c and a in living plant cells. (C) 2004 Elsevier B.V. All rights reserved. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect