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Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex
- Source :
- BMC Cell Biology, Vol 5, Iss 1, p 29 (2004), BMC Cell Biology, BMC Cell Biology, BioMed Central, 2004, 5, pp.29. ⟨10.1186/1471-2121-5-29⟩
- Publication Year :
- 2004
- Publisher :
- BMC, 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.
- Subjects :
- MESH: Fluorescence Resonance Energy Transfer
MESH: Sequence Homology, Amino Acid
MESH: Trans
Arabidopsis
MESH: Onions
MESH: Plant Roots
Caryophyllaceae
MESH: Amino Acid Sequence
MESH: Protein Isoforms
Endoplasmic Reticulum
Plant Roots
Polymerase Chain Reaction
Epitopes
MESH: Protein Structure, Tertiary
Onions
Fluorescence Resonance Energy Transfer
Protein Isoforms
MESH: Arabidopsis
health care economics and organizations
Plant Proteins
MESH: Plant Proteins
lcsh:Cytology
Protoplasts
MESH: Protoplasts
MESH: Protein Subunits
Immunohistochemistry
MESH: Saccharomyces cerevisiae
MESH: Plant Leaves
MESH: Membrane Proteins
Subcellular Fractions
Research Article
Vacuolar Proton-Translocating ATPases
DNA, Complementary
MESH: Epitopes
Recombinant Fusion Proteins
Molecular Sequence Data
MESH: Sequence Alignment
Saccharomyces cerevisiae
Transfection
Zea mays
MESH: Endoplasmic Reticulum
MESH: Recombinant Fusion Proteins
[SDV.BV]Life Sciences [q-bio]/Vegetal Biology
Amino Acid Sequence
lcsh:QH573-671
MESH: Molecular Sequence Data
Sequence Homology, Amino Acid
Membrane Proteins
MESH: Polymerase Chain Reaction
MESH: Immunohistochemistry
MESH: DNA, Complementary
Protein Structure, Tertiary
Plant Leaves
Protein Subunits
MESH: Subcellular Fractions
MESH: Caryophyllaceae
Sequence Alignment
Subjects
Details
- Language :
- English
- ISSN :
- 14712121
- Volume :
- 5
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- BMC Cell Biology
- Accession number :
- edsair.pmid.dedup....ba90d7129dcafcd8f784bed6c9f6e52c
- Full Text :
- https://doi.org/10.1186/1471-2121-5-29⟩