Seventeen a-subunit isoforms of paramecium V-ATPase provide high specialization in localization and function.
In the Paramecium tetraurelia genome, 17 genes encoding the 100-kDa-subunit (a-subunit) of the vacuolar-proton-ATPase were identified, representing by far the largest number of a-subunit genes encountered in any organism investigated so far. They group into nine clusters, eight pairs with >82% amino acid identity and one single gene. Green fluorescent protein-tagging of representatives of the nine clusters revealed highly specific targeting to at least seven different compartments, among them dense core secretory vesicles (trichocysts), the contractile vacuole complex, and phagosomes. RNA interference for two pairs confirmed their functional specialization in their target compartments: silencing of the trichocyst-specific form affected this secretory pathway, whereas silencing of the contractile vacuole complex-specific form altered organelle structure and functioning. The construction of chimeras between selected a-subunits surprisingly revealed the targeting signal to be located in the C terminus of the protein, in contrast with the N-terminal targeting signal of the a-subunit in yeast. Interestingly, some chimeras provoked deleterious effects, locally in their target compartment, or remotely, in the compartment whose specific a-subunit N terminus was used in the chimera.
Wassmer
Thomas
T
Centre de Génétique Moleculaire, Centre National de la Recherche Scientifique, F-91198 Gif-sur-Yvette Cedex, France. thomas.wassmer@uni-konstanz.de
Kissmehl
Roland
R
Cohen
Jean
J
Plattner
Helmut
H
D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D003571:Cytochalasin B / Q000494:pharmacology; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000032:analysis; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016805:Paramecium tetraurelia / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D010588:Phagosomes / Q000187:drug effects; D010802:Phylogeny; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000502:physiology; D021122:Protein Subunits / Q000032:analysis / Q000235:genetics / Q000502:physiology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000032:analysis; D022142:Secretory Vesicles / Q000502:physiology; D025262:Vacuolar Proton-Translocating ATPases / Q000032:analysis / Q000235:genetics / Q000502:physiology*
D020033:Protein Isoforms; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003571:Cytochalasin B; D025262:Vacuolar Proton-Translocating ATPases