ND9P, a novel protein with armadillo-like repeats involved in exocytosis: physiological studies using allelic mutants in paramecium.
In Paramecium, a number of mutants affected in the exocytotic membrane fusion step of the regulated secretory pathway have been obtained. Here, we report the isolation of one of the corresponding genes, ND9, previously suspected to encode a soluble protein interacting with both plasma and trichocyst membranes. Nd9p is a novel polypeptide that contains C-terminal Armadillo-like repeats. Point mutations were found in the first N-terminal quarter of the molecule and in the last putative Armadillo repeat, respectively, for the two thermosensitive mutants, nd9-1 and nd9-2. The different behaviors of these mutants in recovery experiments upon temperature shifts suggest that the N-terminal domain of the molecule may be involved in membrane binding activity, whereas the C-terminal domain is a candidate for protein-protein interactions. The nonsense nd9-3 mutation that produces a short N-terminal peptide has a dominant negative effect on the nd9-1 allele. We show here that, when overexpressed, the dominant negative effect can be produced even on the wild-type allele, suggesting competition for a common target. We suggest that Nd9p could act, like some SNARE proteins, at the membrane-cytosol interface to promote membrane fusion.
Froissard
M
M
Centre de Génétique Moléculaire, CNRS, Av. de le Terrasse, 91198 Gif-sur-Yvette Cedex, France.
Keller
A M
AM
Cohen
J
J
D000465:Algorithms; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D051186:Armadillo Domain Proteins; D015152:Blotting, Northern; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D002569:Cerulenin / Q000494:pharmacology; D003001:Cloning, Molecular; D018389:Codon, Nonsense; D003600:Cytosol / Q000378:metabolism; D029721:Drosophila Proteins; D005089:Exocytosis / Q000235:genetics*; D020868:Gene Silencing; D005799:Genes, Dominant; D005816:Genetic Complementation Test; D015151:Immunoblotting; D019476:Insect Proteins / Q000235:genetics*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010247:Paramecium / Q000235:genetics*; D010641:Phenotype; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins; D011691:Puromycin / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D015534:Trans-Activators
D051186:Armadillo Domain Proteins; D002352:Carrier Proteins; D018389:Codon, Nonsense; D029721:Drosophila Proteins; D019476:Insect Proteins; D047908:Intracellular Signaling Peptides and Proteins; C425766:ND9 protein, Paramecium tetraurelia; D015800:Protozoan Proteins; D015534:Trans-Activators; D002569:Cerulenin; D011691:Puromycin; D012313:RNA