Nd6p, a novel protein with RCC1-like domains involved in exocytosis in Paramecium tetraurelia

Abstract

In Paramecium tetraurelia, the regulated secretory pathway of dense core granules called trichocysts can be altered by mutation and genetically studied. Seventeen nondischarge (ND) genes controlling exocytosis have already been identified by a genetic approach. The site of action of the studied mutations is one of the three compartments, the cytosol, trichocyst, or plasma membrane. The only ND genes cloned to date correspond to mutants affected in the cytosol or in the trichocyst compartment. In this work, we investigated a representative of the third compartment, the plasma membrane, by cloning the ND6 gene. This gene encodes a 1,925-amino-acid protein containing two domains homologous to the regulator of chromosome condensation 1 (RCC1). In parallel, 10 new alleles of the ND6 gene were isolated. Nine of the 12 available mutations mapped in the RCC1-like domains, showing their importance for the Nd6 protein (Nd6p) function. The RCC1 protein is well known for its guanine exchange factor activity towards the small GTPase Ran but also for its involvement in membrane fusion during nuclear envelope assembly. Other proteins with RCC1-like domains are also involved in intracellular membrane fusion, but none has been described yet as involved in exocytosis. The case of Nd6p is thus the first report of such a protein with a documented role in exocytosis.

FIG. 1.

Localization and secretion of trichocysts: the dense core granules of Paramecium. (Left) Phase-contrast image of a living wild-type Paramecium. (Top right) Enlargement of part of this cell showing docked trichocysts under the plasma membrane. (Bottom right) Phase-contrast image of a fixed cell after stimulation of exocytosis by a fixing secretagogue (tannic acid, 0.5%). The contents of the secretory granules expand in contact with external media, allowing easy observation of the secretion. Scale bars, 15 μm.

FIG.2.

Alignment of Nd6 proteins of Paramecium and Tetrahymena. Alignment of the Nd6 homologues of Tetrahymena thermophila (Nd6_Tt1 and Nd6_Tt2) with Nd6 of Paramecium tetraurelia (Nd6_Pt). This alignment was realized using the MultAlin program (http://prodes.toulouse.inra.fr/multalin/multalin.html). Nd6_Pt shares 27% identity and 44% similarity with Nd6_Tt1 and 25% identity and 42% similarity with Nd6_Tt2. Nd6_Tt1 and Nd6_Tt2 share 33% identity and 52% similarity with each other.

FIG. 3.

RCC1 repeats of Nd6p. Alignment of the RCC1 repeats of the human RCC1 protein and the two RLDs of Paramecium tetraurelia (Pt) and Tetrahymena thermophila (Tt) Nd6 proteins. The first line (consensus) represents the simplified structure of RCC1 repeats, where arrows represent β-sheets and A, B, C, and D represent the common names of these β-sheets. Φ, hydrophobic residue. Identities are shown in red, and similarities are shown in blue. In the RCC1 sequence, residues implicated in the Ran-RCC1 interaction have a green background and residues provoking a decrease of k_cat (the overall catalytic rate of the enzyme) and K_m (the concentration of substrate at which the rate of the reaction is one-half of the maximal rate) when mutated are shown, respectively, with a magenta and a gray background (42, 43). In the Nd6p sequence, the positions of the mutations are shown with a cyan background. All these mutations affect conserved residues, suggesting that these amino acids are essential for the structure of the peptide. The residue affected in the nd6-8 mutant (G680E in repeat 10) is at a position corresponding to a mutation in human RCC1 affecting its affinity for Ran, indicating that this residue in Nd6p could also be essential for interaction with a small GTPase.

FIG. 4.

Effects of gene silencing on exocytosis. The symbols +, ɛ, and - indicate the mean exocytotic capacities of silenced populations. +, between 500 and 1,000 trichocysts discharged per cell; ɛ, between 1 and 50 trichocysts discharged; -, no trichocysts discharged. (Left) Paramecia fed with bacteria carrying the L4440 vector without insertion. (Right) ND6 silencing. One day after feeding paramecia with double-strand ND6 RNA-producing bacteria, we can see an alteration of the exocytotic capacity compared to that of control cells fed with nontransformed HT115 bacteria (day 2). The phenotype is stronger on day 3.

FIG. 5.

Monitoring the effect of ND6 silencing on exocytosis. (A) Exocytosis of a wild-type (WT) cell expressing the trichocyst matrix protein 1 fused to GFP (TMP1-GFP) (62) after treatment with a fixing secretagogue solution (2% paraformaldehyde, 1% AED, 1 mM CaCl₂, 10 mM Tris, pH 8.5). (B) The ND6-silenced cell is unable to perform exocytosis. The trichocysts, labeled with TMP1-GFP, are blocked inside the cell under the plasma membrane (arrows). Scale bars, 20 μm.

FIG. 6.

Mutations in the ND6 gene. Overall organization of the 1,925-amino-acid-long Nd6 protein. Gray boxes represent the 14 repeats of the RCC1-like domains, called RLD1 and RLD2. The localization and the nature of the mutations are indicated by the allele numbers.