HAP2(GCS1)-Dependent Gamete Fusion Requires a Positively Charged Carboxy-Terminal Domain

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, United States of America.
PLoS Genetics (Impact Factor: 7.53). 03/2010; 6(3):e1000882. DOI: 10.1371/journal.pgen.1000882
Source: PubMed


Author Summary
Recent studies suggest that HAP2(GCS1) is a deeply conserved protein required for gamete membrane fusion, a critical yet poorly understood step in sexual reproduction. HAP2(GCS1) is present in many plant, protist, and animal genomes, and has been shown to be essential for fertilization in Arabidopsis, Chlamydomonas, and Plasmodium. The loss-of-function phenotype in Chlamydomonas suggests a direct role in gamete plasma membrane fusion. HAP2(GCS1) has no known functional domains, making it difficult to predict how it contributes to gamete fusion. We set out to map the critical features of this protein by testing a series of deletions, substitutions, and interspecific chimeras for their ability to rescue the hap2-1 fertilization defect in Arabidopsis. We found that the N-terminus does not tolerate sequence divergence, but the histidine-rich C-terminus does. We propose that the N-terminus of HAP2(GCS1) functions in part by interacting with proteins on the surface of female gametes. The key feature of the C-terminus is positive charge, a characteristic that could favor interactions with the plasma membrane that promote membrane fusion. Our studies provide a description of HAP2(GCS1) functional domains and provide an important framework for defining the role of this essential component of a conserved reproductive mechanism.

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Available from: Alexander R Leydon, Aug 11, 2014
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    • "It is possible that the truncated GCS1 protein with extracellular and transmembrane domains is responsible for this occasional fertilization, which is suggested by the expression of gcs1 transcript upstream of the T-DNA (Fig. 2c). This is supported by the observation that modified GCS1 variants rescued the gcs1/hap2 phenotype at different levels (Mori et al. 2010; Wong et al. 2010). To examine the possibility that the partial function of the truncated GCS1 protein is responsible for the low-frequency fertilization, analysis using other alleles, such as hap2 allele (von Besser et al. 2006), would provide insights into this possibility. "
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    ABSTRACT: Key message New gametic homozygous mutants. In angiosperms, a haploid male gamete (sperm cell) fuses with a haploid female gamete (egg cell) during fertilization to form a zygote carrying paternally and maternally derived chromosomes. Several fertilization-defective mutants in Arabidopsis thaliana, including a generative cell-specific 1 (gcs1)/hapless 2 mutant, the sperm cells of which are unable to fuse with female gametes, can only be maintained as heterozygous lines due to the infertile male or female gametes. Here, we report successful generation of a gcs1 homozygous mutant by heat-inducible removal of the GCS1 transgene. Using the gcs1 homozygous mutant as male, the defect in gamete fusion was observed with great frequency; in our direct observation by semi-in vivo fertilization assay using ovules, 100 % of discharged sperm cells in culture failed to show gamete fusion. More than 70 % of ovules in the pistil received a second pollen tube as attempted fertilization recovery. Moreover, gcs1 mutant sperm cells could fertilize female gametes at a low frequency in the pistil. This strategy to generate homozygous fertilization-defective mutants will facilitate novel approaches in plant reproduction research. Electronic supplementary material The online version of this article (doi:10.1007/s00497-015-0256-4) contains supplementary material, which is available to authorized users.
    Full-text · Article · Feb 2015 · Plant Reproduction
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    • "The results showed that the C-terminal domain of GCS1 is not essential for gamete fusion but that the highly conserved N-terminal region is indispensable for gamete fusion [15], although there is another report showing that the C-terminal region of GCS1 may also participate in gamete fusion [16]. Importance of the N-terminal region and unnecessariness of the C-terminal region were also clarified in Plasmodium [15]. "
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    ABSTRACT: It has been reported that GCS1 (Generative Cell Specific 1) is a transmembrane protein that is exclusively expressed in sperm cells and is essential for gamete fusion in flowering plants. The GCS1 gene is present not only in angiosperms but also in unicellular organisms and animals, implying the occurrence of a common or ancestral mechanism of GCS1-mediated gamete fusion. In order to elucidate the common mechanism, we investigated the role of GCS1 in animal fertilization using a sea anemone (Cnidaria), Nematostella vectensis. Although the existence of the GCS1 gene in N. vectensis has been reported, the expression of GCS1 in sperm and the role of GCS1 in fertilization are not known. In this study, we showed that the GCS1 gene is expressed in the testis and that GCS1 protein exists in sperm by in situ hybridization and proteomic analysis, respectively. Then we made four peptide antibodies against the N-terminal extracellular region of NvGCS1. These antibodies specifically reacted to NvGCS1 among sperm proteins on the basis of Western analysis and potently inhibited fertilization in a concentration-dependent manner. These results indicate that sperm GCS1 plays a pivotal role in fertilization, most probably in sperm-egg fusion, in a starlet sea anemone, suggesting a common gamete-fusion mechanism shared by eukaryotic organisms.
    Preview · Article · Sep 2014 · Biochemical and Biophysical Research Communications
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    • "In addition, the N-terminus is a domain that is critical in fertilization by interaction with antigens expressed on female gamete, while the C-terminus with positive charge may function through electrostatic interactions with the microgamete plasma membrane (Wong et al., 2010). A short amino acid sequence of about 50 amino acids in the middle of Nterminus , namely HAP2-GCS1 domain, is highly conserved in this protein (Wong et al., 2010). In 2010, Mori et al. showed that although the C-terminal sequence of GCS1 protein is not necessary for gamete fusion, the N-terminus of that is sufficient for gamete fusion, and HAP2-GCS1 domain is critical to successful fertilization in P. berghei. "
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    ABSTRACT: Among vaccines, those that have an impact on transmission are in priority for malaria elimination and eradication. One of the new identified transmission-blocking vaccine (TBV) candidate antigens is Generative Cell Specific 1 (GCS1) located on the male gametocytes of plasmodium species. Since the antigenic diversity could hamper vaccine development, it is essential to determine the gene diversity of gcs1 in global malaria-endemic areas in order to develop efficient TBVs. Therefore, in this study, nucleotide diversity and selection in the P. falciparum GCS1 (PfGCS1) antigen were analyzed in 36 Iranian clinical isolates by using PCR sequencing in order to provide useful information on this TBV candidate antigen. For this purpose, successful sequence analysis was carried out in 36 isolates. The results showed three single-nucleotide polymorphisms including one synonymous (G1475A) and two non-synonymous (A697G and G1479A) mutations leading to 3 distinct haplotypes with different frequencies: GCS1-A (N184/D445, 16.7%), GCS1-B (S184/D445, 63.9%), and GCS1-C (N184/N445, 19.4%). The overall nucleotide diversity (π) for all 36 sequences of Iranian pfgcs1 was 0.00066 ± 0.00012, and the dN-dS value (-0.00028) was negative, suggesting the possible action of purifying selection in this gene. Epitope mapping prediction of PfGCS1 antigen showed that most of the potential linear and conformational B-cell epitopes are located in conserved regions. However, N184S and D445N mutations were also involved in linear and conformational B-cell epitopes, respectively that should be considered in vaccine design. In conclusion, the present study showed a very low genetic diversity of pfgcs1 gene among Iranian isolates. Considering PfGCS1 as a conserved TBV candidate, our data provides valuable information to develop a PfGCS1-based TBV.
    Full-text · Article · Jan 2014 · Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases
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