The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes

Abstract

The cellular and molecular mechanisms that underlie species-specific membrane fusion between male and female gametes remain largely unknown. Here, by use of gene discovery methods in the green alga Chlamydomonas, gene disruption in the rodent malaria parasite Plasmodium berghei, and distinctive features of fertilization in both organisms, we report discovery of a mechanism that accounts for a conserved protein required for gamete fusion. A screen for fusion mutants in Chlamydomonas identified a homolog of HAP2, an Arabidopsis sterility gene. Moreover, HAP2 disruption in Plasmodium blocked fertilization and thereby mosquito transmission of malaria. HAP2 localizes at the fusion site of Chlamydomonas minus gametes, yet Chlamydomonas minus and Plasmodium hap2 male gametes retain the ability, using other, species-limited proteins, to form tight prefusion membrane attachments with their respective gamete partners. Membrane dye experiments show that HAP2 is essential for membrane merger. Thus, in two distantly related eukaryotes, species-limited proteins govern access to a conserved protein essential for membrane fusion.[Keywords: Gamete fusion; cell-cell fusion; malaria; HAP2; Chlamydomonas, Plasmodium] Supplemental material is available at http://www.genesdev.org. Received January 28, 2008; revised version accepted February 22, 2008. Fusion of gametes of opposite sex (or mating type) to form a zygote is the defining moment in the life of a eukaryote. In the first phase of gamete interactions, cell adhesion molecules displayed on the surfaces of the gametes bring the two cells together. In animals, the sperm plasma membrane binds to the extracellular matrix of the egg (the zona pellucida in mammals and the jelly coat in many invertebrates). The interacting gametes use this first-phase adhesion step not only to bind to each other, but also to initiate a signal transduction cascade that activates the sperm and exposes new, fusogenic regions of the sperm plasma membrane. In the second phase of fertilization, the membrane fusion reaction, the plasma membranes of the two gametes come into intimate contact and then fuse, bringing about cytoplasmic continuity (Primakoff and Myles 2002;Rubinstein et al. 2006). Although these two steps-prefusion attachment of the plasma membranes of gametes and merger of their lipid bilayers-have been experimentally separated using in vitro bioassays, gene disruption studies to date have failed to distinguish the two, and no genes have been identified whose disruption allows prefusion attachment and disallows membrane merger. In mice, several proteins involved in gamete membrane interactions have been described, including ADAMS family members and CRISP proteins on sperm and integrins and tetraspanin family members CD9 and CD81 on eggs (for review, see Ellerman et al. 2006;Inoue et al. 2007;Primakoff and Myles 2007). Izumo, an immunoglobulin superfamily sperm protein that appears to be limited to mammals, is gamete-specific and shown by gene disruption to be essential at a late step in ferti...

Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates

“…In spite of the many cellular events common to fertilization and zygote development in almost all eukaryotes, only recently was the first gamete-specific gene identified that is conserved and essential for fertilization in broadly disparate organisms. The gamete membrane protein HAP2(GCS1) is now known to be essential for gamete fusion in the isogamous, unicellular green alga Chlamydomonas; the unicellular malaria organism Plasmodium; and the higher plant Arabidopsis (Mori et al 2006;von Besser et al 2006;Hirai et al 2008;Liu et al 2008). Based on our results from Chlamydomonas and Plasmodium, the current model is that HAP2 is a gamete membrane fusogen in all three organisms and presumably in the many other protists, plants, and multicellular animals that possess it (Mori et al 2006;von Besser et al 2006;Hirai et al 2008;Liu et al 2010;Wong and Johnson 2010).…”

“…The data shown are averages from three independent experiments, and the error bars are standard error of the mean (SEM). Transmission electron microscopy with 4-h and 7-h zygotes was performed as previously described (Liu et al 2008). …”

The development of malaria parasites in the mosquito midgut

“…While the exact proteins involved in initial binding of male and female gametes are yet unknown, gamete fusion is mediated by the microgamete protein GCS1 (generative cell specific 1; also termed HAP2), and disruption of the respective gene in P . berghei resulted in male sterility and blocked fertilization (Hirai et al ., 2008; Liu et al ., 2008). Following gamete fusion, nuclear fusion ensues, and over the next 3 h, meiosis occurs, and the zygote becomes tetraploid (Janse et al ., 1986), a process involving the NIMA‐related kinases Nek‐2 and Nek‐4 (Reininger et al ., 2005, 2009).…”

Seaweed reproductive biology: environmental and genetic controls

“…Interestingly, a generative cell specific 1 gene (GCS1), orthologous to HAP2, which was initially demonstrated to mediate membrane fusion of male and female gametes during fertilization in land plants (Mori et al 2006) and later identified as an ancestral gamete, fusogen, due to its high conservation among distantly related eukaryotes (Liu et al 2008, Wong and Johnson 2010, Speijer et al 2015, seems to be highly conserved among species of Chlorophyta.…”