Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy inChlamydomonas

Abstract

SUMMARYThe plasma membranes of gametes are specialized for fusion, yet, once fusion occurs, in many organisms the new zygote becomes incapable of further membrane fusion reactions. The molecular mechanisms that underlie this loss of fusion capacity (block to polygamy) remain unknown. During fertilization in the green alga Chlamydomonas, the plus gamete-specific membrane protein FUS1 is required for adhesion between the apically localized sites on the plasma membranes of plus and minus gametes that are specialized for fusion, and the minus-specific membrane protein HAP2 is essential for completion of the membrane fusion reaction. HAP2 (GCS1) family members are also required for fertilization in Arabidopsis, and for the membrane fusion reaction in the malaria organism Plasmodium berghei. Here, we tested whether Chlamydomonas gamete fusion triggers alterations in FUS1 and HAP2 and renders the plasma membranes of the cells incapable of subsequent fusion. We find that, even though the fusogenic sites support multi-cell adhesions, triploid zygotes are rare, indicating a fusion-triggered block to the membrane fusion reaction. Consistent with the extinction of fusogenic capacity, both FUS1 and HAP2 are degraded upon fusion. The rapid, fusion-triggered cleavage of HAP2 in zygotes is distinct from degradation occurring during constitutive turnover in gametes. Thus, gamete fusion triggers specific degradation of fusion-essential proteins and renders the zygote incapable of fusion. Our results provide the first molecular explanation for a membrane block to polygamy in any organism.

The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction

“…4A-E). We anticipated that because of its missing TMD and cytoplasmic region, it might be secreted, but we were unable to detect HAP2-ecto in the culture medium, even after 3 h of gamete activation brought about by mixing with wild-type plus gametes (Liu et al, 2010; Fig. 4D).…”

“…4A-C,E). To test whether the absence of the protein at the mating structure was a consequence of the low expression of the upper form of HAP2-ecto+TMD, we incubated the gametes with di-butyryl cAMP (db-cAMP), a treatment that leads to upregulation of HAP2 transcription and translation (Liu et al, 2010;Ning et al, 2013). Such activated cells still lacked HAP2-ecto+TMD at the mating structure, and the activated HAP2-ecto+TMD cells were incapable of fusion with plus gametes (Fig.…”

“…Several C. elegans genes are known to be essential for fertilization, but whether they function directly in the membrane fusion reaction is unknown because of the difficulty of experimentally defining distinct steps in gamete interactions (Singson et al, 2008). To date, the sole, broadly conserved protein shown by gene disruption to be essential for the gamete membrane fusion reaction in any system, including all model organisms, is the membrane protein HAP2 (also called GCS1), which is present in plants and protists, and in some multicellular animals (but notably absent in vertebrates) (Mori et al, 2006;von Besser et al, 2006;Liu et al, 2008Liu et al, , 2010Hirai et al, 2008;Goodman and McFadden, 2008;Cole et al, 2014;Steele and Dana, 2009;Ebchuqin et al, 2014;Kawai-Toyooka et al, 2014).…”

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

“…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).…”

Phospho-Signaling at Oocyte Maturation and Fertilization: Set Up for Embryogenesis and Beyond Part I. Protein Kinases

“…Although it's physiological substrate other than SksC by itself has not yet been identified, adhesion-induced SksC down-regulation and flagellar PTK up-regulation may play important roles simultaneously in gamete fusion and activation of embryogenesis (Pan and Snell 2000;Zhang et al 1996). In this species, a rapid degradation of two gamete-specific proteins, FUS1 and HAP1, occur upon gamete fusion (Liu et al 2010). This event is required for polyspermy block.…”