Membrane proteins driving a cell-cell fusion reaction during fertilization

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Project Summary Membrane fusion between two gametes (e.g., sperm & egg) during fertilization is a crucial step in eukaryotic life. In all organisms, the fusion reaction proceeds in two steps, membrane adhesion and bilayer merger. Remarkably, for no single organism do we yet have the adhesion proteins for both gametes and the fusion protein. Recently, our laboratory and others have shown that the ancient male gamete-specific protein HAP2 is essential for fertilization across a broad range of eukaryotic taxa, including the pathogenic malaria organism Plasmodium, green alga, ciliates, higher plants, and many metazoans. Our collaborative studies have also shown that a key functional motif of Plasmodium HAP2 can be targeted for a transmission-blocking malaria vaccine. Recent work demonstrated that HAP2 is structurally homologous to viral class II fusion proteins (e.g. Dengue and Zika). Class II fusion proteins on enveloped viruses are triggered by the acidic environment of the endosome to undergo a conformational reorganization from homo- or heterodimers into homotrimers that drive bilayer merger and viral entry during infection. HAP2, however, is present at the cell surface and likely regulated differently because it functions in a variety of milieus. Here, I propose to use fertilization in a bi-ciliated green alga as a model system to investigate the mechanisms that regulate a eukaryotic class II fusion protein. For the first time in any system, we now have identified the adhesion receptors on both gametes and the fusion protein. The adhesion protein FUS1 on plus gametes and the adhesion protein MAR1 (which we have just identified) and fusogen HAP2 on minus gametes. In what I feel is a major advance, I have also determined that MAR1 is bifunctional. In addition to binding to FUS1, MAR1 is also biochemically and functionally associated with HAP2 on minus gametes. Moreover, we have determined that FUS1 and MAR1 dependent gamete adhesion is necessary for the reconfiguration of HAP2 from its prefusion form on resting gametes into homotrimers that drive membrane fusion. In this work, I intend to determine the pre-fusion conformation of HAP2 in resting minus gametes, identify the domains of MAR1 and HAP2 that underlie their interactions in resting gametes, identify the domains in FUS1 and MAR1 important for their binding during gametes interactions, and determine the changes that MAR1 and HAP2 undergo during FUS1-MAR1 binding that activate HAP2 for fusion. The long-term objectives of this proposal are to enhance our fundamental understanding of the mechanism of membrane fusion at fertilization and at the same time provide new strategies for development of vaccines to target transmission of pathogenic protozoa.