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

Abstract

Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multicysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction.

Structure-Function Studies Link Class II Viral Fusogens with the Ancestral Gamete Fusion Protein HAP2

“…4D). These results conflict somewhat with data from other systems[8–10] suggesting that the sequence requirements in this region of the protein differ in different organisms.…”

“…A total of 40 HAP2 orthologs[10] were submitted to Phyre2 batch processing[14] to determine the extent to which the predicted structural homology to class II viral fusion proteins is maintained across taxonomic groups. We found that 28 (~70%) had hits to class II viral fusogens (Table S1).…”

“…In short, these studies suggest that species-restricted functions of HAP2 reside within the extracellular region, with numerous residues/motifs including the conserved HAP2-GCS1 domain having a role in HAP2 function[8,9]. By contrast, some studies suggest that the cytosolic region is almost entirely dispensable for activity[8], while others point to positively charged residues as well as multi-cysteine motifs that may be palmitoylated as being important[9,10]. …”

Sex is a ubiquitous, ancient, and inherent attribute of eukaryotic life

“…Highly diverse eukaryotic gametes carrying loss-of-function mutations in HAP2 fail to fuse. This protein family could thus indeed represent an ancestral gamete fusogen in eukaryotes (53,54). Where nuclear fusion is concerned, the GEX1 protein family (fungal orthologs: KAR5) has recently been found to represent a potentially conserved karyogamy factor (55).…”

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

“…In recent years, Chlamydomonas has been increasingly used to study additional biological processes, including lipid biosynthesis (Hu et al, 2008;Wang et al, 2009;Moellering and Benning, 2010;Merchant et al, 2012;Liu and Benning, 2013), pigment biosynthesis and regulation (Lohr et al, 2005;Beale, 2009;Lohr, 2009;Voss et al, 2011), carbon-concentrating mechanisms (Badger et al, 1980;Wang et al, 2011;Brueggeman et al, 2012;Fang et al, 2012), growth during nutrient deprivation (González-Ballester et al, 2010;Miller et al, 2010;Castruita et al, 2011;Boyle et al, 2012;Urzica et al, 2012Urzica et al, , 2013Blaby et al, 2013;Hemschemeier et al, 2013;Toepel et al, 2013;Aksoy et al, 2014;Schmollinger et al, 2014), responses to heat stress (Hemme et al, 2014), photoreception (Beel et al, 2012), fermentation biology and hydrogen gas production (Ghirardi et al, 2007;Mus et al, 2007;Hemschemeier et al, 2008;Dubini et al, 2009;Grossman et al, 2011;Catalanotti et al, 2012Catalanotti et al, , 2013Magneschi et al, 2012;Murthy et al, 2012;Yang et al, 2014), mating (Umen, 2011;Geng et al, 2014;…”