The conserved plant sterility gene HAP2
functions after attachment of fusogenic
membranes in Chlamydomonas
and Plasmodium gametes
Yanjie Liu,1,7Rita Tewari,2,3,7Jue Ning,1Andrew M. Blagborough,2,4Sara Garbom,2Jimin Pei,5
Nick V. Grishin,5Robert E. Steele,6Robert E. Sinden,2William J. Snell,1,8and Oliver Billker2,4,9
1Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA;2Division
of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, United Kingdom;3Institute of Genetics,
University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom;4The Wellcome Trust Sanger
Institute, Hinxton, Cambridge CB10 SA1, United Kingdom;5Howard Hughes Medical Institute, University of Texas
Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA;6Department of Biological Chemistry and the
Developmental Biology Center, University of California, Irvine, California 92697, USA
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 ga-
metes 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 re-
gions of the sperm plasma membrane. In the second
phase of fertilization, the membrane fusion reaction, the
plasma membranes of the two gametes come into inti-
mate 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 pro-
teins 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 es-
sential at a late step in fertilization. Izumo is the best
7These authors contributed equally to this work.
8E-MAIL firstname.lastname@example.org; FAX (214) 648-8694.
9E-MAIL email@example.com; FAX 44-20-7594-5424.
Article published online ahead of print. Article and publication date are
online at http://www.genesdev.org/cgi/doi/10.1101/gad.1656508.
GENES & DEVELOPMENT 22:1051–1068 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org1051
candidate to date for a role in the membrane fusion re-
action in mice. Its specific function has yet to be deter-
mined, however, and the presence of conserved Ig super-
family domains predicts a role in membrane adhesion
(Inoue et al. 2005, 2007; Primakoff and Myles 2007).
In nematodes, several genes have been identified
whose disruption leads to sterility. The Caenorhabditis
elegans gene SPE-9 is essential for gamete interactions,
but it is proposed to be an adhesion and signaling mol-
ecule and probably is not involved in the membrane fu-
sion reaction (Putiri et al. 2004). Other proteins are im-
plicated in gamete interactions in worms, including
EGG-1, EGG-2, SPE-38, and SPE-42 (Chatterjee et al.
2005; Kadandale et al. 2005; Kroft et al. 2005), but their
precise roles in fertilization also are unclear, in large part
because of the difficulty of studying sperm–egg interac-
tions in vitro. The protein Prm1p has been implicated in
cell–cell fusion of Saccharomyces cerevisiae. Its disrup-
tion reduces fusion of a and alpha cells by 50%, but only
when the gene is disrupted in both cell types (Heiman
and Walter 2000; Aguilar et al. 2007; Heiman et al. 2007);
thus, it is not essential for fusion. In invertebrates, at
least two sperm proteins have emerged as candidate ad-
hesion/fusion molecules—an 18-kDa protein in abalone
(Swanson and Vacquier 1995) and the sea urchin protein
bindin (Kamei and Glabe 2003). Because of the difficulty
of generating mutants in these organisms, the functions
of the abalone and urchin proteins in gamete interac-
tions remain unidentified.
A recent screen for sterile mutants in Arabidopsis
identified the male-specific sterility gene HAP2 (Johnson
et al. 2004). A HAP2 family member called GCS1 (for
generative cell-specific) was subsequently identified in a
screen for lily genes whose transcripts were up-regulated
in sperm (generative cells) (Mori et al. 2006). In Arabi-
dopsis, the only organism for which HAP2 mutants are
available, the gene is involved in pollen tube guidance, is
expressed in sperm, and also is essential for seed forma-
tion (Johnson et al. 2004; Mori et al. 2006; von Besser et
al. 2006). Although the gene acts after sperm deposition
in the female gametophyte, its cellular and molecular
functions in seed formation are unknown, since the cel-
lular events subsequent to sperm deposition—sperm mi-
gration, sperm–egg attachment, and sperm–egg fusion—
have not been distinguished experimentally. Interest-
ingly, HAP2 is conserved, and in addition to being in
Arabidopsis and rice (Johnson et al. 2004), members
were found in Chlamydomonas, a red alga, a slime mold,
Plasmodium falciparum, and Leishmania major (Mori
et al. 2006).
In contrast to fertilization in the organisms described
above, fertilization in some protists is highly amenable
to study. In the unicellular, biflagellated green alga
Chlamydomonas reinhardtii, initial adhesion of the fla-
gella of mating type minus and mating type plus gametes
in the first phase of interactions triggers cilium-gener-
ated signaling (Wang et al. 2006) and gamete activation
(for review, see Pan and Snell 2000; Goodenough et al.
2007). Gamete activation prepares the gametes for fusion
and comprises a complex signaling pathway including a
protein tyrosine kinase (Wang and Snell 2003), a cGMP-
dependent protein kinase (Wang et al. 2006), a flagellar
adenylyl cyclase (Saito et al. 1993; Zhang and Snell
1994), and a 10- to 20-fold increase in cAMP. Both ga-
metes shed their glycoproteinaceous cell walls through
the action of a metalloprotease released from each cell
(Matsuda et al. 1985; Buchanan et al. 1989). And sites
specialized for cell fusion—the mating structures—lo-
cated on the apical cell membranes between the two
flagella of both gametes are activated (Goodenough et al.
1982; Wilson et al. 1997). The activated plus mating
structure is a microvillus-like cellular extension ∼3 µm
in length and ∼0.15 µm in diameter. The activated minus
mating structure lacks actin filaments and is shorter and
more bulbous. Continued flagellar adhesion brings the
activated mating structures into intimate contact, and
within seconds after contact, the membranes of the two
mating structure fuse, followed by complete coalescence
of the two gametes into a single, quadriflagellated zy-
gote. The entire process of fertilization, from initial fla-
gellar adhesion of gametes through fusion, can occur
within 30 sec or less.
Previously, we showed that the plus gamete-specific
protein FUS1, which is not found in other species, is
present on the plasma membrane of the mating struc-
ture, the fertilization tubule. Furthermore, in experi-
ments with flagellar adhesion mutants, we demon-
strated that wild-type plus gametes, but not fus1 plus
gametes, were capable of adhering to minus gametes
solely via their activated mating structures. Thus, during
the membrane fusion reaction, plus-specific FUS1 is es-
sential for prefusion attachment between the plus and
minus mating structures (Ferris et al. 1996; Misamore et
Fertilization in the rodent malaria organism, Plasmo-
dium berghei, is also highly amenable to study. Sexual
precursor stages, the gametocytes, form in the vertebrate
host inside infected erythrocytes, but remain quiescent
until ingested by a susceptible Anopheles mosquito. In
the bloodmeal, gametocytes emerge from their host cells
and within minutes differentiate into gametes. Each fe-
male (macro) gametocyte gives rise to a single immotile
female gamete, while male (micro) gametocytes generate
up to eight flagellated male gametes in a process termed
“exflagellation”; within minutes after release, the ga-
metes meet, adhere for a few seconds, and then fuse to
form a zygote (Sinden 1983). Male gamete adhesion to a
female gamete requires the species-limited surface pro-
tein and transmission-blocking vaccine candidate P48/
45 (van Dijk et al. 2001). P48/45 interacts physically
with at least one other gametocyte protein, P230 (Kumar
1987), and in P. falciparum is required to retain the com-
plex on the cell surface once gametes have emerged from
their host cells (Eksi et al. 2006). The male-specific func-
tion of the P48/45–P230 complex is in contrast with its
expression in both male and female gametes, and wheth-
er either protein on male gametes binds directly to a
receptor on female gametes is unknown.
Within 15–20 h, the zygote transforms into a motile
ookinete, which penetrates the midgut epithelium and
Liu et al.
1052 GENES & DEVELOPMENT
establishes the infection in the mosquito by forming an
oocyst between the midgut epithelial cells and their un-
derlying basal lamina. Thus, gamete adhesion and fusion
are obligate steps in mosquito transmission of malaria
and attractive targets for transmission-blocking vac-
cines. In the rodent malaria parasite P. berghei, gameto-
cytes respond efficiently to well-characterized develop-
mental triggers (Billker et al. 1998) in vitro, and gameto-
accessible to analysis in culture. Moreover, targeted gene
disruption is now a routinely used method in Plasmo-
dium (Janse et al. 2006).
To date, no widely conserved mechanism of gamete
fusion has been identified (Chen and Olson 2005; Rubin-
stein et al. 2006; Primakoff and Myles 2007). Thus, it
remains unknown for any organism whether adhesion of
gamete membranes and fusion of the membranes are to-
gether accomplished by a single set of proteins, as with
fusion of many viruses (Earp et al. 2005), or if the two
functions are allocated to distinct sets of proteins. We
also do not understand the molecular basis for the spe-
cies specificity of gamete fusion in many organisms (Fer-
ris et al. 1997; Swanson and Vacquier 2002; Vieira and
Here, in coupled studies of fertilization in Chlamydo-
monas and Plasmodium, we show that gamete fusion
requires the plant sterility gene HAP2. We genetically
distinguish attachment of gamete fusogenic membranes
from membrane merger, and show that the membrane
fusion reaction is governed by species-limited proteins
required for prefusion attachment. In both species, post-
adhesion events resulting in membrane fusion depend on
the conserved HAP2 protein.
A screen for zygote formation mutants
in Chlamydomonas identifies a homolog
of an Arabidopsis male sterility gene
To identify proteins in Chlamydomonas minus gametes
that are essential at a late stage of gamete interactions,
we generated insertional mutants in Chlamydomonas
minus cells (strain B215, mt−) by transformation with a
paromomycin resistance gene (Sizova et al. 2001). Colo-
nies that grew on paromomycin were induced to undergo
gametogenesis, and the gametes were mixed with wild-
type plus gametes and screened for their ability to un-
dergo flagellar adhesion within minutes after mixing and
to form the zygote aggregates that appear ∼4 h after mix-
ing. After screening ∼2500 insertional mutants, we iden-
tified one transformant with the desired phenotype,
63B10. Gametes of 63B10 adhered via their flagella to
wild-type plus gametes and formed small clusters visible
at 10 min after mixing similarly to wild-type minus ga-
metes (Fig. 1A, top panels). On the other hand, the 63B10
minus/wild-type plus mixtures failed to form the large
zygote aggregates characteristic of wild-type/wild-type
gamete mixtures at 4 h and remained in the small clus-
ters (Fig. 1A, bottom panels). In contrast to this easily
observed phenotype when they were gametes, the
growth, motility, phototaxis, and morphology of 63B10
vegetative cells were indistinguishable from those of
wild-type vegetative cells. When we examined wild-type
minus/wild-type plus gamete mixtures and 63B10 mi-
nus/wild-type plus gamete mixtures soon after mixing,
we found that, as expected, most wild-type cells had
fused to form quadriflagellated zygotes, but 63B10 mi-
nus/wild-type plus mixtures failed to form quadriflagel-
lated cells. Instead, the 63B10 minus gametes continued
flagellar adhesion with the wild-type plus gametes,
many visible as pairs of cells with their flagella entwined
and their apical ends closely apposed (Fig. 1B).
After confirming by Southern blotting (Fig. 1C) that
63B10 contained a single insertion of the paromomycin
resistance gene, we used thermal asymmetric interlaced
PCR (TAIL–PCR) to identify 180 base pairs (bp) of geno-
mic sequence adjacent to the inserted plasmid. Searches
of version 2.0 of the Chlamydomonas reinhardtii ge-
nome sequence from the DOE Joint Genome Institute
identified C_530033 as the adjacent gene (Fig. 1D).
Transformation of 63B10 cells with BAC 20L3, which
C_530033, restored their ability to form zygotes (data not
shown), thus confirming that we had identified the ge-
nomic region containing the disrupted gene. To confirm
that C_530033 indeed was sufficient for rescue of zygote
formation, we transformed 63B10 cells with a 20L3 re-
striction fragment whose only full-length gene was
C_530033. As shown in the diagnostic PCR in Figure 1E,
the transformants in which formation of quadriflagel-
lated zygotes was restored contained the rescuing wild-
type gene as well as the disrupted gene. Moreover, only
wild-type gametes and rescued 63B10 gametes formed
the macroscopic aggregates characteristic of zygotes 4 h
after mixing wild-type minus and plus gametes (Fig. 1F).
Thus, C_530033 was essential for gamete fusion. To our
surprise, BLAST searches of NCBI databases (Altschul et
al. 1997) showed that C_530033 encodes the Chlamydo-
monas homolog of HAP2, the Arabidopsis pollen tube
guidance and male sterility gene first reported by Johnson
et al. (2004), Mori et al. (2006), and von Besser et al. (2006).
HAP2 is present in multicellular animals
and in P. berghei
Using PSI-BLAST (Altschul et al. 1997), we extended pre-
vious results (Johnson et al. 2004; Mori et al. 2006) on the
species distribution of HAP2, and found HAP2 family
members in many higher plants whose genome se-
quences are available including maize, wheat, and to-
mato (data not shown), as well as in many other non-
pathogenic and pathogenic protists including Toxo-
plasma gondii, Cryptosporidium hominis, Theileria
parva, Naegleria gruberi, and Trypanosoma brucei (Fig.
1G). The HAP2 gene is also present in a choanoflagellate
(Monosiga brevicollis), one of the closest unicellular
relatives of animals. Consistent with its presence in
Monosiga, HAP2 family members were also present in
multicellular animals including Hydra magnipapillata,
the starlet sea anemone Nematostella vectensis, and in-
HAP2 and gamete membrane fusion
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