Mutant/mutation
The mutant expresses a C-terminal HA-tagged version of ALLC1

Protein (function)
The gene encoding ALLC1 was selected based on a (male-)fertilty mutant screen. This mutant showed normal motility score of male gametes of the mutant.
SUN1 interacts with an allantoicase-like protein. Immunoprecipitation of SUN1-HA from gametocyte lysates eight minutes post-activation identified one of two allantoicase-like proteins encoded in the P. berghei genome, which we designated ALLC1 (PBANKA_1304400). A reciprocal pull-down of ALLC1-HA confirmed the interaction with SUN1. Both proteins also interacted with the essential endoplasmic reticulum chaperone BiP and a DDRGK-motif containing conserved Plasmodium protein expressed most abundantly in male gametocytes. While the disruption of the DDRGK-motif had no effect on fertility, allc1 is a male fertility gene, like sun1 (as determined in the male-fertility screen).

SUN1 is a protein with a carboxy-terminal SUN (Sad1p/UNC-84) domain, PBANKA_1430900. SUN domain proteins were present in the first eukaryote and typically span the inner nuclear envelope, where they form part of the LINC (linker of nucleoskeleton and cytoskeleton) complex that anchors the nucleus by connecting the nuclear lamina to the cytosolic cytoskeleton. The lack of nuclear lamins and canonical LINC complex components in apicomplexa led us to hypothesize that SUN1 has a non-canonical function in male gamete formation

Phenotype
Analysis of the mutant expressing a C-terminal HA-tagged version of ALLC1 showed the following:
SUN1 interacts with an allantoicase-like protein. Immunoprecipitation of SUN1-HA from gametocyte lysates eight minutes post-activation identified one of two allantoicase-like proteins encoded in the P. berghei genome, which we designated ALLC1 (PBANKA_1304400). A reciprocal pull-down of ALLC1-HA confirmed the interaction with SUN1. Both proteins also interacted with the essential endoplasmic reticulum chaperone BiP and a DDRGK-motif containing conserved Plasmodium protein expressed most abundantly in male gametocytes. While the disruption of the DDRGK-motif had no effect on fertility, allc1 is a male fertility gene, like sun1 (as determined in the male-fertility screen).

Additional information
Cellular events of microgametogenesis are well characterized by ultrastructure expansion microscopy (U-ExM) and staining for α-tubulin, the centrosome marker centrin, the general protein label N-hydroxysuccinimide (NHS)-ester highlights microtubule organising centres (MTOCs) and the axonemes and spindles that emanate from them. In expanded sun1-KO gametocytes the two tetrads of forming axonemes were positioned on top of each other, consistent with the absence of a mitotic spindle that would be required for their separation along the nuclear envelope. Often these structures appeared in the cytosol distant from the nuclear envelope, suggesting SUN1 may be required to capture the MTOCs onto the nuclear membrane.

Analysis of a mutant lacking expression of SUN1 (RMgm-5521) showed the following:
Sun1-KO microgametocytes formed exflagellation centres when activated in vitro at the same rate as wild type. Morphologically mature ookinetes are formed, but very few oocysts present on the midguts of infected mosquitoes. Sun1-KO ookinetes contained only half the DNA of wild type ookinetes, probably because most microgametes failed to deliver DNA to
the zygote. This was NOT the result of defective replication during gametogenesis, but mitotic spindles were missing from nuclei, suggesting SUN1 is important for the microgamete nucleus or its genome to connect to the axoneme. Strongly reduced oocyst formation.

Analysis of the mutant expressing a C-terminal HA-tagged version of SUN1 (RMgm-5522)  showed the following:
SUN1-HA was not detected in asexual blood stages and female gametocytes but present in male gametocytes. SUN1-HA was associated with the nuclear envelope of expanded microgametocytes, and two minutes after activation some of the protein formed two rings, one at the centre of each windmill-like tetrad of budding axonemes that were present at the opposite poles of the first endomitotic spindle. Labelling centrin to mark the cytosolic part of the MTOCs corroborated the nuclear envelope localisation of SUN1-HA. In expanded sun1 KO gametocytes the two tetrads of forming axonemes were positioned on top of each other, consistent with the absence of a mitotic spindle that would be required for their separation along the nuclear envelope. Often these structures appeared in the cytosol distant from the nuclear envelope, suggesting SUN1 may be required to capture the MTOCs onto the nuclear membrane. Eight minutes after activation, when WT microgametocytes have completed their third endomitosis, the base of each axoneme was marked by a SUN1-HA ring located between the cytosolic and the nucleoplasmic portion of each bipartite MTOC, precisely where spindle microtubules would need to pass through a nuclear pore to connect a haploid set of chromosomes to the centriolar region that has given rise to the axoneme. At the same time point, sun1 KO microgametocytes also had elongated axonemes, which were however, emanating from MTOCs that remained connected, consistent with an absence of the mitotic events which in WT split the eight MTOCs and distribute them around the nuclear envelope.

Other mutants