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Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway, activating cAMP-dependent protein kinase A (PKA). This requires nine git (glucose insensitive transcription) genes that encode adenylate cyclase, the PKA catalytic subunit, and seven 'upstream' proteins required for glucose-triggered adenylate cyclase activation, including three heterotrimeric G-protein subunits and its associated receptor. We describe here the cloning and characterization of the git[1.sup.+] gene. Girl is distantly related to a small group of uncharacterized fungal proteins, including a second S. pombe protein that is not functionally redundant with Git1, as well as to members of the UNC-13/Munc13 protein family. Mutations in git[1.sup.+] demonstrate functional roles for the two most highly conserved regions of the protein, the C2 domain and the MHD2 Munc homology domain. Cells lacking Git1 are viable, but display phenotypes associated with cAMP-signaling defects, even in strains expressing a mutationally activated G[alpha]-subunit, which activates adenylate cyclase. These cells possess reduced basal cAMP levels and fail to mount a cAMP response to glucose. In addition, Girl and adenylate cyclase physically interact and partially colocalize in the cell. Thus, Git1 is a critical component of the S. pombe glucose/cAMP pathway.

Mutations affecting the Schizosaccharomyces pombe cAMP phosphodiesterase (PDE) gene cgs[2.sup.+] were identified in a screen for suppressors of mutant alleles of the adenylate cyclase gene (git[2.sup.+]/cyr[1.sup.+]), which encode catalytically active forms of the enzyme that cannot be stimulated by extracellular glucose signaling. These mutations suppress both the git[2.sup.-] mutant alleles used in the suppressor selection and mutations in git[1.sup.+], git[3.sup.+], git[5.sup.+], git[7.sup.+], git[10.sup.+], and git[11.sup.+], which are all required for adenylate cyclase activation. Notably, these cgs2 mutant alleles fail to suppress mutations in gpa[2.sup.+], which encodes the G[alpha] subunit of a heterotrimeric G protein required for adenylate cyclase activation, although the previously identified cgs2-2 allele does suppress loss of gpa[2.sup.+]. Further analysis of the cgs2-s1 allele reveals a synthetic interaction with the gpa[2.sup.R176H]-activated allele, with respect to derepression of fbp1-lacZ transcription in glucose-starved cells. In addition, direct measurements of cAMP levels show that cgs2-s1 cells maintain normal basal cAMP levels, but are severely defective in feedback regulation upon glucose detection. These results suggest that PDE activity in S. pombe may be coordinately regulated with adenylate cyclase activity as part of the feedback regulation mechanism to limit the cAMP response to glucose detection.

Apicomplexan parasites replicate by several budding mechanisms with two well-characterized examples being Toxoplasma endodyogeny and Plasmodium schizogony. Completion of budding requires the tapering of the nascent daughter buds toward the basal end, driven by contraction of the basal complex. This contraction is not executed by any of the known cell division associated contractile mechanisms and in order to reveal new components of the unusual basal complex we performed a yeast two-hybrid screen with its major scaffolding protein, TgMORN1. Here we report on a conserved protein with a haloacid dehalogenase (HAD) phosphatase domain, hereafter named HAD2a, identified by yeast two-hybrid. HAD2a has demonstrated enzyme-activity in vitro, localizes to the nascent daughter buds, and co-localizes with MORN1 to the basal complex during its contraction. Conditional knockout of HAD2a in Toxoplasma interferes with basal complex assembly, which leads to incomplete cytokinesis and conjoined daughters that ultimately results in disrupted proliferation. In Plasmodium, we further confirmed localization of the HAD2a ortholog to the basal complex toward the end of schizogony. In conclusion, our work highlights an essential role for this HAD phosphatase across apicomplexan budding and suggests a regulatory mechanism of differential phosphorylation on the structure and/or contractile function of the basal complex. [ABSTRACT FROM AUTHOR]