The Dictyostelium discoideum GPHR ortholog is an endoplasmic reticulum and Golgi protein with roles during development.

Dictyostelium discoideum GPHR (Golgi pH regulator)/Gpr89 is a developmentally regulated transmembrane protein present on the endoplasmic reticulum (ER) and the Golgi apparatus. Transcript levels are low during growth and vary during development, reaching high levels during the aggregation and late developmental stages. The Arabidopsis ortholog was described as a G protein-coupled receptor (GPCR) for abscisic acid present at the plasma membrane, whereas the mammalian ortholog is a Golgi apparatus-associated anion channel functioning as a Golgi apparatus pH regulator. To probe its role in D. discoideum, we generated a strain lacking GPHR. The mutant had different growth characteristics than the AX2 parent strain, exhibited changes during late development, and formed abnormally shaped small slugs and fruiting bodies. An analysis of development-specific markers revealed that their expression was disturbed. The distributions of the endoplasmic reticulum and the Golgi apparatus were unaltered at the immunofluorescence level. Likewise, their functions did not appear to be impaired, since membrane proteins were properly processed and glycosylated. Also, changes in the external pH were sensed by the ER, as indicated by a pH-sensitive ER probe, as in the wild type.

The Dictyostelium discoideum RACK1 orthologue has roles in growth and development.

BACKGROUND: The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes. RESULTS: We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gß function in D. discoideum as developmental and other defects were not rescued in Gß null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development. CONCLUSION: DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins.

The cytohesin paralog Sec7 of Dictyostelium discoideum is required for phagocytosis and cell motility.

BACKGROUND: Dictyostelium harbors several paralogous Sec7 genes that encode members of three subfamilies of the Sec7 superfamily of guanine nucleotide exchange factors. One of them is the cytohesin family represented by three members in D. discoideum, SecG, Sec7 and a further protein distinguished by several transmembrane domains. Cytohesins are characterized by a Sec7-PH tandem domain and have roles in cell adhesion and migration. RESULTS: We study here Sec7. In vitro its PH domain bound preferentially to phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). When following the distribution of GFP-Sec7 in vivo we observed the protein in the cytosol and at the plasma membrane. Strikingly, when cells formed pseudopods, macropinosomes or phagosomes, GFP-Sec7 was conspicuously absent from areas of the plasma membrane which were involved in these processes. Mutant cells lacking Sec7 exhibited an impaired phagocytosis and showed significantly reduced speed and less persistence during migration. Cellular properties associated with mammalian cytohesins like cell-cell and cell-substratum adhesion were not altered. Proteins with roles in membrane trafficking and signal transduction have been identified as putative interaction partners consistent with the data obtained from mutant analysis. CONCLUSIONS: Sec7 is a cytosolic component and is associated with the plasma membrane in a pattern distinctly different from the accumulation of PI(3,4,5)P3. Mutant analysis reveals that loss of the protein affects cellular processes that involve membrane flow and the actin cytoskeleton.

RpkA, a highly conserved GPCR with a lipid kinase domain, has a role in phagocytosis and anti-bacterial defense.

RpkA (Receptor phosphatidylinositol kinase A) is an unusual seven-helix transmembrane protein of Dictyostelium discoideum with a G protein coupled receptor (GPCR) signature and a C-terminal lipid kinase domain (GPCR-PIPK) predicted as a phosphatidylinositol-4-phosphate 5-kinase. RpkA-homologs are present in all so far sequenced Dictyostelidae as well as in several other lower eukaryotes like the oomycete Phytophthora, and in the Legionella host Acanthamoeba castellani. Here we show by immunofluorescence that RpkA localizes to endosomal membranes and is specifically recruited to phagosomes. RpkA interacts with the phagosomal protein complex V-ATPase as proteins of this complex co-precipitate with RpkA-GFP as well as with the GST-tagged PIPK domain of RpkA. Loss of RpkA leads to a defect in phagocytosis as measured by yeast particle uptake. The uptake of the pathogenic bacterium Legionella pneumophila was however unaltered whereas its intra-cellular replication was significantly enhanced in rpkA(-). The difference between wild type and rpkA(-) was even more prominent when L. hackeliae was used. When we investigated the reason for the enhanced susceptibility for L. pneumophila of rpkA(-) we could not detect a difference in endosomal pH but rpkA(-) showed depletion of phosphoinositides (PIP and PIP(2)) when we compared metabolically labeled phosphoinositides from wild type and rpkA(-). Furthermore rpkA(-) exhibited reduced nitrogen starvation tolerance, an indicator for a reduced autophagy rate. Our results indicate that RpkA is a component of the defense system of D. discoideum as well as other lower eukaryotes.