Ji-Sun Kim

Thioredoxin reductase is required for growth and regulates entry into culmination of Dictyostelium discoideum

The thioredoxin system, consisting of thioredoxin, thioredoxin reductase and NADPH, has been well established to be critical for the redox regulation of protein function and signalling. To investigate the role of thioredoxin reductase (Trr) in Dictyostelium discoideum, we generated mutant cells that underexpress or overexpress Trr. Trr‐underexpressing cells exhibited severe defects in axenic growth and development. Trr‐overexpressing (TrrOE) cells formed very tiny plaques on a bacterial lawn and had a lower rate of bacterial uptake. When developed in the dark, TrrOE cells exhibited a slugger phenotype, defined by a prolonged migrating slug stage. Like other slugger mutants, they were hypersensitive to ammonia, which has been known to inhibit culmination by raising the pH of intracellular acidic compartments. Interestingly, TrrOE cells showed defective acidification of intracellular compartments and decreased activity of vacuolar H+‐ATPase which functions in the acidification of intracellular compartments. Moreover, biochemical studies revealed that the thioredoxin system can directly reduce the catalytic subunit of vacuolar H+‐ATPase whose activity is regulated by reversible disulphide bond formation. Taken together, these results suggest that Dictyostelium Trr may be essential for growth and play a role in regulation of phagocytosis and culmination, possibly through the modulation of vacuolar H+‐ATPase activity.

Homeoprotein Hbx4 represses the expression of the adhesion molecule DdCAD-1 governing cytokinesis and development

We investigated the function of homeodomain‐containing protein Hbx4 in Dictyostelium discoideum. Hbx4‐overexpressing cells (Hbx4OE) displayed defects in growth rate and cytokinesis and showed differences in slug motility and cell‐type proportioning from KAx3. Furthermore, the overexpression of Hbx4 inhibited the induction of cadA, which encoded the Ca2+‐dependent cell adhesion molecule DdCAD‐1, despite expression of csaA and gpaB. The electrophoretic mobility shift assay showed that the promoter of cadA contained the Hbx4‐binding site. Moreover, constitutively expressed DdCAD‐1 in Hbx4OE rescued the defects in cytokinesis and development. These results suggest that Hbx4 modulates DdCAD‐1‐mediated cytokinesis and cell‐type proportioning.

Structure of the 34 kDa F‐actin‐bundling protein ABP34 from Dictyostelium discoideum

The crystal structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89 Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions.

Glutathione upregulates cAMP signalling via G protein alpha 2 during the development of Dictyostelium discoideum.

Despite the importance of glutathione in Dictyostelium, the role of glutathione synthetase (gshB/GSS) has not been clearly investigated. In this study, we observed that increasing glutathione content by constitutive expression of gshB leads to mound‐arrest and defects in 3′,5′‐cyclic adenosine monophosphate (cAMP)‐mediated aggregation and developmental gene expression. The overexpression of gpaB encoding G protein alpha 2 (Gα2), an essential component of the cAMP signalling pathway, results in a phenotype similar to that caused by gshB overexpression, whereas gpaB knockdown in gshB‐overexpressing cells partially rescues the above‐mentioned phenotypic defects. Furthermore, Gα2 is highly enriched at the plasma membrane of gshB‐overexpressing cells compared to wild‐type cells. Therefore, our findings suggest that glutathione upregulates cAMP signalling via Gα2 modulation during Dictyostelium development.