A capsule-associated gene of Cryptococcus neoformans, CAP64, is involved in pH homeostasis.

The CAP64 gene is known to be involved in capsule formation in the basidiomycete yeast Cryptococcus neoformans. A null mutant of CAP64, Δcap64, lacks a capsule around the cell wall and its acidic organelles are not stained with quinacrine. In order to clarify whether the Cap64 protein indeed maintains vacuole or vesicle acidification, so that the vesicle containing the capsule polysaccharide or DBB substrate are transported to the cell membrane side, the relationship between CAP64 and intracellular transport genes and between CAP64 and enzyme-secretion activity were analysed. Laccase activity was higher in the Δcap64 strain than in the wild-type strain, and the transcriptional levels of SAV1 and VPH1 were also higher in the Δcap64 strain than in the wild-type strain. The intracellular localization of the Cap64 protein was analysed by overexpressing an mCherry-tagged Cap64 and observing its fluorescence. The Cap64 protein was accumulated within cells in a patch-like manner. The quinacrine-stained cells were observed to analyse the acidified cell compartments; quinacrine was found to be accumulated in a patch-like manner, with the patches overlapping the fluorescence of CAP64-mCherry fusion protein. Quinacrine was thus accumulated in a patch-like fashion in the cells, and the mCherry-tagged Cap64 protein position was consistent with the position of quinacrine accumulation in cells. These results suggest that CAP64 might be involved in intracellular acidification and vesicle secretion via exocytosis.

Structure of sirohydrochlorin ferrochelatase SirB: the last of the structures of the class II chelatase family.

The crystal structure of Bacillus subtilis SirB, which catalyses the insertion of Fe2+ into the substrate sirohydrochlorin (SHC) in siroheme biosynthesis, is reported herein as the last of the structures of class II chelatases. The structure of SirB with Co2+ showed that the active site of SirB is located at the N-terminal domain with metal-binding amino acid residues His10, Glu43, and His76, which was also predicted for CbiX, but is distinct from the C-terminal active sites of CbiK and HemH. The biosynthetic model reactions using SirB, Co2+ and uroporphyrin I or protoporphyrin IX as a SHC analogue revealed that SirB showed chelatase activity for uroporphyrin I, but not for protoporphyrin IX. Simulations of tetrapyrroles docking to SirB provided an insight into its tetrapyrrole substrate recognition: SHC and uroporphyrin I were suitably bound beside the Co2+ ion-binding site at the active site cavity; protoporphyrin IX was also docked to the active site but its orientation was different from those of the other two tetrapyrroles. Summarizing the present data, it was proposed that the key structural features for substrate recognition of SirB could be the hydrophobic area at the active site as well as the substituents of the tetrapyrroles.