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.