Vacuolar Acid Phosphatase Research Articles

Loss of the putative Rab GTPase, Ypt7, impairs the virulence of Cryptococcus neoformans.

Small GTPases of the Rab family coordinate multiple membrane fusion and trafficking events in eukaryotes. In fungi, the Rab GTPase, Ypt7, plays a critical role in late endosomal trafficking, and is required for homotypic fusion events in vacuole biogenesis and inheritance. In this study, we identified a putative YPT7 homologue in Cryptococcus neoformans, a fungal pathogen causing life threatening meningoencephalitis in immunocompromised individuals. As part of an ongoing effort to understand mechanisms of iron acquisition in C. neoformans, we established a role for Ypt7 in growth on heme as the sole iron source. Deletion of YPT7 also caused abnormal vacuolar morphology, defective endocytic trafficking and autophagy, and mislocalization of Aph1, a secreted vacuolar acid phosphatase. Ypt7 localized to the vacuolar membrane and membrane contact sites between the vacuole and mitochondria (vCLAMPs), and loss of the protein impaired growth on inhibitors of the electron transport chain. Additionally, Ypt7 was required for robust growth at 39°C, a phenotype likely involving the calcineurin signaling pathway because ypt7 mutants displayed increased susceptibility to the calcineurin-specific inhibitors, FK506 and cyclosporin A; the mutants also had impaired growth in either limiting or high levels of calcium. Finally, Ypt7 was required for survival during interactions with macrophages, and ypt7 mutants were attenuated for virulence in a mouse inhalation model thus demonstrating the importance of membrane trafficking functions in cryptococcosis.

Identification of Aph1, a phosphate-regulated, secreted, and vacuolar acid phosphatase in Cryptococcus neoformans.

ABSTRACTCryptococcus neoformans strains isolated from patients with AIDS secrete acid phosphatase, but the identity and role of the enzyme(s) responsible have not been elucidated. By combining a one-dimensional electrophoresis step with mass spectrometry, a canonically secreted acid phosphatase, CNAG_02944 (Aph1), was identified in the secretome of the highly virulent serotype A strain H99. We created an APH1 deletion mutant (Δaph1) and showed that Δaph1-infected Galleria mellonella and mice survived longer than those infected with the wild type (WT), demonstrating that Aph1 contributes to cryptococcal virulence. Phosphate starvation induced APH1 expression and secretion of catalytically active acid phosphatase in the WT, but not in the Δaph1 mutant, indicating that Aph1 is the major extracellular acid phosphatase in C. neoformans and that it is phosphate repressible. DsRed-tagged Aph1 was transported to the fungal cell periphery and vacuoles via endosome-like structures and was enriched in bud necks. A similar pattern of Aph1 localization was observed in cryptococci cocultured with THP-1 monocytes, suggesting that Aph1 is produced during host infection. In contrast to Aph1, but consistent with our previous biochemical data, green fluorescent protein (GFP)-tagged phospholipase B1 (Plb1) was predominantly localized at the cell periphery, with no evidence of endosome-mediated export. Despite use of different intracellular transport routes by Plb1 and Aph1, secretion of both proteins was compromised in a Δsec14-1 mutant. Secretions from the WT, but not from Δaph1, hydrolyzed a range of physiological substrates, including phosphotyrosine, glucose-1-phosphate, β-glycerol phosphate, AMP, and mannose-6-phosphate, suggesting that the role of Aph1 is to recycle phosphate from macromolecules in cryptococcal vacuoles and to scavenge phosphate from the extracellular environment.

Biochemical and molecular characterization of AtPAP26, a vacuolar purple acid phosphatase up-regulated in phosphate-deprived Arabidopsis suspension cells and seedlings.

A vacuolar acid phosphatase (APase) that accumulates during phosphate (Pi) starvation of Arabidopsis (Arabidopsis thaliana) suspension cells was purified to homogeneity. The final preparation is a purple APase (PAP), as it exhibited a pink color in solution (A(max) = 520 nm). It exists as a 100-kD homodimer composed of 55-kD glycosylated subunits that cross-reacted with an anti-(tomato intracellular PAP)-IgG. BLAST analysis of its 23-amino acid N-terminal sequence revealed that this PAP is encoded by At5g34850 (AtPAP26; one of 29 PAP genes in Arabidopsis) and that a 30-amino acid signal peptide is cleaved from the AtPAP26 preprotein during its translocation into the vacuole. AtPAP26 displays much stronger sequence similarity to orthologs from other plants than to other Arabidopsis PAPs. AtPAP26 exhibited optimal activity at pH 5.6 and broad substrate selectivity. The 5-fold increase in APase activity that occurred in Pi-deprived cells was paralleled by a similar increase in the amount of a 55-kD anti-(tomato PAP or AtPAP26)-IgG immunoreactive polypeptide and a >30-fold reduction in intracellular free Pi concentration. Semiquantitative reverse transcription-PCR indicated that Pi-sufficient, Pi-starved, and Pi-resupplied cells contain similar amounts of AtPAP26 transcripts. Thus, transcriptional controls appear to exert little influence on AtPAP26 levels, relative to translational and/or proteolytic controls. APase activity and AtPAP26 protein levels were also up-regulated in shoots and roots of Pi-deprived Arabidopsis seedlings. We hypothesize that AtPAP26 recycles Pi from intracellular P metabolites in Pi-starved Arabidopsis. As AtPAP26 also exhibited alkaline peroxidase activity, a potential additional role in the metabolism of reactive oxygen species is discussed.

Rapid increase of vacuolar volume in response to salt stress.

Suspension-cultured cells of mangrove [Bruguiera sexangula (Lour.) Poir.] showed a rapid increase in vacuolar volume under salt stress, although there was no change in the cell volume. The rapid increase in the vacuolar volume was an active process, which followed the activation of the tonoplast H(+)-ATPase and the vacuolar acid phosphatase. The same phenomenon was observed in barley (Hordeum vulgare L. cv. Doriru) root meristematic cells under salt stress but not in pea ( Pisum sativum L.). Increases in vacuolar volume could potentially protect the cytoplasm by decreasing the cytoplasmic volume during the initial phases of salt stress.

PROGRAMMED CELL DEATH IN THE HONEY-BEE (APIS MELLIFERAL.) LARVAE MIDGUT

The histochemical and cytochemical localization of acid phosphatase has been used in an attempt to map the sites of cellular lysis and death. Reaction product was found both in the brush border of the midgut epithelium and in the basal membrane. Vacuolar acid phosphatase activity was found in the regenerative epithelial cells. Extra-cisternal reaction product was associated with the endoplasmic reticulum which was dilated in lysed areas of the cytoplasm. Free acid and alkaline phosphatase activity was found in the basal area of the midgut epithelial cells and the former also occurred in the haemocoel. In the tracheoblastic cells only vacuolar acid phosphatase activity was seen. Chromatin aggregates were distributed throughout the nucleus and the nuclear envelope showed some infolding. Certain mature epithelial cells proved positive for anti-histone associated DNA fragmentation indicative of programmed cell death.

Infectivity of pine and eucalypt isolates of Pisolithus tinctorius (Pers.) Coker & Couch on roots of Eucalyptus urophylla S. T. Blake in vitro

SUMMARYAn ultrastructural comparison of early stages of mycorrhiza formation on Eucalyptus urophylla was carried out using two isolates of P. tinctorius, one cultured from a sporocarp collected under pine and the other cultured from a sporocarp collected under eucalypt. Major differences at the fungus–root interface were observed 2 and 4 d following inoculation. Eucalypt root cells in contact with the isolate from pine developed a thickened cell wall, whereas the isolate from eucalypt was associated with an accumulation of extracellular fibrils. The fibrils stained positively for carbohydrates and protein. They were absent with the isolate from pine. Four days after inoculation with the isolate from eucalypt, acid phosphatase activity was detected along the fungal plasmalemma at the interface with host cells. This enzyme activity was not detected with the isolate from pine. Vacuolar acid phosphatase activity was detected in both fungal isolates when in contact with Eucalyptus roots. It is suggested that host recognition of ectomycorrhizal symbionts can be manifested within hours of inoculation with the fungal partner.

Functional and Regulatory Properties of H+ Pumps at the Tonoplast and Plasma Membranes of Zea mays Coleoptiles

Abstract A microsomal membrane fraction (6000 x g supernatant of a cell homogenate), isolated from coleoptiles of Zea mays, was separated by isopycnic sucrose density gradient centrifugation in the presence of EDTA and without a prior pelleting step to avoid irreversible sticking of different membrane species. The membrane fractions were characterized by assaying commonly used marker enzymes, and the levels of activity investigated of ATP hydrolysis, ATP-dependent H+ transport, and co- and countertransport of ions, such as Cl- , fumarate2-, K+ and Li+. The following results were obtained: (1) ATP hydrolysis is performed by different enzymes associated with different membranes: - vacuolar acid phosphatase (AP; inhibited by molybdate); - Golgi phosphatase, revealing IDPase, pNPase and ATPase activity (not inhibited by molybdate); - ATPase activity of residual submitochondrial particles (sensitive to azide); - a H+-translocating ATPase at tonoplast membranes (Km(ATP) = 0.29 mᴍ; pH 7.5; stimulated by uncouplers and completely inhibited by NO- 3); - the H+-translocating ATPase of the plasmalemma (Km(ATP) = 0.39 mᴍ at pH 6.5, inhibited by vanadate, but not by NO- 3). The latter activity is evident only after an osmotic shock, indicating that PL vesicles primarily exist as inside-in-vesicles. (2) ATP-fueled H+ pumps are localized at tonoplast (TO) and plasmalemma (PL) vesicles, they differ to some extent in their properties: (a) The PLH+ pump has a very narrow pH optimum and exhibits highest levels of activity at pH 6.5 with a pronounced increase of activity between pH 7.5 and 6.5 (properties, obviously important in vivo for the regulation of active H+-extrusion by certain growth substances, which affect the cytoplasmic pH (Hager and Moser, Planta 1984, in press); in contrast, TO H+ pumps show a considerably wider pH optimum with highest levels of activity around pH 7.5. (b) In variance to the PL H+ pump the activity of TO pumps (Km(ATP) = 0.24 mᴍ) is regulated via the oxidation state of essential thiol groups. Their oxidation to the S -S - form (e.g. by blue light in the presence of a flavin) causes an inactivation, whereas a re-reduction by GSH or cystein restores the activity [51]. (c) The ATP-fueled H+ transport into TO vesicles depends on an anion co-transport; most effective is Cl- , but there is also a stimulation by organic ions, C4 and C5 dicarboxylates, such as malate, succinate, fumarate, 2-oxoglutarate and aspartate; NO- 3 is inhibitory. (d) H+-transport into sealed PL vesicles is also anion dependent. In this case, however, NO- 3 is as effective as Cl-. (3) The TO membranes contain a H+/K+ exchange mechanism responsible for a secondary active K+ uptake into the vacuole. This mechanism could be the reason for a lower (ATP dependent) acidification of TO vesicles in the presence of K+ compared with Li+. - Similar effects are observed with plasmamembrane vesicles, but in this case there is still the question whether a H+/K+-exchange, a K+ channel, or both are acting.

Enzymic and Protein Character of Tonoplast from Hippeastrum Vacuoles

The membrane of anthocyanin containing Hippeatrum petal vacuoles was examined for protein and enzyme content after purification by equilibrium density centrifugation. Light scattering, protein, and a Mg(2+)-dependent nucleotide specific ATPase were associated with membrane having a density of 1.08 to 1.12 grams per cubic centimeter. A small amount of acid phosphatase was also present in this region of the gradient, but this activity peaked at about 1.12 grams per cubic centimeter. A component of yeast tonoplast, alpha-mannosidase, was not significantly present. UDP-glucose, anthocyanidin-3-O-glucosyltransferase, thought to be a cytosol enzyme in Hippeastrum, was absent from tonoplast of vacuoles isolated by osmotic shock in 0.2 molar K(2)HPO(4) or 0.35 molar mannitol. Vacuolar acid phosphatase was insensitive to ethylenediaminetetraacetate but was 80% inhibited by 10 millimolar KF, while ATPase was inactivated by 2 millimolar ethylenediaminetetraacetate and only 50% inhibited by 10 millimolar KF. Five major and about 9 minor polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein on 5 to 30 and 6 to 16% gradient gels.