A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis

To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative alpha-glucan synthase, is strongly induced in response to cell wall stress. By placing the agsA promoter region in front of a selectable marker, the acetamidase (amdS) gene of A. nidulans, we reasoned that cell wall mutants with a constitutively active cell wall stress response pathway could be identified by selecting mutants for growth on acetamide as the sole nitrogen source. For the genetic screen, a strain was constructed that contained two reporter genes controlled by the same promoter: the metabolic reporter gene PagsA-amdS and PagsA-H2B-GFP, which encodes a GFP-tagged nuclear protein. The primary screen yielded 161 mutants that were subjected to various cell wall-related secondary screens. Four calcofluor white-hypersensitive, osmotic-remediable thermosensitive mutants were selected for complementation analysis. Three mutants were complemented by the same gene, which encoded a protein with high sequence identity with eukaryotic UDP-galactopyranose mutases (UgmA). Our results indicate that galactofuranose formation is important for fungal cell wall biosynthesis and represents an attractive target for the development of antifungals.     

Involvement of MoVMA11, a Putative Vacuolar ATPase c’ Subunit,in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae

Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaporthe oryzae , subcellular colocalization and quinacrine staining suggested that the V₁V₀ domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c’ subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M . oryzae .     

A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.     

Characterization of Synthetic-Lethal Mutants Reveals a Role for the Saccharomyces Cerevisiae Guanine-Nucleotide Exchange Factor Cdc24p in Vacuole Function and Na(+) Tolerance

Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae. To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24 synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4(ts) at 23°. Five mutants were not complemented by plasmid-borne CDC42, RSR1, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity. The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet(-) vma mutants defective in vacuole function. CSL5 was allelic to VMA5, the vacuolar H(+)-ATPase subunit C, and one third of csl5 cdc24-4(ts) cells were elongated or had misshapen buds. A cdc24-4(ts) Δvma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4(ts) synthetic-lethality was not simply due to altered vacuole function. The cdc24-4(ts) mutant, like Δvma5::LEU2 and csl3 mutants, was sensitive to high levels of Ca(2+) as well as Na(+) in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 M sorbitol. Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na(+) tolerance.     

Vacuolar H+-ATPase is involved in preventing heavy metal-induced oxidative stress in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, vacuolar H⁺-ATPase (V-ATPase) involved in the regulation of intracellular pH homeostasis has been shown to be important for tolerances to cadmium, cobalt and nickel. However, the molecular mechanism underlying the protective role of V-ATPase against these metals remains unclear. In this study, we show that cadmium, cobalt and nickel disturbed intracellular pH balance by triggering cytosolic acidification and vacuolar alkalinization, likely via their membrane permeabilizing effects. Since V-ATPase plays a crucial role in pumping excessive cytosolic protons into the vacuole, the metal-sensitive phenotypes of the Δvma2 and Δvma3 mutants lacking V-ATPase activity were supposed to result from highly acidified cytosol. However, we found that the metal-sensitive phenotypes of these mutants were caused by increased production of reactive oxygen species, likely as a result of decreased expression and activities of manganese superoxide dismutase and catalase. In addition, the loss of V-ATPase function led to aberrant vacuolar morphology and defective endocytic trafficking. Furthermore, the sensitivities of the Δvma mutants to other chemical compounds (i.e. acetic acid, H₂O₂, menadione, tunicamycin and cycloheximide) were a consequence of increased endogenous oxidative stress. These findings, therefore, suggest the important role of V-ATPase in preventing endogenous oxidative stress induced by metals and other chemical compounds.     

Altered distribution of the yeast plasma membrane H+-ATPase as a feature of vacuolar H+-ATPase null mutants

The effect of vacuolar H(+)-ATPase (V-ATPase) null mutations on the targeting of the plasma membrane H(+)-ATPase (Pma1p) through the secretory pathway was analyzed. Gas1p, which is another plasma membrane component, was used as a control for the experiments with Pma1p. Contrary to Gas1p, which is not affected by the deletion of the V-ATPase complex in the V-ATPase null mutants, the amount of Pma1p in the plasma membrane is markedly reduced, and there is a large accumulation of the protein in the endoplasmic reticulum. Kex2p and Gef1p, which are considered to reside in the post-Golgi vesicles, were suggested as required for the V-ATPase function; hence, their null mutant phenotype should have been similar to the V-ATPase null mutants. We show that, in addition to the known differences between those yeast phenotypes, deletions of KEX2 or GEF1 in yeast do not affect the distribution of Pma1p as the V-ATPase null mutant does. The possible location of the vital site of acidification by V-ATPase along the secretory pathway is discussed.     

Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects

The osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature. Here, we show that the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for ste11ssk2ssk22. Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37 degrees C. This phenotype is suppressed by overexpression of LRE1, HLR1 and WSC3, all genes known to influence cell wall composition. The suppression of the temperature-induced hyperosmosensitivity by these genes prompted us to investigate the role of STE11 and other HOG pathway components in cellular integrity and, indeed, we were able show that HOG pathway mutants display sensitivity to cell wall-degrading enzymes. LRE1 and HLR1 were also shown to suppress the cell wall phenotypes associated with the HOG pathway mutants. In addition, the isolated multicopy suppressor genes suppress temperature-induced cell lysis phenotypes of PKC pathway mutants that could be an indication for shared targets of the PKC pathway and high-osmolarity response routes.     

A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of beta1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect beta1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.     

Dependence of Saccharomyces cerevisiae Golgi functions on V-ATPase activity

The V-ATPase of Saccharomyces cerevisiae is an ATP-dependent proton pump responsible for acidification of the vacuole and other internal compartments including the whole secretory pathway. We have studied the behavior of several glycoprotein processing reactions occurring in different Golgi compartments of representative vmaΔ mutants. We found that outer chain initiation is not altered in the mutants while mannosylphosphate transfer, α(1,3)-linked mannoses addition, and α factor maturation seem to be affected. The results suggest a gradation in the dependence of Golgi functions on V-ATPase activity, from early Golgi (unaffected) to late Golgi (significantly reduced). These findings are in agreement with the internal pH of Golgi cisternae measured in mammalian cells, which is more acidic in the late region. The mutant defects can be partially restored by buffering the external medium to pH 6.0, which supports the existence of a mechanism that, in the absence of a functional V-ATPase, could contribute to pH regulation at least in the late Golgi.     

Hph1 and Hph2 are novel components of the Sec63/Sec62 posttranslational translocation complex that aid in vacuolar proton ATPase biogenesis

Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1Δ hph2Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1Δ hph2Δ and hph1Δ hph2Δ sec71Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1Δ hph2Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.     

Degradation of the gluconeogenic enzyme fructose-1, 6-bisphosphatase is dependent on the vacuolar ATPase

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced during glucose starvation. After the addition of glucose, inactivated FBPase is selectively targeted to Vid (vacuolar import and degradation) vesicles and then to the vacuole for degradation. To identify proteins involved in this pathway, we screened various libraries for mutants that failed to degrade FBPase. Via these approaches, subunits of the vacuolar- H+ -ATPase (V-ATPase) have been identified repeatedly. The V-ATPase has established roles in endocytosis, sorting of carboxypeptidase Y and homotypic vacuole fusion. Here, we show that mutants lacking Stv1p, Vph1p, and other subunits of the V-ATPase are defective for FBPase degradation. FBPase was detected in Vid vesicles. However, most FBPase was resistant to proteinase K digestion in the Deltavph1 or vma mutants, whereas the majority of FBPase was sensitive to proteinase K digestion in the Deltastv1 mutant. Therefore, STV1 and VPH1 have distinct functions in FBPase degradation. In cells lacking V0 genes, Vma2p and Vma5p were still detected on Vid vesicles and vacuoles, suggesting that the distribution of V1 proteins is independent of V0 genes. The V0 and V1 domains are assembled following a glucose shift and the assembly is not regulated by protein kinase A and RAV genes. Assembly of the V0 complex is necessary for FBPase trafficking, since mutants that block the assembly and transport of V0 out of the ER were defective in FBPase degradation.     

Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics.     

A genomic screen for yeast vacuolar membrane ATPase mutants

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, approximately 4700 yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl(2). In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36Delta mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34Delta, vps15Delta, vps45Delta, and vps16Delta, caused both Vma- growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1Delta mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a Vma- growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the Vma- growth phenotype.     

Cell wall integrity is dependent on the PKC1 signal transduction pathway in Cryptococcus neoformans

Cell wall biogenesis and integrity are crucial for fungal growth, pathogenesis and survival, and are attractive targets for antifungal therapy. In this study, we identify, delete and analyse mutant strains for 10 genes involved in the PKC1 signal transduction pathway and its regulation in Cryptococcus neoformans. The kinases Bck1 and Mkk2 are critical for maintaining integrity, and deletion of each of these causes severe phenotypes different from each other. In stark contrast to results seen in Saccharomyces cerevisiae, a deletion in LRG1 has severe repercussions for the cell, and one in ROM2 has little effect. Also surprisingly, the phosphatase Ppg1 is crucial for cell integrity. These data indicate that the mechanisms of maintaining cell integrity differ between the two fungi. Deletions in SSD1 and PUF4, potential alternative regulators of cell integrity, also exhibit phenotypes. This is the first comprehensive analysis examining genes involved the maintenance of cell integrity in C. neoformans and sets the foundation for future biochemical and virulence studies.     

Consequences of loss of Vph1 protein-containing vacuolar ATPases (V-ATPases) for overall cellular pH homeostasis

In yeast cells, subunit a of the vacuolar proton pump (V-ATPase) is encoded by two organelle-specific isoforms, VPH1 and STV1. V-ATPases containing Vph1 and Stv1 localize predominantly to the vacuole and the Golgi apparatus/endosomes, respectively. Ratiometric measurements of vacuolar pH confirm that loss of STV1 has little effect on vacuolar pH. Loss of VPH1 results in vacuolar alkalinization that is even more rapid and pronounced than in vma mutants, which lack all V-ATPase activity. Cytosolic pH responses to glucose addition in the vph1Δ mutant are similar to those in vma mutants. The extended cytosolic acidification in these mutants arises from reduced activity of the plasma membrane proton pump, Pma1p. Pma1p is mislocalized in vma mutants but remains at the plasma membrane in both vph1Δ and stv1Δ mutants, suggesting multiple mechanisms for limiting Pma1 activity when organelle acidification is compromised. pH measurements in early prevacuolar compartments via a pHluorin fusion to the Golgi protein Gef1 demonstrate that pH responses of these compartments parallel cytosolic pH changes. Surprisingly, these compartments remain acidic even in the absence of V-ATPase function, possibly as a result of cytosolic acidification. These results emphasize that loss of a single subunit isoform may have effects far beyond the organelle where it resides.     

Mating type regulation of cellular tolerance to DNA damage is specific to the DNA post-replication repair and mutagenesis pathway

In order to help further define DNA post-replication repair (PRR), a conditional synthetic lethal screen was employed to identify new genes involved in the PRR pathway. A synthetic lethal screen with the mms2 mutation resulted in the recovery of two suppressor mutations responsible for regulating PRR. The recovered suppressors are the mating type genes and SIR3. Indeed, controlled expression of both mating type genes or deletion of SIR3 rescued the conditional synthetic lethal mutant phenotypes. Furthermore, comprehensive analyses suggest that mating type heterozygosity confers tolerance to a broad range of DNA damage, and that this effect is limited to all PRR pathway mutations, but does not apply to base excision repair, nucleotide excision repair or recombination repair mutants. In addition, the tolerance conferred to PRR mutants as a result of mating type heterozygosity is dependent on a functional homologous recombination but not the non-homologous end-joining pathway. Thus, mating type status appears to be responsible for signalling DNA content and possibly cell cycle stage, allowing the cell to select the most efficient means to repair the DNA damage.     

Expression of agsA, one of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger, is induced in response to cell wall stress

1,3-alpha-D-Glucan is an important component of the cell wall of filamentous fungi. We have identified a family of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger. The agsA gene was sequenced and the predicted protein sequence indicated that the overall domain structure of 1,3-alpha-D-glucan synthases is conserved in fungi. Using RT-PCR and Northern blot analysis, we found that expression of the agsA gene and to a lesser extent also of agsE were induced in the presence of the cell wall stress-inducing compounds such as Calcofluor White (CFW), SDS, and caspofungin. Loss of agsA function did not result in an apparent phenotype under normal growth conditions but rendered the cells more sensitive to CFW. The induction of 1,3-alpha-D-glucan synthase-encoding genes in response to cell wall stress was not limited to A. niger, but was also observed in Penicillium chrysogenum. We propose that this response to cell wall stress commonly occurs in filamentous fungi.