Identification and characterization of Saccharomyces cerevisiae mutants defective in fluid-phase endocytosis

A mutant library generated by the European Functional Analysis Network (EUROFAN) was screened for strains defective in fluid-phase endocytosis. Accumulation of Lucifer yellow in the vacuole was used as a marker for efficient endocytosis. Fourteen mutants, including ede1Delta, rcy1Delta, sys1Delta and tlg2Delta, previously described to be involved in membrane trafficking, were identified in this screen. alpha-Factor uptake, endocytosis of FM4-64, carboxypeptidase Y secretion, vacuolar morphology, and a vma2 synthetic growth defect were used as criteria to characterize the endocytic defect of the mutant strains obtained. Accordingly, eight mutant strains have endocytic phenotypes in addition to their defect in Lucifer yellow accumulation. These fluid-phase endocytosis mutants are defective at different steps of the endocytic pathway. Interestingly, only two mutants were defective for internalization, two for vacuolar protein sorting and four mutants had aberrant vacuolar morphologies. Some of the mutants identified in this screen that sort carboxypeptidase Y correctly may affect endocytosis at an early post-internalization step before the intersection of the endocytic with the vacuolar protein-sorting pathway.     

Characterization of end4+, a gene required for endocytosis in Schizosaccharomyces pombe

To understand endocytic trafficking in Schizosaccharomyces pombe, we constructed an end4 disruption mutant. The end4+ gene encodes a protein homologous to Sla2p/End4p, which is essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We characterized the fission yeast mutant end4 Delta as well as ypt7 Delta, which is deficient in vacuolar fusion and, hence, endocytosis. The delivery of FM4-64 to the vacuolar membrane, accumulation of Lucifer yellow CH and internalization of plasma membrane protein Map3-GFP were inhibited in the end4 mutant. Deletion of end4 resulted in pleiotropic phenotypes consistent with F-actin depolarization, including high temperature sensitivity, abnormal morphology and mating defects. Extensive missorting of carboxypeptidase Y was detected in the ypt7 mutant; however, little missorting was detected in the end4 mutant. These results indicate that End4p is essential for the internalization process and Ypt7p affects endocytosis at a post-internalization step after the intersection of the endocytic and the vacuolar protein-sorting pathways in fission yeast.     

CaALK8, an alkane assimilating cytochrome P450, confers multidrug resistance when expressed in a hypersensitive strain of Candida albicans

We report the isolation of a novel C. albicans gene designated CaALK8, by its ability to complement drug hypersensitivity of a pdr5 (ABC: ATP-binding cassette drug extrusion pump) null mutant of S. cerevisiae (JG436). CaALK8 in JG436 conferred resistance to drugs such as cycloheximide (CYH), fluconazole (FCZ), O-phenanthroline (PHE) and 4-nitroquinoline oxide (NQO). The gene was so designated because its sequence was identical to a partial sequence entry named as ALK8 in the Candida database (http://alces.med.umn.edu/candida.html). CaALK8 encodes for a putative 515 amino acid protein highly homologous to alkane-inducible cytochromes P450 (CYP52 gene family) of C. maltosa and C. tropicalis. The ability of CaALK8 to confer drug resistance was also established by its expression in another drug-hypersensitive strain of S. cerevisiae (AD 1234568), which was deleted in seven ABC efflux pumps. The homozygous disruption of CaALK8 in a wild-type C. albicans strain (CAI4) did not result in altered drug susceptibilities. The overexpression of CaALK8 in CAI4 resulted in only FCZ resistance. However, a distinct MDR phenotype was evident when CaALK8 was overexpressed in a drug-hypersensitive C. albicans strain disrupted in both CDR1 and CDR2 (ABC drug extrusion pumps of C. albicans). Alk8p, similar to other Alk proteins from C. maltosa and C. tropicalis, could hydroxylate alkanes and fatty acids. In this study we demonstrate that several drugs could compete with the hydroxylation activity by directly interacting with CaAlk8p. Taken together, our results suggest that a member of the CYP52 gene family could mediate MDR in C. albicans, although it does not seem to be involved in the development of azole resistance in clinical isolates. The nucleotide sequence reported in this paper has been submitted to GenBank under Accession No. Y14766.     

Aquaporin in Candida: characterization of a functional water channel protein.

The Candida albicans genome database contains one ORF with homology to aquaporins, AQY1. Xenopus oocytes injected with cRNA encoding C. albicans Aqy1p displayed a coefficient of water permeability (P(f)) that was equivalent to the P(f) for oocytes injected with the cRNA of S. cerevisiae Aqy1p. In addition, as seen in Saccharomyces for Aqy1p and Aqy2p, deletion of AQY1 in C. albicans resulted in cells that were less sensitive than wild-type to osmotic shock. In Saccharomyces, aquaporin null cells also have a cell surface that is more hydrophobic. However, unlike Saccharomyces, there was no effect on the cell surface hydrophobicity, flocculation or cell aggregation in aqy1 null C. albicans cells. Perhaps as a result, there was no difference between the virulence of C. albicans wild-type and aqy1 null strains in a murine model for systemic candidiasis.     

Protective roles of mitochondrial manganese-containing superoxide dismutase against various stresses in Candida albicans

Candida albicans contains copper- and zinc-containing superoxide dismutase but also two manganese-containing superoxide dismutases (MnSODs), one in the cytosol and the other in the mitochondria. Among these, the SOD2 gene encoding mitochondrial MnSOD was disrupted and overexpressed to investigate its roles in C. albicans. The null mutant lacking mitochondrial MnSOD was more sensitive than wild-type cells to various stresses, such as redox-cycling agents, heating, ethanol, high concentration of sodium or potassium and 99.9% O2. Interestingly, the sod2/sod2 mutant was rather more resistant to lithium and diamide than the wild-type, whereas overexpression of SOD2 increased susceptibility of C. albicans to these compounds. The inverse effect of mitochondrial MnSOD on lithium toxicity was relieved when the sod2/sod2 and SOD2-overexpressing cells were grown on the synthetic dextrose medium containing sulphur compounds such as methionine, cysteine, glutathione or sulphite, indicating that mitochondrial MnSOD may affect lithium toxicity through sulphur metabolism. Moreover, disruption or overexpression of SOD2 increased or decreased glutathione reductase activity and cyanide-resistant respiration by alternative oxidase, respectively. Taken together, these findings suggest that mitochondrial MnSOD is important for stress responses, lithium toxicity and cyanide-resistant respiration of C. albicans.     

New phenotypes generated by the G57R mutation of BUD23 in Saccharomyces cerevisiae

BUD23 in Saccharomyces cerevisiae encodes for a class I methyltransferase, and deletion of the gene results in slow growth and random budding phenotypes. Herein, two BUD23 mutants defective in methyltransferase activity were generated to investigate whether the phenotypes of the null mutant might be correlated with a loss in enzymatic activity. Expression at the physiological level of both D77A and G57R mutants was able to rescue the phenotypes of the bud23‐null mutant. The result implied that the methyltransferase activity of the protein was not necessary for supporting normal growth and bud site selection of the cells. High‐level expression of Bud23 (G57R), but not Bud23 or Bud23 (D77A), in BUD23 deletion cells failed to complement these phenotypes. However, just like Bud23, Bud23 (G57R) was localized in a DAPI‐poor region in the nucleus. Distinct behaviour in Bud23 (G57R) could not be originated from a mislocalization of the protein. Over‐expression of Bud23 (G57R) in null cells also produced changes in actin organization and additional septin mutant‐like phenotypes. Therefore, the absence of Bud23, Bud23 (G57R) at a high level might affect the cell division of yeast cells through an as yet unidentified mechanism. Copyright © 2012 John Wiley & Sons, Ltd.     

The cell wall sensor Wsc1p is involved in reorganization of actin cytoskeleton in response to hypo-osmotic shock in Saccharomyces cerevisiae

The cell wall is essential to preserve osmotic integrity of yeast cells. Some phenotypic traits of cell wall mutants suggest that, as a result of a weakening of the cell wall, hypo-osmotic stress-like conditions are created. Consequent expansion of the cell wall and stretching of the plasma membrane trigger a complex response to prevent cell lysis. In this work we examined two conditions that generate a cell wall and membrane stress: one is represented by the cell wall mutant gas1Delta and the other by a hypo-osmotic shock. We examined the actin cytoskeleton and the role of the cell wall sensors Wsc1p and Mid2p in these stress conditions. In the gas1 null mutant cells, which lack a beta(1,3)-glucanosyltransferase activity required for cell wall assembly, a constitutive marked depolarization of actin cytoskeleton was found. In a hypo-osmotic shock wild-type cells showed a transient depolarization of actin cytoskeleton. The percentage of depolarized cells was maximal at 30 min after the shift and then progressively decreased until cells reached a new steady-state condition. The maximal response was proportional to the magnitude of the difference in the external osmolarity before and after the shift within a given range of osmolarities. Loss of Wsc1p specifically delayed the repolarization of the actin cytoskeleton, whereas Wsc1p and Mid2p were essential for the maintenance of cell integrity in gas1Delta cells. The control of actin cytoskeleton is an important element in the context of the compensatory response to cell wall weakening. Wsc1p appears to be an important regulator of the actin network rearrangements in conditions of cell wall expansion and membrane stretching.     

Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

We have screened the EUROFAN (European Functional Analysis Network) deletion strain collection for yeast mutants defective in secretory/vacuolar pathways and/or associated biochemical modifications. We used systematic Western immunoblotting to analyse the electrophoretic pattern of several markers of the secretory/vacuolar pathways, the soluble alpha-factor, the periplasmic glycoprotein invertase, the plasma membrane GPI-anchored protein Gas1p, and two vacuolar proteins, the soluble carboxypeptidase Y and the membrane-bound alkaline phosphatase, which are targeted to the vacuole by different pathways. We also used colony immunoblotting to monitor the secretion of carboxypeptidase Y into the medium, to identify disruptants impaired in vacuolar targeting. We identified 25 mutants among the 631 deletion strains. Nine of these mutants were disrupted in genes identified in recent years on the basis of their involvement in trafficking (VPS53, VAC7, VAM6, APM3, SYS1), or glycosylation (ALG12, ALG9, OST4, ROT2). Three of these genes were identified on the basis of trafficking defects by ourselves and others within the EUROFAN project (TLG2, RCY1, MON2). The deletion of ERV29, which encodes a COPII vesicle protein, impaired carboxypeptidase Y trafficking from the endoplasmic reticulum to the Golgi apparatus. We also identified eight unknown ORFs, the deletion of which reduced Golgi glycosylation or impaired the Golgi to vacuole trafficking of carboxypeptidase Y. YJR044c, which we identified as a new VPS gene, encodes a protein with numerous homologues of unknown function in sequence databases.     

ACTIVITY OF THE VACUOLAR PROTEASES OF YEAST AND THE SIGNIFICANCE OF THE CYTOSOLIC PROTEASE INHIBITORS DURING THE POST-FERMENTATION DECLINE PHASE

The vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y, together with inhibitors for proteinase B and carboxypeptidase Y, have been identified in a brewing strain of Saccharomyces cerevisiae fermenting a malt extract wort at 30°C. The specific activity on a protein basis measured in freshly prepared extracts increased for all three enzymes during the post-fermentation decline phase but on a per cell basis the total activity, measured after removal of any inhibitor by autocatalysis, fell steadily. No evidence was found for the presence of proteinase A inhibitor at any point but the cells contained considerable amounts of proteinase B inhibitor especially during the growth and fermentation phases. Proteinase B only became detectable in freshly prepared extracts after a distinct loss in cell viability had become obvious. The situation for carboxypeptidase Y was intermediate between that for proteinase A and proteinase B. At high cell concentrations leakage of proteinase B and its inhibitor into the medium occurred but neither of the other two proteases were detectable outside the cells .     

Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans

A role for the cAMP-dependent pathway in regulation of the cell wall in the model yeast Saccharomyces cerevisiae has recently been demonstrated. In this study we report the results of a phenotypic analysis of a Candida albicans mutant, characterized by a constitutive activation of the cAMP pathway due to deletion of PDE2, the gene encoding the high cAMP-affinity phosphodiesterase. Unlike wild-type strains, this mutant has an increased sensitivity to cell wall and membrane perturbing agents such as SDS and CFW, and antifungals such as amphotericin B and flucytosine. Moreover, the mutant is characterized by an altered sensitivity and a significantly reduced tolerance to fluconazole. The mutant's membrane has around 30% higher ergosterol content and the cell wall glucan was 22% lower than in the wild-type. These cell wall and membrane changes are manifested by a considerable reduction in the thickness of the cell wall, which in the mutant is on average 60-65 nm, compared to 80-85 nm in the wild-type strains as revealed by electron microscopy. These results suggest that constitutive activation of the cAMP pathway affects cell wall and membrane structure, and biosynthesis, not only in the model yeast S. cerevisiae but also in the human fungal pathogen C. albicans.     

Effect of ethanol on the production of carboxypeptidase Y using the GAL10 promoter in a Saccharomyces cerevisiae gal80 mutant

In the course of studying carboxypeptidase Y (CPY) production, we found that the expression level of the gene, which is under the control of the GAL10 promoter, increased in a Saccharomyces cerevisiae gal80 mutant grown in a medium containing ethanol as the sole carbon source. In the cultivation of the gal80 mutant KS58-2D/pCY303 carrying a multicopy plasmid, which contains the PRC1 gene fused to the GAL10 promoter, CPY production continued after the consumption of galactose. In this phase, the cells utilized ethanol as the carbon source. To increase the CPY production level, we examined the effect of carbon source feeding in a fed-batch culture. The production level in the fed-batch culture using ethanol was 1.3-fold higher than that in a batch culture and 1.6-fold higher than that in a fed-batch culture using galactose. By 5'-deletion analysis of the GAL10 promoter, the region between -256 and -232 was found to be important for the promoter activity in the gal80 mutant growing in the presence of ethanol.     

Characterization of Candida albicans orthologue of the Saccharomyces cerevisiae signal-peptidase-subunit encoding gene SPC3

The Candida albicans orthologue of the SPC3 gene, which encodes one of the subunits essential for the activity of the signal peptidase complex in Saccharomyces cerevisiae, was isolated by complementation of a thermosensitive mutation in the S. cerevisiae SEC61 gene. The cloned gene (CaSPC3) encodes a putative protein of 192 amino acids that contains one potential membrane-spanning region and shares significant homology with the corresponding products from mammalian (Spc22/23p) and yeast (Spc3p) cells. CaSPC3 is essential for cell viability, since a hemizygous strain containing a single copy of CaSPC3 under control of the methionine-repressible MET3 promoter did not grow in the presence of methionine and cysteine. The cloned gene could rescue the phenotype associated with a spc3 mutation in S. cerevisiae, indicating that it is the true C. albicans orthologue of SPC3. However, in contrast with results previously described for its S. cerevisiae orthologue, CaSPC3 was not able to complement the thermosensitive growth associated with a mutation in the SEC11 gene. The heterologous complementation of the sec61 mutant suggests that Spc3p could play a role in the interaction that it is known to occur between the translocon (Sec61 complex) and the signal peptidase complex, at the endoplasmic reticulum membrane.     

Glucose metabolism and ethanol production in adh multiple and null mutants of Kluyveromyces lactis

Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten-fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen-limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants.     

Molecular cloning of CaYRB1, the Candida albicans RanBP1/YRB1 homologue.

The yeast Ran binding protein 1 (Yrb1p) is a small protein of 23 kDa that is highly conserved among eukaryotes. It stimulates the GTPase activity of Gsp1p in the presence of the GTPase activating protein Rna1p. In addition to its role in nucleocytoplasmic transport of macromolecules, YRB1/RanBP1 could be involved in the regulation of microtubules structure and dynamics. Since microtubules are tightly associated with morphological changes, we have been interested to study the role and function of YRB1 in the pathogenic fungus Candida albicans, where there is regulated change in cellular morphology. The gene product of CaYRB1 encodes a 212 amino acid protein displaying 73% homology to the S. cerevisiae homologue. The bacterially expressed gene product has an apparent molecular weight of 35.7 kDa. We show that it can complement a S. cerevisiae yrb1 null mutant and that its mRNA does not appear to be regulated in response to conditions inducing morphological changes in C. albicans.     

Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173.