| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan
2 Research Center, Asahi Glass Co. Ltd, Yokohama, Kanagawa 221-8755, Japan
3 National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi Hiroshima, Hiroshima 739-0046, Japan
4 Department of Chemistry, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi Hiroshima, Hiroshima 739-8526, Japan
Correspondence
Kaoru Takegawa
takegawa{at}ag.kagawa-u.ac.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in erg6 but not in other erg mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the erg mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in Saccharomyces cerevisiae. Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins.
Three supplementary figures are available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Like cholesterol in mammalian cells, it is responsible for membrane fluidity and permeability (Parks et al., 1995).
|
). Multiple genetic and biochemical studies have culminated in the virtually complete elucidation of the pathway leading to ergosterol (Lees et al., 1995; Parks et al., 1995). The enzymic reactions have been largely defined by identification of erg mutants defective in ergosterol biosynthesis, and by their complementation based on sterol auxotrophy or altered sterol composition. Most genes involved in the early part of the pathway to lanosterol are essential for growth, because yeasts require sterols and no sterol molecule is synthesized up to this point. In contrast, mutations in the steps from zymosterol to ergosterol (Fig. 1b
) are not essential for growth because the intermediates produced can partially substitute for ergosterol. The latter part of the ergosterol biosynthetic pathway is not linear in the sense of consecutive reactions, because the enzymes converting lanosterol to ergosterol do not show a strict substrate preference. Thus, null mutants impaired in the steps from zymosterol to ergosterol accumulate characteristic sterols, which are not defined substrates of the respective enzymes.
Recent studies have identified a membrane microdomain rich in sterols and sphingolipids (Simons & van Meer, 1988; Simons & Ikonen, 1997; Sturley, 2000). This membrane microdomain is called the lipid raft, which corresponds to a related structure, the caveola, in mammalian membranes. Many functions have been attributed to these structures, including cholesterol transport, endocytosis and signal transduction (Alonso & Millán, 2001; Pelkmans, 2005; Maguy et al., 2006; Wachtler & Balasubramanian, 2006). The most widely used assay for rafts is based on the observation that a subset of associated plasma membrane components is resistant to nonionic detergents, such as Triton X-100, at 4 °C (Bagnat et al., 2000). Lipid rafts are also called detergent-resistant membranes (DRMs) because of their detergent resistance. However, lipid rafts and DRMs can theoretically be separated, as detergent solubilization may involve the formation of nonphysiological structures (Munro, 2003; Lichtenberg et al., 2005).
In Sacch. cerevisiae and Candida species, many experiments have demonstrated a role for ergosterol in physiological functions, such as membrane permeability, resistance to drugs, protein transport to the plasma membrane, sporulation and endocytosis (Geber et al., 1995; Parks et al., 1999; Young et al., 2003; Sanglard et al., 2003; Pasrija et al., 2005a, b; Mukhopadhyay et al., 2002; Proszynski et al., 2005; Enyenihi & Saunders, 2003; Munn et al., 1999; Heese-Peck et al., 2002; Kishimoto et al., 2005). In addition, an association between many plasma membrane proteins and DRMs has been studied and some DRM-associated proteins have been identified (Dupré & Haguenauer-Tsapis, 2003; Bagnat et al., 2000, 2001; Umebayashi & Nakano, 2003; Malinska et al., 2004; Grossmann et al., 2006; Lauwers & André, 2006). While there is no doubt that DRMs are rich in ergosterol, it is not at all clear if ergosterol is essential for the association between DRMs and plasma membrane proteins (Eisenkolb et al., 2002; Gaigg et al., 2005).
In Schizosaccharomyces pombe, a physiological change caused by a genetic defect in ergosterol metabolism is known only in the sts1 mutant, encoding an Erg4 homologue (Shimanuki et al., 1992). Nonetheless, genes encoding the latter part of the ergosterol biosynthetic pathway have been predicted through studies of transcriptional activation under anaerobic conditions (see Fig. 1b and Table 1 of Todd et al., 2006). It is not known whether these genes are truly involved in the ergosterol biosynthetic pathway, nor is it clear what specific defects are caused by loss of ergosterol in fission yeast. To address these questions, disruption mutants of six sterol biosynthesis genes were constructed. These mutants were found to be deficient in ergosterol and to be resistant to polyene drugs, although no striking defects in endocytosis were observed.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
(Takegawa et al., 1995) and MTD2 (cpy1; Tabuchi et al., 1997) were used as positive and negative controls, respectively, for the colony blot assay. Strains were grown in standard rich medium (YES), and in synthetic minimal medium (MM). Sporulation medium (ME) was used as described by Moreno et al. (1991). Schiz. pombe cells were transformed by the lithium acetate method or by electroporation (Suga & Hatakeyama, 2001; Morita & Takegawa, 2004). Standard genetic methods have been described previously (Alfa et al., 1993).
Gene disruptions.
Genes encoding ergosterol biosynthesis proteins were cloned into pGEM T-EASY (erg31+, erg32+ and erg5+) or pGEM T (erg6+ and sts1+) vectors (Promega) following PCR amplification using the primers listed in Table 1. The resulting plasmids were digested with restriction enzymes, followed by insertion of a ura4+ gene cassette to generate gene disruption constructs. For erg2+, 0.6 kb sequences of promoter and terminator regions were amplified by PCR, and sequentially cloned into KpnI–XhoI sites (promoter) and EcoRI–BamHI sites (terminator) of pBluescript II-KS (Stratagene) carrying a ura4+ cassette at the ClaI site. Wild-type strain ARC039 was transformed with the PCR products amplified from these constructs. Gene disruptions were confirmed by PCR using appropriate primers.
|
. Harvested cells were washed twice with distilled water and then resuspended in 3 ml KOH/methanol solution (20 % KOH in 100 % methanol) with a small amount of pyrogallol. After heating for 2 h at 85 °C, total sterols in the reaction mixture were extracted twice with 2 ml petroleum ether. The organic phases were combined, dried under nitrogen atmosphere, and stored at –20 °C before use. Before separation by reverse-phase HPLC, dried sterols were resuspended in 0.5 ml methanol. HPLC analysis was performed using a reverse-phase HPLC column (TSKgel ODS80Ts; Tosoh) with methanol as eluent at 40 °C, 1 ml min–1 solvent flow, and detection at 280 nm.
Resistance assays.
Cells cultured overnight in 5 ml YES medium were diluted with water to an OD600 of 0.5, corresponding to about 107 cells ml–1 and used as inoculum. Cell suspensions were serially diluted 1 : 10 and 1 : 100, and 5 µl aliquots were spotted onto plates containing potential inhibitors at the indicated concentrations. Growth was scored after 3 days.
Analyses of carboxypeptidase Y (CPY).
CPY missorted to the medium was detected by colony blot assay as described by Cheng et al. (2002). Briefly, yeast strains were replica-plated on a YES plate in contact with a nitrocellulose membrane and then incubated at 30 °C for 2 days. The nitrocellulose membranes were washed several times with water and then subjected to immunodetection with rabbit antiserum against CPY (Tabuchi et al., 1997) and anti-rabbit IgG–horseradish peroxidase (GE Healthcare). Visualization was enhanced by the ECL system (GE Healthcare).
Pulse–chase analysis and immunoprecipitation of vacuolar CPY from Schiz. pombe were carried out as described by Tabuchi et al. (1997). Antibody incubations were carried out using rabbit polyclonal antibody against Schiz. pombe Cpy1p (Tabuchi et al., 1997).
Visualization of sterol-rich plasma membrane domain.
Staining with filipin was carried out as described by Wachtler et al. (2003). Filipin was added to the medium at a final concentration of 5 µg ml–1 and cells were observed immediately using a fluorescence microscope (model BX-60; Olympus).
Vacuole staining.
Vacuolar membranes were labelled with FM4-64 (Iwaki et al., 2003). Cells were grown to exponential phase in YES medium at 30 °C, and 500 µl of cells was then incubated in medium containing 8 µM FM4-64 for 30 min at 30 °C. Cells were then centrifuged at 13 000 g for 1 min, washed by resuspending in YES to remove free FM4-64, and collected by centrifugation at 13 000 g for 1 min. The cells were resuspended in YES and incubated for 90 min at 30 °C before microscopic observation. Stained cells were observed using a fluorescence microscope. For measurement of vacuoles after fusion in response to hypotonic stress, stained cells were incubated in distilled water for 6 h to ensure full fusion. The diameter of every vacuole visible in one focal plane per cell was measured using NIH-image software and downloaded to Microsoft Excel for analysis (more than 100 vacuoles were counted).
Analysis of fluid-phase endocytosis.
Fluid-phase endocytosis was observed microscopically after cells were treated with Lucifer Yellow CH (LY, Sigma). Staining with LY was performed as described by Murray & Johnson (2001). Briefly, 1 ml of exponentially growing cells in YES medium was collected by centrifugation, washed twice with fresh medium, and resuspended in 0.5 ml YES medium containing 5 mg LY ml–1. Cells were incubated at 30 °C for 60 min with shaking and then washed three times with fresh medium. Labelled cells were then examined by microscopy.
Fluorescence microscopy.
Cells were observed with an Olympus BX-60 fluorescence microscope using appropriate filter sets (Olympus). Images were captured with a Sensys Cooled CCD camera using MetaMorph (Roper Scientific), and were saved as Adobe Photoshop files on a Macintosh G4 computer.
Plasmid constructs.
To tag the C terminus of Pma1p with green fluorescent protein (GFP), the pma1+ ORF was amplified by PCR and cloned into pTN197, a derivative of the thiamine-repressible expression vector pREP41 (Nakamura et al., 2001). To tag SPBC359.03c with GFP, the SPBC359.03c ORF was amplified by PCR and subcloned into pTN197, resulting in plasmid pTN197/ SPBC359.03c.
DRM isolation and immunoblotting.
DRM was isolated as described by Bagnat et al. (2000). One hundred OD600 units of cells was collected, washed twice with distilled water, and stored at –80 °C. The cell pellet was then lysed in 0.5 ml TNE buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA] containing a protease inhibitor mix (Nakarai tesque) by vortexing with glass beads five times for 1 min with 1 min intervals on ice. The lysate was cleared by centrifugation at 500 g for 5 min. A 0.75 ml aliquot of TNE buffer was added to 0.25 ml of the lysate, and then incubated with Triton X-100 (TX-100; 1 % final) for 30 min on ice. After extraction of TX-100, the lysate (1 ml) was adjusted to 40 % Optiprep by addition of 2 ml Optiprep solution (Nycomed) and overlaid with 4.8 ml 30 % Optiprep in TXNE (TNE with 0.1 % TX-100) and 0.8 ml TXNE. The samples were centrifuged at 200 000 g for 2 h, and six fractions of equal volume were collected from the top. The top fraction was subjected to a second incubation with TX-100, loaded onto a second Optiprep gradient, and centrifuged again. Fractions from gradients were precipitated with 10 % (w/v) TCA, dissolved in appropriate volumes of cracking buffer [8 M urea, 5 % (w/v) SDS, 1 mM EDTA, 50 mM Tris/HCl (pH 6.8), 5 % (v/v) 2-mercaptoethanol], and incubated at 65 °C for 20 min. Samples (10 µl) were separated by SDS-PAGE and transferred to PVDF filters. Rabbit polyclonal antibody against GFP, a generous gift from Dr R. Sugiura (Kinki University), was used at a 1 : 10 000 dilution. Protein–antibody complexes were visualized by chemiluminescence using the Amersham ECL plus system (GE Healthcare).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, see also Supplementary Fig. S1, available with the online version of this paper). In order to determine whether the products of these genes catalysed conversion of zymosterol to ergosterol, gene disruption mutants were constructed. Total sterols extracted from stationary-phase cells of wild-type and the gene disruption mutants were compared by reversed-phase HPLC analyses. These analyses revealed that the single disruption mutants, erg31 and erg32, could synthesize ergosterol (Supplementary Fig. S2), indicating that these proteins were functionally redundant. Therefore, a double disruption mutant, erg31erg32 was also constructed.
HPLC elution patterns are shown in Fig. 2. Ergosterol was not detected in the erg6, erg5 or sts1 mutants. No clear peaks were detected in the erg2 and erg31erg32 mutants by absorption at 280 nm (data not shown). The sts1 mutant accumulated ergosta-5,7,22,24(28)-tetraenol, while the other mutants accumulated other sterols.
|
cells could not be stained with filipin (Malathi et al., 2004). In Schiz. pombe erg cells, fluorescence was detected at the plasma membrane and sterols were enriched at the growing cell tips, as observed in the wild-type (Fig. 3). Although the intensity of fluorescence was lower in the erg mutants, except for sts1, the staining patterns were similar to wild-type (Fig. 3). These results indicated that ergosterol derivatives partly substitute for ergosterol and were transported to the plasma membrane. On the other hand, erg6 cells had an abnormal cellular morphology, and more than one septum was found in some of the erg2 cells.
|
mutants mutant exhibited sensitivity to 15 µg cycloheximide (CHX) ml–1 (Fig. 4), 100 mM CaCl2 and 0.01 % (w/v) SDS (data not shown), as previously reported (Shimanuki et al., 1992). All the ergosterol-deficient mutants were found to be sensitive to CHX and to 1 µg staurosporine ml–1, and tolerant to 10 µg nystatin ml–1 and to 1 µg amphotericin B ml–1 (Fig. 4). The erg31 single mutant grew almost as well as wild-type, while the erg32 single mutant exhibited slight sensitivity to staurosporine and tolerance to nystatin and amphotericin B. These results indicated that Erg31p and Erg32p have redundant functions and that Erg32p might be a major C-5 sterol desaturase. The polyene drugs nystatin and amphotericin B bind ergosterol, creating pores and causing cell lysis. Therefore, the tolerance to these drugs reflects the ergosterol deficiency of erg mutants. However, the sts1 mutant was not resistant to nystatin, suggesting that nystatin binds ergosta-5,7,22,24(28)-tetraenol.
|
; Munn et al., 1999; Heese-Peck et al., 2002), even though vacuolar morphology and CPY maturation are known to be closely related (Raymond et al., 1992). Therefore, we determined if these vesicular trafficking events had a sterol requirement. Missorting of CPY was determined by colony blot assay, because some mutants defective in CPY transport to the vacuole missort significant amounts of CPY to the cell surface. A fraction of CPY was detected in erg6 and erg2 cells, although CPY secretion was not detected in erg31erg32, erg5 or sts1 cells (Fig. 5a). In order to determine whether CPY secretion in erg6 and erg2 cells was caused by lytic release, pulse–chase analysis was performed. During the initial 15 min of labelling, the endoplasmic reticulum- and Golgi-specific precursor form (proCPY) and a small amount of the vacuole-specific mature form (mCPY) were produced in the wild-type. After a 30 min chase, proCPY was almost completely converted to the mature form (Fig. 5b). Processing defects were not found in erg6 and erg2 cells. These results indicate that CPY secretion was due to lytic release, because it has been suggested that CPY is processed to a mature form by unidentified proteases in the vacuole (Tabuchi et al., 1997).
|
cells other than erg5 had slightly smaller vacuoles (Fig. 6a, c). Vacuoles in wild-type cells had a mean diameter of 0.80±0.14 µm, while the means in erg cells were as follows: erg6, 0.70±0.18 µm; erg2, 0.52±0.09 µm; erg31erg32, 0.60±0.11 µm; erg5, 0.7±0.15 µm; erg4, 0.60±0.09 µm. When cells were transferred to water, a smaller number of much larger vacuoles were observed as a result of vacuolar fusion (Bone et al., 1998). All of the ergosterol-deficient mutants were able to undergo normal vacuolar fusion in response to hypotonic stress (Fig. 6b, c), in contradiction with a previous study reporting an ergosterol requirement for homotypic vacuolar fusion (Kato & Wickner, 2001). Vacuoles in wild-type cells had a mean diameter of 1.24±0.43 µm, while the means in erg cells were as follows: erg31erg32, 1.07±0.28 µm; sts1, 1.08±0.34 µm. Vacuolar fusion in erg5 occurred to the same extent as in wild-type cells (Fig. 6a, b), so measurement of erg5 vacuoles was omitted. Vacuoles of erg6 and erg2 could not be measured because some were not circular in shape. The difference in mean diameter between pre-fusion and post-fusion was 0.44 (wild-type) to 0.48 µm (sts1). Therefore, we conclude that homotypic vacuolar fusion was not affected by loss of ergosterol.
|
; Heese-Peck et al., 2002; Pichler & Riezman, 2004). In order to determine whether erg mutants are impaired in endocytosis, vacuolar accumulation of the soluble fluorescent dye Lucifer Yellow CH (LY) was assayed. In assays for fluid-phase endocytosis, cells were incubated with LY at 30 °C for 1 h and observed by fluorescence microscopy. Endocytic uptake of dye resulted in LY accumulation in vacuoles. Vacuolar accumulation of LY in all the erg mutants was similar to that observed in wild-type cells (Fig. 7).
|
mutants mutants. Pma1p is a P-type H+-ATPase that localizes to the plasma membrane and has been reported to associate with DRM (Takeda et al., 2004). Pma1-GFP was found to localize to the cell surface in all erg cells (Fig. 8a). Crude membrane was solubilized with 1 % Triton X-100 and fractionated after discontinuous gradient centrifugation using Optiprep. Each fraction was analysed by Western blotting using rabbit anti-GFP serum. In wild-type cells, Pma1-GFP was detected in the TX-100-insoluble fraction even after the second gradient centrifugation, in agreement with a previous report (Takeda et al., 2004). In most of the erg mutants, the distribution of Pma1-GFP was similar to that in wild-type cells, although it was found in a broad range of fractions in erg6 cells (Fig. 8b). These results indicated that association of Pma1-GFP to DRM was weakened in erg6 cells.
|
mutants. GFP-tagged ATP-binding cassette (ABC) transporters Bfr1-GFP and Pmd1-GFP were found to localize normally in the plasma membrane (Iwaki et al., 2006) (Supplementary Fig. S3). One of the amino acid permease homologues, SPBC359.03c, has been reported to localize to the plasma membrane when expressed as a C-terminal GFP-tagged allele (Matsumoto et al., 2002). GFP-tagged SPBC359.03c (SPBC359.03c-GFP) localized mainly to the plasma membrane in wild-type cells (Fig. 9). In erg mutants, it localized to numerous tiny dots within the cytoplasm and could not be found in the plasma membrane in stationary-phase cells (Fig. 9). These observations indicate that mutations in the erg genes have a modest affect on membrane structure and may alter localization of some, but not all, plasma membrane proteins. Moreover, these observations indicate that plasma membrane proteins can be divided into two classes, i.e. sensitive or resistant to changes in sterol composition, although the cause of these differences is currently unknown.
|
mutants cells sporulate inefficiently (Enyenihi & Saunders, 2003). In order to determine the sporulation efficiency in Schiz. pombe erg mutants, some of the erg mutants were crossed to the wild-type homothallic strain KJ100-7B, and homothallic erg mutants were obtained. The strains were then streaked onto ME medium, and observed microscopically after incubation for 3 days, after which cells were counted. As can be seen from the morphology of spores under these conditions (Fig. 10), the asci of erg5 cells resembled those of wild-type cells, while the asci of erg2 cells appeared aberrant. The erg2 cells formed zygotes but did not differentiate into mature spores (asci : zygotes : vegetative cells 6 : 77 : 122). In sts1 cells, the numbers of asci and zygotes were quite low: asci : zygotes : vegetative cells 49 : 11 : 609. These ratios in wild-type and erg5 cells were 105 : 8 : 200 and 121 : 20 : 183, respectively. When erg6 cells were crossed to wild-type, viable Ura autotrophic mutants were not obtained. Thus, this observation indicates that ergosterol is involved in sporulation or zygote formation and that some sterol derivatives produced in erg5 cells can completely substitute for ergosterol with respect to sporulation function.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). However, there is no evidence to date that these conserved genes are truly required for ergosterol synthesis in fission yeast. In the present study, it was shown that the Schiz. pombe erg mutants, with the exception of the single mutants erg31 and erg32, are deficient in ergosterol synthesis and are resistant to polyene drugs, indicating that the proteins encoded by the erg genes are responsible for ergosterol biosynthesis.
As shown in Fig. 2, ergosterol was not detected in all erg mutants, while other sterol derivatives were. These detected peaks were identified as cholesta-5,7,22,24-tetraenol (main peak of erg6), cholesta-5,7,24-trienol (a small peak at 10.6 min in erg6), and ergosta-5,7-dienol (main peak of erg5), inferred from studies of Sacch. cerevisiae (Munn et al., 1999; Skaggs et al., 1996; Shobayashi et al., 2005). Although none of the sterols in erg2 or erg31erg32 could be detected by HPLC with a UV-detector, several unidentified sterols were detected by gas chromatography, and the molecular masses of some of these sterols were estimated to be 398 and 396 by gas chromatography-mass spectrometry (data not shown). These molecular masses contain multiple candidates. Identification of the sterols produced in these erg mutants is the subject of ongoing work.
Two ERG3 homologues are specific to Schiz. pombe. These proteins share 57 % identity and 71 % similarity. A single ERG3 gene is found in Sacch. cerevisiae, Candida albicans and Candida glabrata, while Aspergillus fumigatus has three ERG3 homologues (Arthington et al., 1991; Geber et al., 1995; Miyazaki et al., 1999; Alcazar-Fuoli et al., 2006). While ergosterol was not detected in the double disruption mutant erg31erg32, it was present in the single mutants. Schiz. pombe Erg31p and Erg32p were found to be functional during aerobic growth, similar to the situation forA. fumigatus, in which all three ERG3-encoded proteins have been suggested to function as C-5 sterol desaturases (Alcazar-Fuoli et al., 2006). Although fission yeast Erg31p and Erg32p share redundant functions, these two proteins seem to have distinct roles in ergosterol synthesis. erg31+ is induced about fourfold under anaerobic conditions, while transcription of erg32+ decreases (Todd et al., 2006). Tolerance of erg mutants to nystatin and amphotericin B (Fig. 4) suggests that Erg32p is the major C-5 sterol desaturase under aerobic conditions. A shift from aerobic to anaerobic conditions might result in a change in induction of the major C-5 sterol desaturase from Erg32p to Erg31p. In order to understand why the major C-5 sterol desaturase changes due to transcriptional regulation, the enzymic properties of these proteins need to be determined.
Interestingly, Schiz. pombe erg cells were not found to be deficient in endocytosis or vacuolar fusion processes (Figs 6 and 7). In Sacch. cerevisiae, erg3 and erg2 have been reported to be impaired in endocytosis (Munn et al., 1999; Heese-Peck et al., 2002). It has also been reported that vacuoles isolated from these erg mutants are incapable of fusion in vitro, and that vacuolar fusion is partially restored by addition of sterol (Kato & Wickner, 2001). These reports suggest that ergosterol may be transported from the plasma membrane to vacuoles via endocytosis, and provide a binding site for certain vacuolar peripheral membrane proteins required for vacuolar fusion. If ergosterol has a similar function in Schiz. pombe, normal vacuolar fusion is then consistent with the normal endocytosis in erg cells, and the sterols in these mutants can apparently substitute for ergosterol in this process.
Phenotypic analysis revealed that ergosterol deficiency causes pleiotropic phenotypes. Sensitivity to certain reagents and abnormal localization of SPBC359.03c-GFP might be a consequence of the change in sterol composition, and aberrant sterol composition might alter plama membrane structures, causing mislocalization and/or changes in activities of membrane pumps. ERG genes have been shown to be required for correct delivery of a chimeric marker protein to the cell surface in Sacch. cerevisiae (Proszynski et al., 2005), and correct localization of tryptophan permease, Tat2p, has also been shown to be dependent on ergosterol (Umebayashi & Nakano, 2003). A recent study in higher eukaryotes indicated that sterol directly binds part of a multiprotein-channel complex and partly regulates the function of a cholesterol–protein supercomplex (Huber et al., 2006). Although changes in localization of plasma membrane proteins (Pma1-GFP, Pmd1-GFP and Bfr1-GFP) were hardly detected, some effects on activity were apparent as tolerance or sensitivity to drugs (Fig. 4). Bfr1p and Pmd1p are involved in CHX tolerance (Nishi et al., 1992; Turi & Rose, 1995; Nagao et al., 1995), and sensitivity to CHX in erg mutants might reflect a decrease in activity of these ABC transporters. The multidrug resistance pump Pdr5p has been reported to function with reduced efficiency in Sacch. cerevisiae erg mutants (Kaur & Bachhawat, 1999). Among our mutants, only erg2 showed Li+ sensitivity (data not shown); this might be due to decreased activity of the Na+/H+ exchanger Sod2p, which has been shown to be essential for Na+ and Li+ tolerance (Jia et al., 1992). Abnormal cell morphology and multiseptum formation in erg6 and erg2 might reflect mislocalizations or lowered activities of the cytoskeleton, cell wall synthesizing enzymes, and degrading enzymes, such as glucan synthase, chitin synthase, and glucanases. Inefficient sporulation in erg2 and sts1 (Fig. 10) and the failure to isolate homothallic erg6 mutants might be due to decreased activity of proteins required for formation of zygotes and spores, including mating pheromone transporters and receptors.
These phenotypes might correlate with the membrane microdomain that is rich in sterols and sphingolipids. Protein association with this microdomain is commonly assayed by solubilization with TX-100. Among tested proteins, Pma1-GFP was detected solely in the TX-100-insoluble fraction (DRM), while all other proteins were distributed in a broader range of fractions (data not shown). Loose association of Pma1-GFP was detected in erg6, but not in the other erg mutants. These results suggest that proteins loosely associated with the DRM may be difficult to detect in Schiz. pombe, and that many ergosterol derivatives can substitute functionally for ergosterol in the DRM. Similar to our findings in Schiz. pombe, the P-type ATPase Pma1-GFP localized to the plasma membrane in Sacch. cerevisiae erg mutants (Gaigg et al., 2005) and Pma1p associated with the DRM in an erg3 mutant (Kishimoto et al., 2005). Technical difficulties may limit the ability to detect small amounts of solubilized DRM-associated protein and very subtle mislocalization of DRM-associated protein.
Alternatively, changes in sterol composition may cause increased membrane permeability, resulting in increased uptake of drugs. Sacch. cerevisiae erg mutants have altered phospholipids as well as sterols, resulting in increased fluidity due to membrane disorder (Sharma, 2006). In Sacch. cerevisiae, deletion of ERG6 increases the rate of passive diffusion of small lipophilic drugs (Emter et al., 2002). Budding yeast cells lacking Pdr16p and Pdr17p have altered membrane sterol and lipid composition, display increased rates of passive drug diffusion, and are hypersensitive to many drugs (van den Hazel et al., 1999). The CPY colony blot assay indicated that cell integrity might be reduced in erg2 and erg6 cells (Fig. 5), although solubilization of DRM was hardly detected in erg2 cells (Fig. 8). Cell lysis might be caused by non-DRM sterol function.
Further studies will be required to distinguish between DRM- and non-DRM sterol functions in Schiz. pombe. It is possible that once the genes required for sphingolipid biosynthesis are identified, studies using sphingolipid biosynthetic mutants will be informative in relation to DRM function.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: D. Burke
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alfa, C., Fantes, P., Hyams, J., McLoed, M. & Warbrick, E. (1993). Experiments with Fission Yeast: a Laboratory Course Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Alonso, M. A. & Millán, J. (2001). The role of lipid rafts in signaling and membrane trafficking in T lymphocytes. J Cell Sci 114, 3957–3965.[Medline]
Arthington, B. A., Bennett, L. G., Skatrud, P. L., Guynn, C. J., Barbuch, R. J., Ulbright, C. E. & Bard, M. (1991). Cloning, disruption and sequence of the gene encoding yeast C-5 sterol desaturase. Gene 102, 39–44.[CrossRef][Medline]
Bagnat, M., Keränen, S., Shevchenko, A., Shevchenko, A. & Simons, K. (2000). Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc Natl Acad Sci U S A 97, 3254–3259.
Bagnat, M., Chang, A. & Simons, K. (2001). Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast. Mol Biol Cell 12, 4129–4138.
Bone, N., Millar, J. B. A., Toda, T. & Armstrong, J. (1998). Regulated vacuole fusion and fission in Schizosaccharomyces pombe: an osmotic response dependent on MAP kinases. Curr Biol 8, 135–144.[CrossRef][Medline]
Cheng, H., Sugiura, R., Wu, W., Fujita, M., Lu, Y., Sio, S. O., Kawai, R., Takegawa, K., Shuntoh, H. & Kuno, T. (2002). Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway, and its connection to calcineurin function. Mol Biol Cell 13, 2963–2976.
Daum, G., Lees, N. D., Bard, M. & Dickson, R. (1998). Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14, 1471–1510.[CrossRef][Medline]
Dupré, S. & Haguenauer-Tsapis, R. (2003). Raft partitioning of the yeast uracil permease during trafficking along the endocytic pathway. Traffic 4, 83–96.[CrossRef][Medline]
Eisenkolb, M., Zenzmaier, C., Leitner, E. & Schneiter, R. (2002). A specific structural requirement for ergosterol in long-chain fatty acid synthesis mutants important for maintaining raft domains in yeast. Mol Biol Cell 13, 4414–4428.
Emter, R., Heese-Peck, A. & Kralli, A. (2002). ERG6 and PDR5 regulate small lipophilic drug accumulation in yeast cells via distinct mechanisms. FEBS Lett 521, 57–61.[CrossRef][Medline]
Enyenihi, A. H. & Saunders, W. S. (2003). Large-scale functional genomic analysis of sporulation and meiosis in Saccharomyces cerevisiae. Genetics 163, 47–54.[Medline]
Gaigg, B., Timischl, B., Corbino, L. & Schneiter, R. (2005). Synthesis of sphingolipids with very long chain fatty acids but not ergosterol is required for routing of newly synthesized plasma membrane ATPase to the cell surface of yeast. J Biol Chem 280, 22515–22522.
Geber, A., Hitchcock, C. A., Swartz, J. E., Pullen, F. S., Marsden, K. E., Kwon-Chung, K. J. & Bennett, J. E. (1995). Deletion of the yeast Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility. Antimicrob Agents Chemother 39, 2708–2717.[Abstract]
Grossmann, G., Opekarova, M., Novakova, L., Stolz, J. & Tanner, W. (2006). Lipid raft-based membrane compartmentation of a plant transport protein expressed in Saccharomyces cerevisiae. Eukaryot Cell 5, 945–953.
Heese-Peck, A., Pichler, H., Zanolari, B., Watanabe, R., Daum, G. & Riezman, H. (2002). Multiple functions of sterols in yeast endocytosis. Mol Biol Cell 13, 2664–2680.
Huber, T. B., Schermer, B., Müller, R. U., Höhne, M., Bartram, M., Calixto, A., Hagmann, H., Reinhardt, C., Koos, F. & other authors (2006). Posocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels. Proc Natl Acad Sci U S A 103, 17079–17086.
Iwaki, T., Osawa, F., Onishi, M., Koga, T., Fujita, Y., Hosomi, A., Tanaka, N., Fukui, Y. & Takegawa, K. (2003). Characterization of vps33+, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe. Yeast 20, 845–855.[CrossRef][Medline]
Iwaki, T., Giga-Hama, Y. & Takegawa, K. (2006). A survey of all 11 ABC transporters in fission yeast: two novel ABC transporers are required for red pigment accumulation in a Schizosaccharomyces pombe adenine biosynthetic mutant. Microbiology 152, 2309–2321.
Jia, Z. P., McCullough, N., Martel, R., Hemmingsen, S. & Young, P. G. (1992). Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast. EMBO J 11, 1631–1640.[Medline]
Kato, M. & Wickner, W. (2001). Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion. EMBO J 20, 4035–4040.[CrossRef][Medline]
Kaur, R. & Bachhawat, A. K. (1999). The yeast multidrug resistance pump, Pdr5p, confers reduced drug resistance in erg mutants of Saccharomyces cerevisiae. Microbiology 145, 809–818.
Kishimoto, T., Yamamoto, T. & Tanaka, K. (2005). Defects in structural integrity of ergosterol and the Cdc50p-Drsp putative phospholipids translocase cause accumulation of endocytic membranes, onto which actin patches are assembled in yeast. Mol Biol Cell 16, 5592–5609.
Lauwers, E. & André, B. (2006). Association of yeast transporters with detergent-resistant membranes correlates with their cell-surface location. Traffic 7, 1045–1059.[CrossRef][Medline]
Lees, N. D., Skaggs, B., Kirsch, D. R. & Brad, M. (1995). Cloning of the late genes in the ergosterol biosynthetic pathway of Saccharomyces cerevisiae – a review. Lipids 30, 221–226.[Medline]
Lichtenberg, D., Goñi, F. M. & Heerklotz, H. (2005). Detergent-resistant membranes should not be identified with membrane rafts. Trends Biochem Sci 30, 430–436.[CrossRef][Medline]
Maguy, A., Hebett, T. E. & Nattel, S. (2006). Involvement of lipid rafts and caveolae in cardiac ion channel function. Cardiovasc Res 69, 798–807.
Malathi, K., Higaki, K., Tinkelenberg, A. H., Balderes, D., Almanzar-Paramio, D., Wilcox, L. J., Erdeniz, N., Redican, F., Padamsee, M. & other authors (2004). Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution. J Cell Biol 164, 547–556.
Malinska, K., Malinsky, J., Prekarova, M. & Tanner, W. (2004). Distribution of Can1p into stable domains reflects lateral protein segregation within the plasma membrane of living S. cerevisiae cells. J Cell Sci 117, 6031–6041.
Matsumoto, S., Bandyopadhyay, A., Kwiatkowski, D. J., Maitra, U. & Matsumoto, T. (2002). Role of the Tsc1-Tsc2 complex in signaling and transport across the cell membrane in the fission yeast Schizosaccharomyces pombe. Genetics 161, 1053–1063.
Miyazaki, Y., Geber, A., Miyazaki, H., Falconer, D., Parkinson, T., Hitchcock, C., Grimberg, B., Nyswaner, K. & Bennett, J. E. (1999). Cloning, sequencing, expression and allelic sequence diversity of ERG3 (C-5 sterol desaturase gene) in Candida albicans. Gene 236, 43–51.[CrossRef][Medline]
Moreno, S., Klar, A. & Nurse, P. (1991). Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol 194, 795–823.[Medline]
Morita, T. & Takegawa, K. (2004). A simple and efficient procedure for transformation of Schizosaccharomyces pombe. Yeast 21, 613–617.[CrossRef][Medline]
Mukhopadhyay, K., Kohli, A. & Prasad, R. (2002). Drug susceptibilities of yeast cells are affected by membrane lipid composition. Antimicrob Agents Chemother 46, 3695–3705.
Munn, A. L., Heese-Peck, A., Stevenson, B. J., Pichler, H. & Riezman, H. (1999). Specific sterols required for the internalization step of endocytosis in yeast. Mol Biol Cell 10, 3943–3957.
Munro, S. (2003). Lipid rafts: elusive or illusive? Cell 115, 377–388.[CrossRef][Medline]
Murray, J. M. & Johnson, D. I. (2001). The Cdc42p GTPase and its regulators of Nrf1p and Scd1p are involved in endocytic trafficking in the fission yeast Schizosaccharomyces pombe. J Biol Chem 276, 3004–3009.
Nagao, K., Taguchi, Y., Arioka, M., Kadokura, H., Takatsuki, A., Yoda, K. & Yamasaki, M. (1995). bfr1+, a novel gene of Schizosaccharomyces pombe which confers brefeldin A resistance, is related to the ATP-binding cassette superfamily. J Bacteriol 177, 1536–1543.
Nakamura, T., Nakamura-Kubo, M., Hirata, A. & Shimoda, C. (2001). The Schizosaccharomyces pombe spo3+ gene is required for assembly of the forespore membrane and genetically interacts with psy1+-encoding syntaxin-like protein. Mol Biol Cell 12, 3955–3972.
Nishi, K., Yoshida, M., Nishimura, M., Nishikawa, M., Nishiyama, M., Horinouchi, S. & Beppu, T. (1992). A leptomycin B resistance gene of Schizosaccharomyces pombe encodes a protein similar to the mammalian P-glycoproteins. Mol Microbiol 6, 761–769.[Medline]
Parks, L. W., Smith, S. J. & Crowley, J. H. (1995). Biochemical and physiological effects of sterol alterations in yeast – a review. Lipids 30, 227–230.[Medline]
Parks, L. W., Crowley, J. H., Leak, F. W., Smith, S. J. & Tomeo, M. E. (1999). Use of sterol mutants as probes for sterol functions in the yeast, Saccharomyces cerevisiae. Crit Rev Biochem Mol Biol 34, 399–404.[CrossRef][Medline]
Pasrija, R., Prasad, T. & Prasad, R. (2005a). Membrane raft lipid constituents affect drug susceptibilities of Candida albicans. Biochem Soc Trans 33, 1219–1223.[CrossRef][Medline]
Pasrija, R., Krishnamurthy, S., Prasad, T., Ernst, J. F. & Prasad, R. (2005b). Squalene epoxidase encoded by ERG1 affects morphologenesis and drug susceptibilities of Candida albicans. J Antimicrob Chemother 55, 905–913.
Pelkmans, L. (2005). Secrets of caveolae- and lipid raft-mediated endocytosis revealed by mammalian viruses. Biochim Biophys Acta 1746, 295–304.[Medline]
Pichler, H. & Riezman, H. (2004). Where sterols are required for endocytosis. Biochim Biophys Acta 1666, 51–61.[Medline]
Proszynski, T. J., Klemm, R. W., Gravert, M., Hsu, P. P., Gloor, Y., Wagner, J., Kozak, K., Graner, H., Walzer, K. & other authors (2005). A genome-wide visual screen reveals a role for sphingolipids and ergosterol in cell surface delivery in yeast. Proc Natl Acad Sci U S A 102, 17981–17986.
Raymond, C. K., Howald-Stevenson, I., Vater, C. A. & Stevens, T. H. (1992). Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell 3, 1389–1402.[Abstract]
Sanglard, D., Ischer, F., Parkinson, T., Falconer, D. & Bille, J. (2003). Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrob Agents Chemother 47, 2404–2412.
Sharma, S. C. (2006). Implications of sterol structure for membrane lipid composition, fluidity and phospholipids asymmetry in Saccharomyces cerevisiae. FEMS Yeast Res 6, 1047–1051.[CrossRef][Medline]
Shimanuki, M., Goebl, M., Yanagida, M. & Toda, T. (1992). Fission yeast sts1+ gene encodes a protein similar to the chicken lamin B receptor and is implicated in pleiotropic drug-sensitivity, divalent cation-sensitivity, and osmoregulation. Mol Biol Cell 3, 263–273.[Abstract]
Shobayashi, M., Mitsueda, S., Ago, M., Fujii, T., Iwashita, K. & Iefuji, H. (2005). Effects of culture conditions on ergosterol biosynthesis by Saccharomyces cerevisiae. Biosci Biotechnol Biochem 69, 2381–2388.[CrossRef][Medline]
Simons, K. & Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 569–572.[CrossRef][Medline]
Simons, K. & van Meer, G. (1988). Lipid sorting in epithelial cells. Biochemistry 27, 6197–6202.[CrossRef][Medline]
Skaggs, B. A., Alexander, J. F., Pierson, C. A., Schweitzer, K. S., Chun, K. T., Koegel, C., Barbuch, R. & Bard, M. (1996). Cloning and characterization of the Saccharomyces cerevisiae C-22 sterol desaturase gene, encoding a second cytochrome P-450 involved in ergosterol biosynthesis. Gene 169, 105–109.[CrossRef][Medline]
Sturley, S. L. (2000). Conservation of eukaryotic sterol homeostasis: new insights from studies in budding yeast. Biochim Biophys Acta 1529, 155–163.[Medline]
Suga, M. & Hatakeyama, T. (2001). High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation. Yeast 18, 1015–1021.[CrossRef][Medline]
Tabuchi, M., Iwaihara, O., Ohtani, Y., Ohuchi, N., Sakurai, J., Morita, T., Iwahara, S. & Takegawa, K. (1997). Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe. J Bacteriol 179, 4179–4189.
Takeda, T., Kawate, T. & Chang, F. (2004). Organization of a sterol-rich membrane domain by cdc15p during cytokinesis in fission yeast. Nat Cell Biol 6, 1142–1144.[CrossRef][Medline]
Takegawa, K., DeWald, D. B. & Emr, S. D. (1995). Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology. J Cell Sci 108, 3745–3756.[Abstract]
Todd, B. L., Stewart, E. V., Burg, J. S., Hughes, A. L. & Espenshade, P. (2006). Sterol regulatory element binding protein is a principal regulator of anaerobic gene expression in fission yeast. Mol Cell Biol 26, 2817–2831.
Turi, T. G. & Rose, J. K. (1995). Characterization of a novel Schizosaccharomyces pombe multidrug resistance transporter conferring brefeldin A resistance. Biochem Biophys Res Commun 213, 410–418.[CrossRef][Medline]
Umebayashi, K. & Nakano, A. (2003). Ergosterol is required for targeting of tryptophan permease to the yeast plasma membrane. J Cell Biol 161, 1117–1131.
van den Hazel, H. B., Pichler, H., do Valle Matta, M. A., Leitner, E., Goffeau, A. & Daum, G. (1999). PDR16 and PDR17, two homologous genes of Saccharomyces cerevisiae, affect lipid biosynthesis and resistance to multiple drugs. J Biol Chem 274, 1934–1941.
Wachtler, V. & Balasubramanian, M. K. (2006). Yeast lipid rafts? – an emerging view. Trends Cell Biol 16, 1–4.[CrossRef][Medline]
Wachtler, V., Rajagopalan, S. & Balasubramanian, M. K. (2003). Sterol-rich plasma membrane domains in the fission yeast Schizosaccharomyces pombe. J Cell Sci 116, 867–874.
Young, L. Y., Hull, C. M. & Heitman, J. (2003). Disruption of ergosterol biosynthesis confers resistance to amphotericin B in Candida lusitaniae. Antimicrob Agents Chemother 47, 2717–2724.
Received 2 July 2007;
revised 26 November 2007;
accepted 7 December 2007.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
