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Cryptococcus neoformans Yop1, an endoplasmic reticulum curvature-stabilizing protein, participates with Sey1 in influencing fluconazole-induced disomy formation

Popchai Ngamskulrungroj, Yun Chang, Bryan Hansen, Cliff Bugge, Elizabeth Fischer, Kyung J. Kwon-Chung
DOI: http://dx.doi.org/10.1111/j.1567-1364.2012.00824.x 748-754 First published online: 1 November 2012


Cryptococcus neoformans, an opportunistic fungal pathogen, manifests an intrinsic adaptive mechanism of resistance toward fluconazole (FLC) termed heteroresistance. Heteroresistance is characterized by the emergence of minor resistant subpopulations at levels of FLC that are higher than the strain's minimum inhibitory concentration. The heteroresistant clones that tolerate high concentrations of FLC often contain disomic chromosome 4 (Chr4). SEY1, GLO3, and GCS2 on Chr4 are responsible for endoplasmic reticulum (ER) integrity and important for Chr4 disomy formation under FLC stress. We sought an evidence of a direct relationship between ER morphology and Chr4 disomy formation. Deletion of the YOP1 gene on Chr7, which encodes an ER curvature-stabilizing protein that interacts with Sey1, perturbed ER morphology without affecting FLC susceptibility or the frequency of FLC-induced disomies. However, deletion of both YOP1 and SEY1, not only perturbed ER morphology more severely than in sey1Δ or yop1Δ strains, but also abrogated the FLC-induced disomy. Although the heteroresistance phenotype was retained in the sey1Δyop1Δ strains, tolerance to FLC appeared to have resulted not from chromosome duplication but from gene amplification restricted to the region surrounding ERG11 on Chr1. These data support the importance of ER integrity in C. neoformans for the formation of disomy under FLC stress.

  • Cryptococcus neoformans
  • disomy formation
  • fluconazole
  • antifungal resistance
  • heteroresistance
  • endoplasmic reticulum


Cryptococcosis has been effectively treated with amphotericin B induction therapy followed by maintenance regimens with azole drugs that target the biosynthetic pathway of ergosterol, an essential component of the fungal membrane (Kwon-Chung et al., 2000; Akins, 2005; Sullivan et al., 2006). Ergosterol is synthesized in the endoplasmic reticulum (ER) starting from acetyl-CoA through a series of enzymes encoded by various ERG genes (Akins, 2005). The sterol is then delivered to the cell membrane via both vesicular and nonvesicular routes (Sullivan et al., 2006; Schulz & Prinz, 2007).

Fluconazole (FLC), a triazole drug, has been the most widely used azole antifungal in the maintenance therapy of cryptococcosis (Washton, 1989; Perfect et al., 2010). Because the report on drug resistant cases of cryptococcosis has been infrequent, studies on the molecular mechanism of drug resistance in Cryptococcus neoformans have been considerably limited compared to pathogenic species of other yeasts. Only few cases of missense mutations in ERG11 were reported from azole-resistant clinical strains (Rodero et al., 2003; Sionov et al., 2012). A laboratory-constructed C. neoformans strain that overexpressed an ABC transporter, AFR1. was reported to be resistance to FLC (Posteraro et al., 2003). In 1999, a novel azole resistance termed heteroresistance was first reported in C. neoformans based on the behavior of two strains, one isolated from a patient in Italy and the other from a patient in Israel (Mondon et al., 1999). Recently, heteroresistance was described as an intrinsic, adaptive resistance to FLC, and it was found to be universal in the strains of C. neoformans and Cryptococcus gattii thus far tested (Mondon et al., 1999; Sionov et al., 2009; Varma & Kwon-Chung, 2010). The phenomenon of heteroresistance was characterized as the emergence of minor subpopulations within a single colony of the susceptible strain that could adapt to FLC concentrations higher than the strain's minimum inhibitory concentration. The acquired resistance in these subpopulations is transient because it is lost upon release from drug stress (Sionov et al., 2009). The level of heteroresistance to fluconazole (LHF) of each strain was defined as the lowest concentration of FLC at which resistant subpopulations emerge. For the genome-sequenced strain H99, the LHF is 32 μg mL−1 FLC, and the heteroresistant subpopulations (0.3–0.6%) that emerged at 32 μg mL−1 FLC contained disomic Chr1 (Sionov et al., 2010). When the FLC concentration was increased to 64 μg mL−1 or higher, Chr4 also became disomic (Sionov et al., 2010; Ngamskulrungroj et al., 2012). These extra copies of duplicated chromosomes were lost in a stepwise manner upon daily transfer in drug-free media (Sionov et al., 2010). Chr1 contains ERG11, the target of FLC, and the ABC drug transporter AFR1. which play a major role in C. neoformans azole resistance (Sionov et al., 2010). It has been proposed that the increases in dosage of these genes enable the strains to overcome the drug stress. Moreover, a recent study showed that SEY1, GLO3. and GCS2 genes on Chr4, which are essential for ER integrity and/or function, are important for the FLC-induced Chr4 disomy (Ngamskulrungroj et al., 2012).

In eukaryotic cells, ER plays an important role in several core processes of cell biology such as biosynthesis of secretory and membrane proteins, lipid synthesis, and Ca2+ storage. Unlike the extensive reticular structure of mammalian ER, the yeast tubular ER is closely located to the plasma membrane, thus, named as cortical ER (reviewed in Hu et al., 2011). In addition to the key cellular processes, cryptococcal ER is believed to play an important role in azole resistance by influencing the process of disomy formation in response to FLC stress (Ngamskulrungroj et al., 2012). However, the mechanism of such influence on disomy formation remains unknown. In this study, we show that YOP1 which encodes a protein that interacts with Sey1 (Brands & Ho, 2002; Hu et al., 2009) plays an important role in ER integrity. While deletion of YOP1 alone did not affect FLC susceptibility or the formation of chromosome disomy, the double deletion of sey1Δ/yop1Δ resulted in severe aberration of ER morphology and also abolished FLC-induced disomy formation.

Materials and methods

Strains media and level of FLC resistance

All deletant strains used in the study were constructed in the C. neoformans H99 background (Perfect et al., 1993) and are listed in Table 1. Strains were maintained on YPD agar (1% yeast extract, 2% peptone, 2% glucose, 2% agar) or YPD supplemented with 8, 16, 32, 64, or 128 μg mL−1 FLC. For spot assays, a 2 μL cell suspension with an optical density of 2 at 600 nm (OD600) and its 10-fold serial dilutions were spotted on YPD agar with or without drug supplements. The plates were incubated at 30 °C for 3–5 days and photographed. As in the previous study (Ngamskulrungroj et al., 2012), we defined the first level of heteroresistance to FLC (1LHF) as the lowest concentration of FLC at which minor resistant subpopulations emerge. We used arbitrary twofold increments of FLC concentration to define the subsequent LHFs. For instance, 1 LHF of the wild-type strain H99 is 32 μg mL−1, 2LHF is 64 μg mL−1 and 3LHF is 128 μg mL−1 (Table 1).

View this table:
Table 1

List of strains and their levels of heteroresistance

StrainDescriptionConcentrations of FLC at 1, 2, and 3LHF (μg mL−1)
H99Wild type32, 64, 128
yop1Δ ER curvature-stabilizing protein32, 64, 128
sey1Δ GTPase interacts with ER-shaping protein16, 32, 64
sey1Δyop1Δ Double deletion of SEY1 and YOP1 16, 32, 64
yop1Δ::YOP1 Homologous complementation of yop1Δ 32, 64, 128
sey1Δ::SEY1 Homologous complementation of sey1Δ32, 64, 128
sey1Δyop1Δ::YOP1 Homologous complementation of yop1Δ in sey1Δyop1Δ 16, 32, 64
sey1Δyop1Δ::SEY1Homologous complementation of sey1Δ in sey1Δyop1Δ 32, 64, 128

Gene manipulations

The YOP1 homolog of Saccharomyces cerevisiae in the C. neoformans strain H99 was identified by a blastp search of the H99 genome database (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html) and was disrupted by biolistic transformation (Toffaletti et al., 1993). Briefly, disruption constructs were created by overlapping PCR technique (Davidson et al., 2000) with nourseothricin (NAT1), G418 (NEO), or hygromycin (HGR) resistance genes as dominant selectable markers. These constructs were transformed into H99 cells by a BioRad model PDS-1000/He biolistic particle delivery system. Homologous integrations were confirmed by PCR and Southern hybridization. Complementation of each deletant was accomplished by homologous integration at the disrupted locus.

Quantitation of gene dosage

Quantitative real-time PCR (qPCR) assays were performed to quantitate the copy number of genes on specific chromosomes as previously described (Sionov et al., 2010). Six to eight independent clones of each strain were tested at 3LHF (128 μg mL−1 FLC; Ngamskulrungroj et al., 2012).

Comparative genome hybridization (CGH)

Genomic DNA was extracted from 3 to 5-day-old cultures grown on YPD plates with or without the drug as described previously (Sionov et al., 2010). DNA was labeled using the BioPrime®Array CGH Genomic Labeling System Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. Briefly, genomic DNA was digested with DpnII (New England Biolabs, Ipswich). Experimental and control samples were labeled with Alexa647 and Alexa555 dyes, respectively. The labeled samples were hybridized to JEC21-based 70mer slides (http://genome.wustl.edu/services/microarray/cryptococcus_neoformans) and analyzed as previously described (Sionov et al., 2010).

Fluorescence microscopy

To visualize the ER within yeast cells, the ER-protein Sec61β/Sbh1 homolog, CNAG_06351, was tagged with GFP (Hu et al., 2009) as described in the previous study (Ngamskulrungroj et al., 2012). The GFP signals enabled visualization of the ER morphology using the fluorescence Zeiss Axiovert microscope and Axiovision (version 4.0) software.

Transmission electron microscopy

Cells were prepared and stained as previously described (Yoneda & Doering, 2006). Briefly, cells were grown overnight in YPD broth. Mid-log phase cells were harvested and washed once in 1 mL of the primary fixative (0.1 M sorbitol, 1 mM MgCl2, 1 mM CaCl2, 2% glutaraldehyde in 0.1 M PIPES buffer, pH 6.8). Then, the cells were fixed with 2% KMnO4, dehydrated using a series of graded ethanol and propylene oxide prior to infiltrating and embedding in Epon. Cells were prepared for visualization of the membrane structures under Transmission electron microscopy (TEM) as described (Yoneda & Doering, 2006; Ngamskulrungroj et al., 2012).

Focused ion beam-scanning electron microscopy

Cells were prepared as they were for the TEM mentioned earlier. Specimens were mounted on the focused ion beam-scanning electron microscopy (FIB-SEM). All data sets were collected by a FEI Helios NanoLab 650 DualBeam™ equipped with Ga+ LMIS FIB used for milling (FEI, Hillsboro, OR) according to the method described previously (Ngamskulrungroj et al., 2012).


Deletion of YOP1 causes severe alterations in ER morphology

A previous study showed that deletions of the genes responsible for ER integrity reduced the frequency of Chr4 disomy. However, the correlation between normal ER morphology and chr4 disomy is controversial. To study the impact of ER morphology on FLC-induced disomy, we identified and deleted the homolog of Yop1, an ER curvature-stabilizing and Sey1 interacting protein (YOP1, CNAG_06646; Brands & Ho, 2002; Hu et al., 2009). The YOP1 gene resides on Chr7 which has not been observed to undergo changes in copy number in response to elevated FLC concentrations irrespective of the tested strain's genetic background [(Ngamskulrungroj et al., 2012), data not shown]. The yop1 disruptants were characterized and compared with our previous data obtained with the wild-type, sey1Δ and sey1Δ::SEY1 strains (Ngamskulrungroj et al., 2012). When the GFP-Sec61β ER protein was used as a reporter, the wild-type strain showed the GFP signal to be located near the nuclear envelope and an element near the plasma membrane connected with some strands in the cytoplasm, a typical ER pattern seen in S. cerevisiae and C. neoformans (Fig. 1a; Hu et al., 2009; Ngamskulrungroj et al., 2012). In contrast, yop1Δ displayed an aberrant ER morphology similar to the sey1Δ strain under a fluorescent microscope. The ER strands of the sey1Δ and yop1Δ strains were portrayed as smooth threads in contrast to bead-like strands of the wild type. This ER aberrance was even more evident in the case of double deletant where the nuclear envelope was also distorted (Fig. 1a). Like in sey1Δ, the ER in yop1Δ was clearly elongated as observed under TEM, which was almost never the case in the wild-type strain (Fig. 1b). These losses of the bead-like chartacteristics and elongation of the ER suggested the possible loss of ER reticulation. Finally, because ER is known to be a reticular network throughout the entire cell (English et al., 2009), a FIB-SEM that provides a view of three dimensional structure was used to examine the ER network. The wild-type cells showed the typical tubular ER extending throughout the cytoplasm. The ER (artificially labeled in green color) morphology in both yop1Δ and sey1Δ was aberrant and was observed mostly as an expanded sheet (Fig. 2) in contrast to the reticulate ER of the wild-type cell. Moreover, a double deletion of both YOP1 and SEY1 caused a more severe morphological abnormality of the ER as well as nuclear membrane (artificially labeled in blue color) than either of yop1Δ or sey1Δ (Figs 1 and 2).

Figure 1

Disruption of YOP1 causes ER abnormality as was seen in sey1Δ. (a) Localization of the ER protein GFP-Sec61β in each indicated strain. Cells were grown on YPD agar at 30 °C and subsequently visualized by fluorescence microscopy. White arrow head = ER; gray arrow head = nucleus [determined by co-localization with Hoechst 3342 dye as published previously (Ngamskulrungroj et al., 2012)]. (b) TEM. Overnight cell cultures of indicated strains grown to mid-log phase were prepared and examined by TEM. Arrow = ER.

Figure 2

The FIB-SEM shows aberration in ER morphology. Orthoslices representing central sections are shown in the left column. Data collected by serial 3–10 nm cuts of each sample through the cell were used to generate 3D reconstructions. ER and nuclei were psuedo-colored with green and blue, respectively.

YOP1is not important for FLC tolerance

Because YOP1 was shown to be required for the maintenance of ER structural integrity like SEY1, we investigated the effect of YOP1 deletion on FLC sensitivity. Figure 3 shows that sensitivity of yop1Δ to FLC is similar to that of the wild-type H99. Disruption of YOP1 in combination with SEY1 exhibited FLC sensitivity similar to the levels in sey1Δ. Complementation of SEY1 alone restored FLC sensitivity to the wild-type level. These results suggested that YOP1 does not play a role in FLC tolerance despite its similar function with SEY1 in the maintenance of ER morphology.

Figure 3

Spot assay for FLC tolerance. Cells of the indicated strains were spotted on YPD alone or YPD supplemented with either 8 or 16 μg mL−1 FLC and incubated for 5 days at 30 °C. sy = sey1Δyop1Δ.

Deletion of YOP1 alone does not affect disomic chromosome formation in response to FLC stress

In this study, we used 3LHF (arbitrary fourfold increments of FLC concentration from the 1LHF) to determine the frequency of disomic chromosomes because this FLC concentration was consistently shown to induce disomies of Chr1 and Chr4 in wild-type strains (Ngamskulrungroj et al., 2012). It has been shown that among the clones resistant to 3LHF derived from sey1Δ. only one of 11 clones (9%) showed the presumed Chr4 disomy (Fig. 3, Ngamskulrungroj et al., 2012). Surprisingly, although yop1Δ had a clearly altered ER morphology, the deletion of YOP1 alone did not have any impact on Chr1 or Chr4 disomy (Fig. 4). However, if YOP1 was deleted in the sey1Δ background, Chr4 disomy was absent in the FLC-resistant clones (Fig. 4). Furthermore, the type-2 clones (see Fig. 4 legend) of yop1Δsey1Δ resistant to 3LHF had a partial duplication of Chr1 exclusively in the region that contained ERG11. and no alteration was detected in the copy number of other chromosomes (Fig. 5 and data not shown). These results suggested that, although not important for FLC tolerance, YOP1 plays a critical role in combination with SEY1 to form disomic chromosome in C. neoformans under FLC stress.

Figure 4

Frequency of Chr4 disomy formation in the deletants of the genes associated with ER integrity. Clones resistant to 3LHF derived from each strain were isolated, and the copy number of Chr1 and Chr4 in the genomes was inferred by qPCR of resident genes specific to each chromosome. The figure displays the percentage of each type of disomy in the FLC-resistant strains. Type 1 denotes strains that contain disomy of both Chr1 and Chr4, Type 2 denotes strains that contain disomy of Chr1 only, and Type 3 denotes strains that contain no disomies of either Chr1 or Chr4. Six to eight independent clones of each strain were tested. sy = sey1Δyop1Δ.

Figure 5

CGH analysis of the sey1Δyop1Δ double deletants isolated from 3LHF. Genomic DNA extracted from a sey1Δyop1Δ double deletant with Type 2 duplication pattern at 3LHF shown in Fig. 4 was used for CGH analysis. H99 and the resistant clones of H99 grown at 3LHF were included as controls. Only the Chr1 status is shown. Except for the 3LHF clone from H99, no disomy was found in any of the double mutant clones grown at 3LHF (data not shown). Arrows indicate the location of ERG11. Each bar represents the copy number of each gene residing on Chr1 in a log2 scale.


In C. neoformans, the association between occurrence of aneuploidy and azole resistance by either partial or whole chromosome duplication has been documented (Sionov et al., 2010; Ngamskulrungroj et al., 2012). A number of genes on Chr1 and Chr4 that play critical roles in FLC-induced disomy formation have been characterized (Sionov et al., 2010; Ngamskulrungroj et al., 2012). Our previous study showed the functional importance of a SEY1, a GTPase, in FLC-induced disomy. Deletion of SEY1 decreased the frequency of Chr4 disomy and this disomy was abrogated when the gene was deleted in combination with GLO3. encoding an ADP-ribosylation factor GTPase activating protein (Ngamskulrungroj et al., 2012). Interestingly, the inability to form FLC-induced disomy appears to be correlated with morphologically aberrant ER. Here, we show that the alteration in ER morphology resulting from the deletion of YOP1, an ER-shaping protein, did not affect the frequency of FLC-induced disomy. This result indicates that intact ER morphology is not always required for disomy formation under FLC stress. However, severe alterations in ER morphology caused by deletion of both SEY1 and YOP1 resulted in a total loss of FLC-induced disomy.

Maintenance of ER integrity is essential for cellular homeostasis in eukaryotes. ER is often tightly associated with almost every other membrane-bound compartment in the cell including the plasma membrane, nuclear envelope, mitochondria, golgi, vacuoles, and peroxisomes. Interactions between these organelles have shown to be functionally important (reviewed in English et al., 2009). For example, proper alignment of the ER and mitochondria is indispensable for the function and survival of cells (Csordas et al., 2006). Typically, the ER is comprised of ER tubules, ER sheets, and the nuclear envelope (Shibata et al., 2006). During mitosis, the ER and nuclear envelope undergo large structural and functional modifications to redistribute the organelles and the associated proteins to the daughter cells (Puhka et al., 2007; English et al., 2009). In yeast, the nuclear envelope has a direct role in chromosome segregation (Schmitt, 2010). In fact, a possible association between ER and disomy formation of C. neoformans has been shown in the present as well as in a previous study (Ngamskulrungroj et al., 2012). Our present work underscores the importance of ER in chromosome disomy by showing that, in the sey1Δyop1Δ strain where the ER malformation is even more severe than in sey1Δ alone, FLC-induced disomy is further decreased. However, the deletion of the YOP1 alone did not show any defect in FLC-induced disomy despite the severe ER morphological defect. Because Sey1 is a GTPase and mediates homotypic ER fusion (Brands & Ho, 2002; Hu et al., 2009, 2011), it is possible that function of ER is more important than morphology alone in the formation of FLC-induced disomy in C. neoformans.

Yop1 is a member of curvature-stabilizing proteins along with Dp1 and reticulons (Rtn; Shibata et al., 2006). These proteins are required to form tubular structure of the ER. Depletion of these proteins in mammalian cells causes transformation of tubular ER mostly into the sheets (Voeltz et al., 2006). In S. cerevisiae, a deletion of either of these proteins did not alter the ER morphology when grown in regular yeast media (Voeltz et al., 2006). Similarly, a deletion of only the interacting protein, Sey1. had no effect on S. cerevisiae ER morphology, suggesting the existence of additional factor(s) required for the maintenance of ER morphology (Hu et al., 2009). In contrast, deletion of either YOP1 or SEY1 resulted in a significant loss of tubular ER in C. neoformans. Because no homolog of Rtn was found in the C. neoformans genome by Blast search, our results suggest that the role of these proteins in ER shaping may be more restrictive in C. neoformans.

It has been shown that all yeast strains carrying extra copies of chromosome exhibit genomic instability (Niwa et al., 2006). In 2007, Torres et al. reported that aneuploid strains of S. cerevisiae shared a number of defective traits including cell cycle progression (Torres et al., 2007). Likewise, disomy formation in C. neoformans may result in some degree of genomic instability. Moreover, maintenance of extra chromosome copies would be significant burden to cells. Perhaps, the sey1Δyop1Δ strain with the severe defects in ER integrity is not able to overcome the burden of extra chromosome maintenance. Thus, only cells that amplified the region of ERG11 gene on Chr1 managed to survive the FLC stress without disomy formation.

Both Chr1 and Chr4, the two chromosomes most frequently found to be duplicated under FLC stress, contain genes that play critical roles in FLC-induced disomy (Sionov et al., 2010). For example, relocation of ERG11 to Chr3 increases the frequency of Chr3 disomy while decreasing the frequency of Chr1 disomy under FLC stress. Similarly, relocation of SEY1 and/or GLO3 to Chr3 from Chr4 increased FLC-induced Chr3 disomy and reduced the frequency of Chr4 disomy ((Ngamskulrungroj et al., 2012), unpublished data). However, the relationship between the importance of certain genes in FLC-induced disomy formation and the gene's impact on FLC sensitivity is incongruent. For example, ERG11, AFR1, GLO3. and SEY1 all contribute to resistance of C. neoformans to FLC and are important for FLC-induced disomy (Sionov et al., 2010), while GCS2 is involved in FLC-induced Chr4 disomy without any impact on FLC resistance (Ngamskulrungroj et al., 2012). The mechanism by which azoles cause disomy remains elusive. Our results provide further evidence of the relationship between ER integrity and chromosomal disomy under FLC stress. How ER integrity dictates disomy formation remains unclear and requires further studies to resolve this important question of cell biology.


This study was supported by funds from the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank A. Varma for critical discussions and reading of the manuscript.


  • Editor: Richard Calderone


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