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

Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
FEMS Yeast Research (Impact Factor: 2.82). 06/2012; 12(7):748-54. DOI: 10.1111/j.1567-1364.2012.00824.x
Source: PubMed
ABSTRACT
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 sey1∆ or yop1∆ strains, but also abrogated the FLC-induced disomy. Although the heteroresistance phenotype was retained in the sey1∆yop1∆ 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.

Full-text

Available from: Kyung J Kwon-Chung, Nov 19, 2015
RESEARCH ARTICLE
Cryptococcus neoformans Yop1, an endoplasmic reticulum
curvature-stabilizing protein, participates with Sey1
in influencing fluconazole-induced disomy formation
Popchai Ngamskulrungroj
1,2
, Yun Chang
1
, Bryan Hansen
3
, Cliff Bugge
4
, Elizabeth Fischer
3
& Kyung J. Kwon-Chung
1
1
Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH, Bethesda, MD, USA;
2
Department of Microbiology,
Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand;
3
Electron Microscopy Unit, Rocky Mountain Laboratories, NIAID, NIH,
Hamilton, MT, USA; and
4
FEI company, Hillsboro, OR, USA
Correspondence: Kyung J. Kwon-Chung,
Molecular Microbiology Section, Laboratory
of Clinical Infectious Diseases, NIAID, NIH,
Building 10, Bethesda, MD 20892, USA.
Tel.: +1 301 496 1602; fax: +1 301 480
3240; e-mail: June_kwon-chung@nih.gov
Received 4 May 2012; revised 7 June 2012;
accepted 19 June 2012.
Final version published online 18 July 2012.
DOI: 10.1111/j.1567-1364.2012.00824.x
Editor: Richard Calderone
Keywords
Cryptococcus neoformans; disomy formation;
fluconazole; antifungal resistance;
heteroresistance; endoplasmic reticulum.
Abstract
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.
Introduction
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
ª 2012 Federation of European Microbiological Societies FEMS Yeast Res 12 (2012) 748–754
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YEAST RESEARCH
Page 1
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. neofor-
mans and Cryptococcus gattii thus far tested (Mondon
et al., 1999; Sionov et al., 2009; Varma & Kwon-Chung,
2010). The phenomenon of heteroresistance was character-
ized 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 lgmL
1
FLC, and
the heteroresistant subpopulations (0.30.6%) that
emerged at 32 lgmL
1
FLC contained disomic Chr1
(Sionov et al., 2010). When the FLC concentration was
increased to 64 lgmL
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 tar-
get 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
Ca
2+
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 lgmL
1
FLC. For spot assays, a 2 lL cell suspension
with an optical density of 2 at 600 nm (OD
600
) 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 lgmL
1
, 2LHF is 64 lgmL
1
and 3LHF is
128 lgmL
1
(Table 1).
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/Multi-
Table 1. List of strains and their levels of heteroresistance
Strain Description Concentrations of FLC at 1, 2, and 3LHF (lgmL
1
)
H99 Wild type 32, 64, 128
yop1Δ ER curvature-stabilizing protein 32, 64, 128
sey1Δ GTPase interacts with ER-shaping protein 16, 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 sey1Δ 32, 64, 128
sey1Δyop1Δ::YOP1 Homologous complementation of yop1Δ in sey1Δyop1Δ 16, 32, 64
sey1Δyop1Δ::SEY1 Homologous complementation of sey1Δ in sey1Δ yop1Δ 32, 64, 128
FEMS Yeast Res 12 (2012) 748–754 ª 2012 Federation of European Microbiological Societies
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Characterization of YOP1 in C. neoformans 749
Page 2
Home.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 hygro-
mycin (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 chro-
mosomes as previously described (Sionov et al., 2010). Six
to eight independent clones of each strain were tested at
3LHF (128 lgmL
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 (Invi-
trogen, 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/ser-
vices/microarray/cryptococcus_neoformans) and analyzed
as previously described (Sionov et al., 2010).
Fluorescence microscopy
To visualize the ER within yeast cells, the ER-protein
Sec61b/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 over-
night 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 MgCl
2
, 1 mM CaCl
2
, 2% glutaraldehyde
in 0.1 M PIPES buffer, pH 6.8). Then, the cells were fixed
with 2% KMnO
4
, 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
DualBeamTM equipped with Ga
+
LMIS FIB used for
milling (FEI, Hillsboro, OR) according to the method
described previously (Ngamskulrungroj et al., 2012).
Results
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 mor-
phology 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 curva-
ture-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-Sec61b 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
ª 2012 Federation of European Microbiological Societies FEMS Yeast Res 12 (2012) 748–754
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750 P. Ngamskulrungroj et al.
Page 3
(Fig. 1b). These losses of the bead-like chartacteristics and
elongation of the ER suggested the possible loss of ER retic-
ulation. Finally, because ER is known to be a reticular net-
work throughout the entire cell (English et al., 2009), a
FIB-SEM that provides a view of three dimensional struc-
ture 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 con-
trast 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).
YOP1 is not important for FLC tolerance
Because YOP1 was shown to be required for the mainte-
nance 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 combina-
tion 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.
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). Surpris-
ingly, although yop1Δ had a clearly altered ER morphol-
ogy, 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.
Discussion
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.,
(a) (b)
Fig. 1. Disruption of YOP1 causes ER
abnormality as was seen in sey1Δ.
(a) Localization of the ER protein GFP-Sec61b
in each indicated strain. Cells w ere 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.
FEMS Yeast Res 12 (2012) 748–754 ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Characterization of YOP1 in C. neoformans 751
Page 4
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
Fig. 3. Spot assay for FLC tolerance. Cells of the indicated strains
were spotted on YPD alone or YPD supplemented with either 8 or
16 lgmL
1
FLC and incubated for 5 days at 30 °C.
sy = sey1Δyop1Δ.
Fig. 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.
Fig. 2. The FIB-SEM shows aberration in ER morphology. Orthoslices
representing central sections are shown in the left column. Data
collected by serial 310 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.
Fig. 5. CGH analysis of the sey1Dyop1D double deletants isolated
from 3LHF. Genomic DNA extracted from a sey1Dyop1D 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 log
2
scale.
ª 2012 Federation of European Microbiological Societies FEMS Yeast Res 12 (2012) 748–754
Published by Blackwell Publishing Ltd. All rights reserved
752 P. Ngamskulrungroj et al.
Page 5
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 mitochon-
dria 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). Simi-
larly, 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 mainte-
nance 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). Like-
wise, disomy formation in C. neoformans may result in
some degree of genomic instability. Moreover, mainte-
nance 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, con-
tain 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 forma-
tion and the gene’s impact on FLC sensitivity is incongru-
ent. 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 with-
out 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.
Acknowledgement
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.
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FEMS Yeast Res 12 (2012) 748–754 ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
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ª 2012 Federation of European Microbiological Societies FEMS Yeast Res 12 (2012) 748–754
Published by Blackwell Publishing Ltd. All rights reserved
754 P. Ngamskulrungroj et al.
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    • "With these techniques, a resolution of 20–40 nm is achieved for thick 3-D samples without a need for manual generation of serial sections. These technologies have also become useful for understanding a general picture of a microbial cell, and there have been several reports in which fungal cells were observed by FIB-SEM (Kamino et al. 2004, Ngamskulrungroj et al. 2012, Kralj Kunčič et al. 2010). Our results revealed that the hyphae partially expanded only in the cellulase hyper-producing mutant of T. reesei, PC- 3-7 when cultured in Avicel and that the abundance of the hypertrophic vacuoles might have caused cell expansion and a reduction in the thickness of the cell membrane. "
    [Show abstract] [Hide abstract] ABSTRACT: The cellulolytic fungus Trichoderma reesei is a potent cellulase producer and, therefore, cellulase hyper-producing mutants have been developed. However, morphological feature has still remained to be analyzed for understanding phenotypic change of T. reesei mutants. In this review, we show an electron microscopic observation of T. reesei to obtain new insights of morphological phenotypes of T. reesei mutants. We also successfully reconstructed the three dimensional structure of T. reesei hypha by using focused ion beam SEM technique.
    Preview · Article · Jan 2015
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    • "Genes on Chr4 also were linked to drug resistance. These encode a GTPase and two GTPase-activating proteins that are involved in the regulation of endoplasmic reticulum morphogenesis and trafficking (Ngamskulrungroj et al. 2012b ). The endoplasmic reticulum is a site for sterol synthesis, but the mechanism(s) by which these genes increase drug resistance is not known. "
    [Show abstract] [Hide abstract] ABSTRACT: Human fungal pathogens can exist in a variety of ploidy states, including euploid and aneuploid forms. Ploidy change has a major impact on phenotypic properties, including the regulation of interactions with the human host. In addition, the rapid emergence of drug-resistant isolates is often associated with the formation of specific supernumerary chromosomes. Pathogens such as Candida albicans and Cryptococcus neoformans appear particularly well adapted for propagation in multiple ploidy states with novel pathways driving ploidy variation. In both species, heterozygous cells also readily undergo loss of heterozygosity (LOH), leading to additional phenotypic changes such as altered drug resistance. Here, we examine the sexual and parasexual cycles that drive ploidy variation in human fungal pathogens and discuss ploidy and LOH events with respect to their far-reaching roles in fungal adaptation and pathogenesis.
    Full-text · Article · Jul 2014 · Cold Spring Harbor Perspectives in Medicine
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    • "Those double deletants were extremely sensitive to FLC, and the clones resistant to levels of FLC higher than their MIC revealed a monosomic Chr1 with segmental amplification only in the small region surrounding the ERG11 gene [27] . Similar results were obtained when SEY1 and the Chr7- inhabiting YOP1 gene, which encodes a Sey1-interacting ER curvature maintenance protein, were both deleted [30]. Since FLC disrupts the biosynthesis of ergosterol, which is produced in the ER and delivered to plasma membrane [31,32], it is likely that increases in dosage of the genes relevant for ER integrity provide increased fitness under FLC stress. "
    Full-text · Article · Nov 2012 · PLoS Pathogens
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