EUKARYOTIC CELL, Dec. 2011, p. 1714–1723
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 10, No. 12
The C2 Domain Protein Cts1 Functions in the Calcineurin Signaling
Circuit during High-Temperature Stress Responses in
Eanas F. Aboobakar, Xuying Wang, Joseph Heitman, and Lukasz Kozubowski*
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710
Received 27 June 2011/Accepted 30 September 2011
Calcineurin is a conserved calcium/calmodulin-dependent serine/threonine-specific protein phosphatase
that acts in cell stress responses. Calcineurin is essential for growth at 37°C and for virulence of the human
fungal pathogen Cryptococcus neoformans, but its substrates remain unknown. The C2 domain-containing,
phospholipid-binding protein Cts1 was previously identified as a multicopy suppressor of a calcineurin
mutation in C. neoformans. Here we further characterize the function of Cts1 and the links between Cts1 and
calcineurin. GFP-Cts1 localizes to cytoplasmic puncta and colocalizes with the endosomal marker FM4-64. The
cts1? mutant shows a distinct FM4-64 staining pattern, suggesting involvement of Cts1 in endocytic trafficking.
In large budded cells, GFP-Cts1 localizes transiently at the mother bud neck, as a single ring that undergoes
contraction. mCherry-Cts1 colocalizes with the GFP-tagged calcineurin catalytic subunit Cna1 at sites of
mRNA processing at 37°C, suggesting that Cts1 and calcineurin function coordinately during thermal stress.
GFP-Cts1 exhibits slower electrophoretic mobility for cells grown at 37°C than for cells grown at 24°C, and the
shift to a higher molecular weight is more pronounced in the presence of the calcineurin inhibitor FK506. In
vitro treatment with calf intestinal alkaline phosphatase (CIP) restores faster electrophoretic mobility to
GFP-Cts1, suggesting that Cts1 is phosphorylated at 37°C and may be dephosphorylated in a calcineurin-
dependent manner. mCherry-Cts1 also coimmunoprecipitates with GFP-Cna1, with greater complex formation
at 37°C than at 24°C. Taken together, these findings support potential roles for Cts1 in endocytic trafficking,
mRNA processing, and cytokinesis and suggest that Cts1 is a substrate of calcineurin during high-temperature
Cryptococcus neoformans is a human fungal pathogen that
causes life-threatening meningitis, primarily in immunocom-
promised patients (5, 17). Cryptococcal meningitis is one of the
most important HIV-related opportunistic infections, with ?1
million cases occurring each year (36). Identification of novel
therapeutic targets for cryptococcosis is essential given the
toxic effects of existing therapies and the rising prevalence of
drug-resistant strains (38).
The ability to survive, grow, and divide at human physiolog-
ical temperature is an important C. neoformans virulence at-
tribute and is orchestrated by elaborate signaling pathways that
are not yet fully elucidated (3, 19). Calcineurin, a calcium/
calmodulin-dependent serine/threonine-specific protein phos-
phatase, is essential for C. neoformans growth at 37°C and for
its virulence (1, 21, 35). Calcineurin consists of two subunits,
the catalytic A (Cna1) and the regulatory B (Cnb1) subunits,
both of which are necessary for enzymatic function and survival
of C. neoformans at 37°C (1, 12, 35). Studies of both pathogenic
and nonpathogenic fungi have revealed important roles for
calcineurin in myriad physiological processes, including mor-
phogenesis, cell cycle progression, cytokinesis, septation, hy-
phal elongation during mating, cell wall biogenesis, and ion
homeostasis (7, 8, 15, 16, 18, 28, 30, 40). The calcineurin
signaling pathway is the target of the immunosuppressive drugs
cyclosporine and FK506 (26). While the importance of cal-
cineurin in fungal virulence has been established, its down-
stream targets in C. neoformans remain largely unknown (6).
The CTS1 gene (calcineurin temperature suppressor 1) was
identified in a multicopy suppressor screen of a calcineurin
mutant of C. neoformans (11). Cts1 contains a C2 domain,
which binds phospholipids and is found in a number of eukary-
otic proteins involved in membrane trafficking, generation of
lipid second messengers, activation of GTPases, and control of
protein phosphorylation (32, 41). Fox et al. previously reported
that overexpression of Cts1 restores growth at 37°C in cna1?
and cnb1? calcineurin mutant strains and confers resistance to
FK506 and cyclosporine at 37°C in wild-type (WT) cells (11).
Like the cna1? mutant, the cts1? mutant is inviable at 37°C
and avirulent in a murine model of cryptococcosis. Deletion of
CTS1 also confers defects in septation during vegetative
growth and in hyphal elongation during mating. Transcription
of CTS1 is increased in the cna1? mutant, and the cts1? cna1?
double mutant is synthetically lethal, suggesting that Cts1 and
calcineurin operate in parallel pathways with shared functions
in high-temperature growth and virulence.
However, this does not rule out the possibility that Cts1 and
calcineurin cooperate in certain processes necessary for sur-
vival at 37°C. Our studies sought to elucidate the interconnec-
tions between Cts1 and the calcineurin signaling pathway, in-
* Corresponding author. Mailing address: Department of Molecular
Genetics and Microbiology, Duke University Medical Center, 322
CARL Building, Box 3546 DUMC, Durham, NC 27710. Phone: (919)
684-3036. Fax: (919) 684-5458. E-mail: email@example.com.
† Supplemental material for this article may be found at http://ec
?Published ahead of print on 14 October 2011.
cluding examination of whether Cts1 is a phosphoprotein and
if its phosphorylation state is dependent on calcineurin phos-
phatase activity. Additionally, we sought to further character-
ize the role of Cts1 in C. neoformans high-temperature growth
and virulence. Here we report that GFP-Cts1 localizes to cy-
toplasmic puncta and colocalizes with the endosomal marker
FM4-64. The cts1? mutant shows a distinct FM4-64 staining
pattern, revealing a role for Cts1 in endocytic trafficking. GFP-
Cts1 also appears transiently at the mother bud neck in large
budded cells, which suggests a role in cytokinesis. Western blot
analysis reveals that Cts1 is phosphorylated at 37°C and is
hyperphosphorylated in the presence of the calcineurin inhib-
itor FK506 at 37°C, suggesting that Cts1 is likely a substrate of
calcineurin during high-temperature stress. Colocalization and
coimmunoprecipitation of mCherry-Cts1 and GFP-Cna1 at
37°C further support this hypothesis.
MATERIALS AND METHODS
Strains, medium, and growth conditions. All C. neoformans strains used in this
study are derived from the congenic serotype A (Cryptococcus neoformans var.
grubii) strains H99 (MAT?) and KN99 (MATa). Table 1 lists the strains and
plasmids used in this study. Unless otherwise indicated, strains were grown on
standard yeast medium.
Disruption of the CTS1 gene. Table S1 in the supplemental material includes
primer sequences used for generating deletions and fluorescent protein chime-
ras. The 5? and 3? regions of CTS1 were amplified from H99? or KN99a genomic
DNA, and the dominant selectable markers NAT (nourseothricin) and NEO
(G418) were amplified from plasmids pNATSTM#209 and pJAF1 (14), respec-
tively. The CTS1 gene replacement cassettes were generated by overlap PCR as
previously described (9). The cassettes were then precipitated onto gold micro-
carrier beads, and strain H99? or KN99a was transformed biolistically as previ-
ously described (10). Stable transformants were selected on YPD medium con-
taining nourseothricin or G418. To screen for cts1? mutants, diagnostic PCR was
performed. Positive transformants were further confirmed by Southern blotting.
Generation of fluorescent protein chimeras. Primers used to generate fluo-
rescent protein chimeras are listed in Table S1 in the supplemental material. To
generate a strain expressing GFP-Cts1, a plasmid encoding GFP-Cts1 and con-
taining a NAT resistance gene was engineered. The CTS1 open reading frame
(ORF) together with 139 bp of the 3?-untranslated region (3?-UTR) (containing
the flanking restriction sites for BamHI) was amplified from strain H99?, di-
gested with BamHI, and cloned into BamHI-digested and calf intestinal alkaline
phosphatase (CIP)-treated plasmid pCN19 (kindly provided by Connie Nichols
and Andrew Alspaugh, Duke University). The resulting plasmid, pLKB44, ex-
presses GFP-Cts1 under the control of a constitutive histone promoter. Strain
KN99a or the cts1? strain EA83 was transformed biolistically with plasmid
pLKB44 as described previously (10), and positive clones were screened based on
the presence of a fluorescence signal. Positive transformants were further con-
firmed by Western blotting.
To generate a strain expressing GFP-C2, a sequence containing a stop codon
followed by the 3?-UTR was ligated at an XmaI cleavage site within the CTS1
ORF in the plasmid pLKB44. This resulted in a chimera where green fluorescent
protein (GFP) was followed by the first 265 amino acids (aa) of Cts1 (161 aa of
the predicted C2 domain plus ?100 aa of downstream sequence). The cts1?
strain EA83 was transformed biolistically with the resulting plasmid, pLKB93.
Positive clones were screened based on the presence of a fluorescence signal and
further confirmed by Western blotting.
To generate a strain expressing GFP-Cts1C2?, amino acids 162 to 813 of Cts1
along with 139 bp of 3?-UTR sequence (containing flanking restriction sites for
BamHI) were amplified from strain H99?, digested with BamHI, and cloned into
BamHI-digested and CIP-treated plasmid pCN19. The cts1? strain EA83 was
transformed biolistically with the resulting plasmid, pLKB94. Positive clones
were screened based on the presence of a fluorescence signal and further con-
firmed by Western blotting.
To generate plasmids expressing mCherry N-terminal fusion chimeras, plas-
mids pLKB49 (NEO) and pLKB55 (HYG) were used as described previously
Microscopy. For imaging of yeast cells, ?0.5 ?l of cell suspension was placed
on a thin 2% agar complete medium patch on the slide and covered with a
For the FM4-64 experiments, a working concentration of 80 ?M was used
(from a 16 mM stock). In pulse-chase experiments, cells were pulsed with
FM4-64 on ice, washed, and analyzed immediately under a microscope. Images
were taken every 5 min. Strain LK221, expressing the putative septin GFP-Cns5
(23), was used as a wild-type control with a GFP signal. LK221 grows as well as
the wild type, and FM4-64 dynamics in this strain were similar to those of the
H99 wild-type strain.
Bright-field, differential interference contrast (DIC), and fluorescence images
were captured with either a Zeiss Axio Scope 2 Plus fluorescence microscope
(Carl Zeiss, Thornwood, NY) equipped with an AxioCam MRm digital camera
or a Zeiss Axio Scope microscope equipped with an Orca cooled charge-coupled
device camera (Hamamatsu, Bridgewater, NJ) and interfaced with MetaMorph
software (Universal Imaging, Silver Spring, MD). Images were processed using
Immunoblotting. Cells expressing GFP-Cts1 were grown overnight at room
temperature in liquid yeast extract-peptone-dextrose (YPD). Cultures were re-
freshed in the morning to a starting optical density at 600 nm (OD600) of ?0.3
and grown at room temperature until reaching an OD600of ?0.7. At this point,
each of the cultures was divided into four samples, which were subjected to the
following four different conditions for 1 h: 24°C, 24°C plus FK506 treatment,
37°C, and 37°C plus FK506 treatment. In samples containing FK506, 1 ?g/ml
FK506 was added.
Approximately 1 ? 107cells were resuspended in 225 ?l of pronase buffer (25
mM Tris-HCl, pH 7.5, 1.4 M sorbitol, 20 mM NaN3, and 2 mM MgCl2). Tri-
chloroacetic acid (TCA) was added to a final concentration of 17% (vol/vol), and
samples were stored at ?80°C. Cells were homogenized by vortexing with glass
beads at 4°C for 10 min. The lysates were collected, and the beads were washed
two times with 5% TCA to recover the remaining protein lysates. Precipitated
proteins were collected by centrifugation at 4°C. Pellets were dried and resus-
pended in approximately 30 ?l of Thorner buffer (40 mM Tris-HCl, pH 6.8, 8 M
urea, 5% SDS, 0.1 mM EDTA, 143 mM ?-mercaptoethanol, and 0.4 mg ml?1
bromophenol blue). Residual TCA was neutralized by the addition of 2 M
unbuffered Tris base. Prior to SDS-PAGE, samples were heated for 2 min at
42°C and then centrifuged at 14,000 rpm, and supernatants were loaded onto 4
to 20% polyacrylamide gels.
To detect GFP fusion proteins, an anti-GFP polyclonal antibody (Santa Cruz
TABLE 1. Strains and plasmids used in this study
a cts1::NEO GFP-CTS1:NAT
a cts1::NEO GFP-C2:NAT
a cts1::NEO GFP-CTS1C2?:NAT
a GFP-CTS1:NAT mCH-MYO1:HYG
? cts1::NAT mCH-MYO1:HYG
a GFP-CTS1:NAT mCH-RAB5:NEO
? mCH-CTS1:NEO GFP-CNA1:NAT
? cts1::NAT mCH-CNA1:NEO
? mCH-CTS1:NEO GFP-DCP1:NAT
a GFP-CTS1:NAT mCH-PUB1:NEO
VOL. 10, 2011Cts1 COOPERATES WITH CALCINEURIN IN C. NEOFORMANS1715
Biotech, Santa Cruz, CA) was used at a 1:1,000 dilution. The secondary antibody
was an anti-rabbit horseradish peroxidase (HRP)-conjugated antibody used at a
1:10,000 dilution. A PSTAIR antibody (Abcam, Cambridge, MA) was used at a
1:2,000 dilution as a loading control.
CIP assay. An overnight culture of cells expressing GFP-Cts1 was diluted to an
OD600of ?0.3 and grown until reaching an OD600of ?0.7. FK506 was added to
a concentration of 1 ?g/ml, and cells were shifted to 37°C for 1 h. One hundred
milliliters of the culture was then harvested. All subsequent steps were per-
formed at 4°C. Cells were collected by centrifugation and resuspended in 3
volumes of ice-cold breaking buffer (10 mM Tris, 150 mM NaCl, 0.5 mM EDTA,
pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease
inhibitor cocktail. An equivalent volume of glass beads was added, and cells were
shaken on a bead beater eight times for 90 s each, with 2-min interludes. Cell
lysates were transferred to fresh tubes. Four volumes of breaking buffer was used
to wash the beads. Lysates were cleared by centrifugation for 20 min at 14,000
rpm (4°C), and supernatants were transferred to fresh tubes. Lysates were incu-
bated for 1 h with 90 ?l of the antibody-conjugated protein A agarose beads (2
?l of anti-GFP antibody). The beads were then pelleted and washed four times
with the breaking buffer and once with CIP buffer (50 mM Tris-HCl, pH 8.0, 1
mM MgCl2, 0.1 mM ZnCl2) containing 1 mM PMSF and protease inhibitor
After immunoprecipitation, beads were resuspended in 30 ?l of CIP buffer.
Reaction mixtures were then incubated with or without the addition of 15 U of
CIP (NEB) at 37°C for 15 min. A parallel reaction was carried out in phosphatase
buffer containing CIP and 10 mM sodium orthovanadate as a phosphatase
inhibitor. The beads were then resuspended in an equivalent volume of 2?
loading buffer, boiled for 5 min, and electrophoresed in polyacrylamide-SDS gels.
Immunoprecipitation. Cells coexpressing mCherry-Cts1 and GFP-Cna1 were
grown overnight in liquid YPD, diluted in the morning to a starting OD600of
?0.3, and grown until reaching an OD600of ?0.7. The same procedure was also
used for the negative control, in which cells expressed only GFP-Cna1. At this
point, each of the cultures was divided into two samples, which were grown at
either 24°C or 37°C. All subsequent steps were performed at 4°C. Cells were
collected by centrifugation and resuspended in 3 volumes of ice-cold breaking
buffer (10 mM Tris, 150 mM NaCl, 0.5 mM EDTA, pH 7.5) containing 1 mM
PMSF and a protease inhibitor cocktail (Roche). An equivalent volume of glass
beads was added, and cells were shaken on a bead beater 10 times for 90 s each,
with 2-min periods of cooling. Cell lysates were transferred to fresh tubes. Four
volumes of breaking buffer was used to wash the beads. Lysates were cleared by
centrifugation for 20 min at 14,000 rpm (4°C), and supernatants were transferred
to fresh tubes.
RFP-Trap beads (Chromotek GmbH) were then added to each of the samples,
and mixtures were tumbled at 4°C for 1 h. The beads were then pelleted and
washed four times with the breaking buffer. After immunoprecipitation, the
beads were resuspended in an equivalent volume of 2? loading buffer, boiled for
5 min, and electrophoresed in polyacrylamide-SDS gels.
To detect mCherry-Cts1, an anti-DsRed polyclonal antibody (Invitrogen) was
used at a 1:1,000 dilution. The secondary antibody was an anti-rabbit HRP-
conjugated antibody used at a 1:10,000 dilution.
Other methods. For analysis of cell viability, 10-fold serial dilutions from
overnight liquid cultures were performed, and 2 ?l of each dilution was spotted
on an appropriate medium as indicated in the figure legends. The largest number
of spotted cells was 104cells.
To perform matings of C. neoformans, cells of opposite mating types were
mixed in water, spotted on V8 or MS agar medium, and cultured at 25°C in the
dark. Formation of hyphae, basidia, and chains of basidiospores was examined by
Phenotype of the serotype A cts1? mutant strain. Deletion
of the CTS1 gene was previously shown to cause inviability at
37°C and to confer defects in septation during vegetative
growth and in hyphal elongation during mating in serotype D
strains (Cryptococcus neoformans var. neoformans) (11). Our
studies examined the role of Cts1 in strains of serotype A
(Cryptococcus neoformans var. grubii), which is the predomi-
nant serotype among clinical isolates (31, 45). Given that the
serotype A and serotype D lineages have considerable genetic
and genomic differences, we examined the phenotype of the
serotype A cts1? mutant strain to gain insights into its function
in this variety (13).
The C2 domain of Cts1 was previously shown to be essential
for high-temperature growth, septation, and phospholipid
binding in serotype D strains (11). However, the phenotype
and viability at 37°C of a strain expressing only the C2 domain
of Cts1, which would provide insights into whether the C2
domain of Cts1 alone is sufficient to complement the growth
defects of the cts1? mutant, remained unknown. In addition to
examining the phenotype of the cts1? mutant, we therefore
also sought to examine the phenotypes of strains lacking the
C2 domain of Cts1 or expressing only the C2 domain of Cts1.
The CTS1 gene was disrupted by homologous recombination
with a cassette containing either the nourseothricin or neomy-
cin resistance gene. Mutation of the CTS1 gene was confirmed
by PCR and Southern blot analysis (data not shown). The
cts1? strain was complemented with CTS1 fused to the GFP
gene at the N terminus and ectopically expressed under the
control of a constitutive histone H3 promoter. Similarly, the C2
domain of Cts1 and a truncation allele lacking the C2 domain
were expressed ectopically from the histone H3 promoter as
GFP fusions in the cts1? background (Fig. 1A). Sequencing
and Western blot analysis were used to confirm that these
fusion proteins were stably expressed (see Fig. S1 in the sup-
plemental material). However, we noted that a small fraction
(5 to 10%) of cells expressing mutant versions of Cts1 fused to
GFP did not show a fluorescence signal based on microscopic
observations (data not shown).
Growth of the cts1? strain was largely affected at 37°C in a
spot dilution growth assay, consistent with the phenotype pre-
viously reported for serotype D (11) (Fig. 1B). GFP-Cts1 im-
proved growth in the cts1? background, indicating that the
GFP tag did not significantly affect the function of Cts1. While
the cts1? strain exhibited partial growth at 37°C, the cts1?
GFP-C2 and cts1? GFP-Cts1C2? strains did not grow at 37°C,
which suggests that both the C2 domain and the C-terminal
domain of Cts1 are essential for high-temperature growth and
that expression of these constructs may have a negative effect
on cell growth at 37°C. Strikingly, the cts1? GFP-Cts1C2?
strain also grew slower than the cts1? strain at 24°C, suggesting
that expression of GFP-Cts1C2? may have a negative effect on
cell growth even at 24°C.
The serotype A cts1? strain did not grow in a spot dilution
growth assay on medium containing the calcineurin inhibitor
FK506 at 24°C (Fig. 1B). This drug-imposed phenotype indi-
cates that Cts1 and calcineurin likely function in parallel or
branched pathways in serotype A, which is consistent with
previous findings on serotype D (11). Strikingly, the GFP-C2
allele was able to partially complement the growth defect of
the cts1? strain in the presence of FK506 at 24°C, although
significantly less than GFP-Cts1. The cts1? GFP-Cts1C2?
strain was not viable in the presence of FK506 at 24°C, which
suggests that the fragment containing the C2 domain may be
essential for survival in the absence of calcineurin at 24°C.
Interestingly, chains of cells were not observed in the sero-
type A cts1? deletion strain, in contrast to previous findings on
serotype D (11), suggesting that deletion of CTS1 does not
confer a cell separation defect in the serotype A strain back-
ground. However, a cytokinesis defect was observed in the
serotype A cts1? strain, as most cells displayed unusually large
1716ABOOBAKAR ET AL.EUKARYOT. CELL
buds, multiple buds, and/or misshapen buds (Fig. 1C and D).
The cytokinesis defect was rescued by the expression of GFP-
Cts1, as the morphology of the cts1? GFP-Cts1 cells was sim-
ilar to that of WT GFP-Cts1 cells (Fig. 1C; see Fig. S2 in the
supplemental material). Both the cts1? GFP-C2 and cts1?
GFP-Cts1C2? strains also exhibited a cytokinesis defect, indi-
cating that neither fragment is able to fully complement the
cts1? mutant phenotype. However, the cts1? GFP-C2 strain
had a less severe phenotype than the cts1? GFP-Cts1C2?
strain, as a smaller percentage of aberrant cells (defined as
cells with unusually large buds, multiple buds, and/or mis-
shapen buds) was observed (Fig. 1C and D). The morpholog-
ical defect of the cts1? GFP-Cts1C2? strain was also more
severe than that of the cts1? strain, which further supports the
hypothesis that expression of GFP-Cts1C2? has a negative
effect on cell morphology.
We also examined whether the serotype A cts1? strain ex-
hibits mating defects similar to those observed in serotype D
(Fig. 1E). No defects were observed when either the ?
cts1::NAT or a cts1::NEO strain was cocultured with wild-type
tester strains in a unilateral cross on MS medium. However, no
hyphae, basidia, or spores were observed when ? cts1::NAT
and a cts1::NEO mutant strains were cocultured in a bilateral
cross on MS medium, which is consistent with previous findings
in serotype D (11). To determine whether Cts1 is necessary for
cell-cell fusion, the mating patches were resuspended in dis-
tilled water and plated on nourseothricin and G418 double-
selection medium. No doubly resistant isolates were obtained,
indicating that Cts1 is necessary for cell fusion during mating
(data not shown). It is important that while calcineurin is
essential for production of recombinant basidiospores, it is not
necessary for cell fusion, suggesting that involvement in cell
fusion is a calcineurin-independent function of Cts1 (7).
Cts1 localizes to cytoplasmic puncta and appears tran-
siently at the mother bud neck during cytokinesis. To gain
further insights into the function of Cts1, localization of GFP-
Cts1 was examined. In some large budded cells, a single ring
corresponding to GFP-Cts1 was observed at the mother bud
neck (Fig. 2). The fact that the vast majority of cells did not
show localization of GFP-Cts1 to the mother bud neck sug-
gested that this localization might be transient. To address this
question, we performed time-lapse analysis of cells expressing
GFP-Cts1 (Fig. 2A). GFP-Cts1 underwent constriction at the
mother bud neck similar to that of Myo1, which is part of the
actomyosin ring and plays a critical role in cytokinesis (2).
GFP-Cts1 constricted as a single ring and later appeared on
FIG. 1. Serotype A cts1? mutants exhibit defects in growth during stress, cytokinesis, and hyphal elongation during mating. (A) Diagram
showing the GFP-Cts1 fusion proteins used in this study. The putative calcineurin-binding motifs LPVP, LAPP, and LAVP are indicated. (B) Spot
dilution growth assays of cts1? mutants. (C) Deletion of CTS1 confers a cytokinesis defect during high-temperature growth in C. neoformans
serotype A. Neither the GFP-C2 nor GFP-Cts1C2? allele was able to complement the cytokinesis defect. The cts1? GFP-Cts1C2? strain has a
more severe phenotype than the cts1? strain. Bar, 5 ?m. (D) Graphs showing the proportions of aberrant cells in the images in panel C (n ? 50
cells for each panel). (E) Hyphal elongation and sporulation were observed when either the MAT? cts1? or MATa cts1? strain was coincubated
with a wild-type tester strain in a unilateral cross on MS medium. However, when the MAT? cts1? and MATa cts1? strains were cocultured in a
bilateral cross, no hyphae were produced.
VOL. 10, 2011Cts1 COOPERATES WITH CALCINEURIN IN C. NEOFORMANS1717
both sides of the neck as either two single spots or a double
ring. To examine whether Cts1 may also be a part of the
actomyosin ring, mCherry-Myo1 was coexpressed with GFP-
Cts1. In large budded cells, the constriction of GFP-Cts1
closely followed that of mCherry-Myo1 but occurred later,
suggesting that Cts1 is not part of the actomyosin ring (Fig.
2B). We also examined the possibility that Cts1 may be nec-
essary for Myo1 function by expressing mCherry-Myo1 in a
cts1? mutant strain. mCherry-Myo1 appearance at the bud
neck during cytokinesis was not affected by the absence of
CTS1 (data not shown).
In addition to the bud neck localization, GFP-Cts1 was also
observed at cytoplasmic puncta in all cells. At 24°C, GFP-Cts1
localized to an average of 2.9 cytoplasmic puncta per cell in the
cts1? background (Fig. 3A and B). Because CTS1 is essential
for high-temperature growth, we also examined whether Cts1
undergoes a change in localization during high-temperature
stress. The cts1? GFP-Cts1 strain was incubated on a micro-
FIG. 2. Cts1 localizes to the mother bud neck during cytokinesis. (A) Time-lapse analysis of cells expressing GFP-Cts1. Cts1 transiently appears
at the mother bud neck as a single ring that undergoes constriction (arrows). Bar, 5 ?m. (B) mCherry-Myo1 (red) was coexpressed with GFP-Cts1
(green). Time-lapse analysis shows that the constriction of GFP-Cts1 closely follows that of mCherry-Myo1 but occurs later, suggesting that Cts1
is not part of the actomyosin ring.
FIG. 3. Cts1 localizes to cytoplasmic puncta that increase in number during thermal stress. (A) GFP-Cts1 localizes to cytoplasmic puncta at
both 24°C and 37°C and to the mother bud neck (arrow). In contrast, the GFP-C2 protein largely showed only diffuse cytoplasmic localization;
cytoplasmic puncta were observed in only some cells, primarily at 37°C. The GFP-C2 protein did not localize to the mother bud neck. The
GFP-Cts1C2? fusion protein, on the other hand, showed a localization pattern that was similar to that of GFP-Cts1. The GFP-Cts1C2? protein
localized to cytoplasmic puncta at both 24°C and 37°C. Additionally, it also localized to the mother bud neck in some large budded cells (arrow).
(B) Average number of cytoplasmic puncta per cell for each of the three strains (n ? 50 cells for each panel).
1718ABOOBAKAR ET AL.EUKARYOT. CELL
scope slide that was placed on a heating block at 37°C. While
GFP-Cts1 still localized to cytoplasmic puncta and to the
mother bud neck in some large budded cells, the number of
cytoplasmic puncta at which GFP-Cts1 localized increased
from an average of 2.9 per cell to 4.7 per cell after only 30 min
of incubation at 37°C (Fig. 3A and B). This suggests that the
function of Cts1 at cytoplasmic puncta may be upregulated and
become more important during high-temperature stress re-
We also examined whether the C2 domain of Cts1 is either
necessary or sufficient for localization to cytoplasmic puncta
and the mother bud neck (Fig. 3A). In the cts1? GFP-Cts1C2?
strain, the GFP tag signal showed localization to puncta and
the mother bud neck, similar to that of the cts1? GFP-Cts1
strain. However, the average number of GFP-Cts1C2? cyto-
plasmic puncta was ?2-fold higher at 24°C and ?1.2-fold
higher at 37°C than that of GFP-Cts1 cytoplasmic puncta (Fig.
3B). On the other hand, the cts1? GFP-C2 strain exhibited
mainly diffuse cytoplasmic localization, with only a few cells
showing localization to puncta. However, the average number
of GFP-C2 puncta per cell increased when cells were shifted to
37°C. Localization of GFP-C2 to the mother bud neck was not
observed. This indicates that the C2 domain is neither neces-
sary nor sufficient for localization to cytoplasmic puncta or the
mother bud neck. Proper localization, however, is not sufficient
for Cts1 functions to support high-temperature growth, as the
growth of the cts1? GFP-Cts1C2? strain was clearly affected at
37°C, despite the ability of the Cts1C2? protein to localize to
cytoplasmic puncta and the mother bud neck.
Cts1 colocalizes with the endosomal marker FM4-64 and
may play a role in endocytic trafficking. The localization of
GFP-Cts1 to both cytoplasmic puncta and the mother bud neck
in large budded cells suggested two distinct functions for Cts1.
Previous work implicated a role for Cts1 in septation, which is
consistent with localization of GFP-Cts1 to the mother bud
neck (11). We also further explored the cytoplasmic localiza-
tion of Cts1 to determine the subcellular compartments or
structures to which Cts1 localizes, which would provide insights
into other potential roles of Cts1. Colocalization was not ob-
served when cells expressing GFP-Cts1 were stained with Mi-
toTracker (data not shown), which indicates that Cts1 puncta
do not correspond to mitochondria.
To determine whether the cytoplasmic puncta of Cts1 rep-
resent endosomes or vacuolar membranes, cells expressing
GFP-Cts1 were pulsed on ice with the endocytic tracker dye
FM4-64, incubated at 30°C, and analyzed every 15 min. After
45 min of incubation at 30°C, colocalization was observed at
cytoplasmic puncta that may represent late endosomes (Fig.
4A). This indicates that Cts1 is localized to and may be in-
volved in the endocytic pathway. To further explore this pos-
sibility, GFP-Cts1 was coexpressed with the mCherry-tagged
early endosomal marker Rab5. Although colocalization was
not observed in the majority of cells, the GFP and mCherry
signals did overlap in some cells (out of 50 cells, 13 showed at
least one punctum exhibiting colocalization) (Fig. 4B). This
further supports the hypothesis that Cts1 may play a role in
To further investigate a possible role for Cts1 in endocytosis,
we examined the FM4-64 staining pattern in the cts1? mutant
strain. A vacuolar staining pattern was observed in the cts1?
mutant, whereas the wild type showed punctate staining (Fig.
4C). The punctate staining pattern of FM4-64 observed in
wild-type cells is strikingly different from that seen in Saccha-
romyces cerevisiae, which typically shows sustained vacuolar
staining. In fact, the staining in cts1? cells more closely resem-
bled that in S. cerevisiae cells. To further examine this distinct
phenotype, time-lapse analysis of FM4-64 localization was per-
formed in a pulse-chase experiment. The cts1? mutant and a
wild-type GFP-expressing strain, LK221, were mixed at a 1:1
ratio and examined simultaneously on the same slide to ensure
identical conditions. The GFP signal was used to distinguish
between the wild type and the cts1? mutant (Fig. 4D). Cells
were pulsed with FM4-64 on ice, washed, and immediately
analyzed by time-lapse microscopy to test the progression of
endocytosis. In both wild-type and mutant cells, FM4-64
reached the vacuole, similar to the staining described for S.
cerevisiae. However, in contrast to the case in S. cerevisiae,
FM4-64 eventually disappeared from the vacuolar membrane.
In the GFP-expressing wild-type strain, the percentage of cells
showing vacuolar staining of FM4-64 increased initially and
then declined to ?6% after 60 min. In the cts1? mutant, the
percentage of cells showing vacuolar staining increased ini-
tially, but the decline was delayed compared to that of the wild
type and the proportion of cells showing a vacuolar signal
remained at 30% after 30 min, compared to 13% of the wild
type (Fig. 4D). We eliminated the possibility that this differ-
ence was due to the GFP tag by examining the FM4-64 staining
pattern in a 1:1 culture of wild-type GFP-expressing and non-
GFP-expressing cells. Both wild-type strains showed similar
dynamics of FM4-64 progression through endocytosis (Fig.
4D). These findings suggest that Cts1 plays a role in endocytic
trafficking and may be involved specifically in recycling of the
mCherry-Cts1 colocalizes with GFP-Cna1 at sites of mRNA
processing. We previously reported that the catalytic subunit
of calcineurin, GFP-Cna1, undergoes a relocalization to cyto-
plasmic puncta during high-temperature stress in C. neofor-
mans (22). Because GFP-Cts1 also localizes to cytoplasmic
puncta at 37°C, we examined whether calcineurin and Cts1
colocalize, which would be consistent with concerted functions
in processes necessary for high-temperature growth. Cts1 was
tagged with mCherry at the N terminus and expressed ectop-
ically under the control of a constitutive GPD1 promoter.
GFP-Cna1 was coexpressed with mCherry-Cts1, and strikingly,
colocalization was observed at 37°C but not at 24°C (Fig. 5A).
While the average number of puncta corresponding to
mCherry-Cts1 was greater than the number corresponding to
GFP-Cna1, colocalization was observed at every cytoplasmic
punctum at which GFP-Cna1 was present.
To test whether Cts1 localization to cytoplasmic puncta is
dependent on calcineurin catalytic activity, cells expressing
GFP-Cts1 were treated with the calcineurin inhibitor FK506.
GFP-Cts1 localization did not change significantly at either
24°C or 37°C, suggesting that calcineurin catalytic activity is not
necessary for the localization of Cts1 (data not shown). We
also examined whether calcineurin localization depended on
Cts1 by expressing GFP-Cna1 in the cts1? mutant strain. Cal-
cineurin localization at both 24°C and 37°C was similar to that
previously reported for the wild type (22), indicating that cal-
VOL. 10, 2011 Cts1 COOPERATES WITH CALCINEURIN IN C. NEOFORMANS 1719
cineurin localization is not dependent on Cts1 (data not
We also previously reported that the P-body component
Dcp1 and the stress granule constituent Pub1 colocalize with
Cna1 at 37°C, suggesting a role for calcineurin in regulation of
protein synthesis (22). To test whether Cts1 puncta also colo-
calize with these proteins, mCherry-Cts1 was coexpressed with
GFP-Dcp1 and GFP-Cts1 was coexpressed with mCherry-
Pub1. GFP-Dcp1 and mCherry-Cts1 colocalized at 24°C, but
not at all cytoplasmic puncta (Fig. 5B). On the other hand,
mCherry-Cts1 largely colocalized with GFP-Dcp1 and GFP-
Cts1 largely colocalized with mCherry-Pub1 at 37°C (Fig. 5B
and C). At 24°C, however, mCherry-Pub1 showed diffuse cy-
toplasmic localization, and colocalization with GFP-Cts1 was
not observed. These findings suggest a potential role for Cts1
in regulation of mRNA processing at P-bodies and stress gran-
ules, especially during the high-temperature stress response.
Cts1 is a phosphoprotein and is hyperphosphorylated in the
presence of FK506. Colocalization of mCherry-Cts1 and GFP-
Cna1 at 37°C suggested that Cts1 and calcineurin function
coordinately in processes necessary for high-temperature
growth. To further examine the interconnection between Cts1
and the calcineurin signaling pathway, we investigated whether
Cts1 may be a substrate of the protein phosphatase calcineurin.
Cells expressing GFP-Cts1 were grown at 24°C and 37°C in
both the presence and absence of the calcineurin inhibitor
FK506. GFP-Cts1 was subsequently detected by Western blot
analysis, which revealed an additional band at 37°C with a
slower electrophoretic mobility than at 24°C (Fig. 6A). Strik-
ingly, the signal corresponding to the higher-molecular-weight
band of GFP-Cts1 was even more abundant in the presence of
FK506 at 37°C.
To determine whether the shift to a higher molecular weight
was due to phosphorylation, cells expressing GFP-Cts1 were
grown at 37°C in the presence of FK506 and immunoprecipi-
tated using GFP-Trap beads. The beads were subsequently
treated with CIP. Western blot analysis revealed a faster elec-
trophoretic mobility of GFP-Cts1 for the sample incubated
with CIP (Fig. 6B). On the other hand, the sample incubated
with both CIP and sodium orthovanadate, a phosphatase in-
FIG. 4. Cts1 colocalizes with the endosomal marker FM4-64 and may play a role in endocytosis. (A) Cells expressing GFP-Cts1 were pulsed
with the endosomal marker FM4-64 on ice, washed, and incubated at 30°C. After a 45-min incubation period, FM4-64 colocalized with GFP-Cts1
at cytoplasmic puncta that may represent endosomes. (B) GFP-Cts1 was coexpressed with the mCherry-tagged early endosomal marker Rab5.
Colocalization was not observed in the majority of cells, but the GFP and mCherry signals did overlap in some cells (arrow). (C) The FM4-64
staining pattern was examined in the cts1? mutant and compared to that of the wild type after 20 min of incubation at 24°C. FM4-64 displayed
vacuolar staining in the cts1? mutant but not in the wild-type KN99a strain. (D) The distinct FM4-64 staining pattern of the cts1? mutant was
further examined in a pulse-chase experiment. A wild-type GFP-expressing strain, LK221 and the cts1? mutant were pulsed with FM4-64 on ice,
washed, and immediately analyzed by time-lapse microscopy to test the progression of endocytosis. To ensure identical conditions, the two strains
were mixed at a 1:1 ratio prior to the experiment and examined simultaneously on the same slide. The GFP tag allowed us to distinguish between
the wild type and the cts1? mutant, as indicated in the upper panels. The graphs show the percentages of cells with a vacuolar signal over time
for FM4-64 staining in a control experiment (left) and an experiment where the GFP-expressing wild type and the cts1? mutant were compared
1720 ABOOBAKAR ET AL.EUKARYOT. CELL
hibitor, did not show faster electrophoretic mobility. These
data indicate that Cts1 is phosphorylated during high-temper-
ature stress and is hyperphosphorylated in the presence of
FK506, suggesting that Cts1 may be dephosphorylated in a
mCherry-Cts1 and GFP-Cna1 coimmunoprecipitate at
37°C. Colocalization of mCherry-Cts1 and GFP-Cna1 at 37°C
and the hyperphosphorylated state of GFP-Cts1 in the pres-
ence of FK506 at 37°C suggested that Cts1 may be a substrate
of calcineurin. To further test this hypothesis, we examined
whether Cts1 and calcineurin are part of the same complex in
vivo at either 24°C or 37°C. Cells coexpressing mCherry-Cts1
and GFP-Cna1 were grown at either 24°C or 37°C. mCherry-
Cts1 and any interacting proteins were subsequently immuno-
precipitated from cell lysates by using RFP-Trap beads.
Strikingly, GFP-Cna1 was detected on the immunoblot for
both the 24°C and 37°C immunoprecipitation samples, with a
significantly greater abundance detected at 37°C (Fig. 6C). The
abundance of mCherry-Cts1, on the other hand, was roughly
equivalent in both samples, indicating that greater complex
formation between GFP-Cna1 and mCherry-Cts1 occurs dur-
ing high-temperature stress. Moreover, the difference in the
abundance of GFP-Cna1 in the two samples argues against the
possibility that the detected coimmunoprecipitation was due to
nonspecific binding between the GFP and mCherry fluorescent
tags. Taken together, these findings provide evidence that Cts1
and calcineurin are part of the same complex, with greater
complex formation occurring at 37°C than at 24°C.
The importance of calcineurin phosphatase activity in eu-
karyotic transcriptional regulation during stress has been well
established. Important targets of calcineurin include members
of the nuclear factor of activated T cell (NFAT) family of
transcription factors, which activate immune responses in mul-
ticellular eukaryotes (20). In the budding yeast Saccharomyces
cerevisiae, dephosphorylation of Crz1 by calcineurin leads to
elevated transcription of more than 160 genes that promote
remodeling of the cell surface in response to stress (8). Impor-
tantly, no clear functional homologue of the CRZ1 gene has
been identified in C. neoformans serotype A or D, suggesting
FIG. 5. Cts1 and calcineurin colocalize at sites of mRNA process-
ing during high-temperature stress. (A) Cells coexpressing mCherry-
Cts1 and GFP-Cna1 were examined at both 24°C and 37°C. At 24°C,
GFP-Cna1 showed diffuse cytoplasmic localization and colocalization
with mCherry-Cts1 was not observed. Strikingly, at 37°C, calcineurin
underwent a relocalization to cytoplasmic puncta, where it largely
colocalized with mCherry-Cts1. (B) mCherry-Cts1 was coexpressed
with the P-body component GFP-Dcp1 to test for colocalization at
24°C and 37°C. Cts1 and Dcp1 mostly colocalized at 37°C. However,
significant colocalization was not observed at 24°C. While mCherry-
Cts1 and GFP-Dcp1 did colocalize at some puncta (arrow 1), only
partial colocalization was observed at others (arrow 2). There were
also puncta where only GFP-Dcp1 appeared (arrow 3) and some
where only mCherry-Cts1 localized (arrow 4). (C) GFP-Cts1 was co-
expressed with the mCherry-tagged stress granule component Pub1. At
24°C, mCherry-Pub1 exhibited diffuse cytoplasmic localization, and
colocalization with GFP-Cts1 was not observed. However, at 37°C,
mCherry-Pub1 localized to cytoplasmic puncta that mostly colocalized
with GFP-Cts1 (arrow).
FIG. 6. Cts1 appears to be a substrate of calcineurin during high-
temperature stress response. (A) Western blot analysis of GFP-Cts1
showing slower electrophoretic mobility at 37°C, which was more pro-
nounced in the presence of the calcineurin inhibitor FK506. (B) CIP
treatment of immunoprecipitated GFP-Cts1 confirmed that the slower
electrophoretic mobility at 37°C in panel A was due to phosphoryla-
tion. (C) mCherry-Cts1 was immunoprecipitated using RFP-Trap
beads in samples grown at either 24°C or 37°C. Coimmunoprecipita-
tion of GFP-Cna1 and mCherry-Cts1 was detected at both 24°C and
37°C and was significantly more pronounced at 37°C. To better display
the input, a shorter exposure is shown in the lower left panel. Similarly,
a longer exposure of the immunoprecipitation is shown in the lower
right panel to illustrate the difference between 24 and 37°C.
VOL. 10, 2011Cts1 COOPERATES WITH CALCINEURIN IN C. NEOFORMANS1721
that the effects of calcineurin in C. neoformans could be me-
diated by different transcription factors or may be partly or
entirely posttranscriptional (24).
Overexpression of the C2 domain protein Cts1 was previ-
ously shown to restore growth at 37°C in a calcineurin cna1?
mutant of C. neoformans, which suggested that Cts1 may be a
substrate and possibly an effector of calcineurin during high-
temperature stress (11). Here we report three findings that
support this hypothesis: (i) mCherry-Cts1 and GFP-Cna1 co-
localize specifically during thermal stress at 37°C; (ii) Cts1 is
hyperphosphorylated in the presence of the calcineurin inhib-
itor FK506 at 37°C, which suggests that Cts1 is dephosphoryl-
ated in a calcineurin-dependent manner; and (iii) mCherry-
Cts1 and GFP-Cna1 coimmunoprecipitate, with stronger
complex formation at 37°C than at 24°C. It is important that
Cts1 also contains the LAVP calcineurin-binding motif found
in NFATc1 and NFATc4, as well as the LAPP calcineurin-
binding motif found in the calcineurin regulatory protein
RCAN1 (25, 27, 29, 37, 43) (Fig. 1A). Taken together, these
findings reveal a novel posttranscriptional role of calcineurin
during high-temperature stress responses in C. neoformans.
Cts1 was first isolated and described for serotype D, so in
this study we examined the function of Cts1 in serotype A.
Interestingly, the CTS1 ORF in serotype A lacks the sequence
for a putative leucine zipper motif that was previously de-
scribed for serotype D (11). This motif was dispensable for
growth at 37°C but was found to be essential for virulence,
proper hyphal elongation, and viability in the absence of cal-
cineurin function in serotype D (11). The absence of the leu-
cine zipper motif in serotype A extends previous findings in-
dicating that this domain is not essential for growth at 37°C and
suggests that the effects of the C-terminal truncation of Cts1 in
serotype D may be due to a lack of other important motifs
within the C terminus of Cts1.
What is the role of Cts1 in C. neoformans high-temperature
growth and virulence? It was previously reported that deletion
of CTS1 confers defects in septation during vegetative growth
and in hyphal elongation during mating (11). Here we exam-
ined the localization of Cts1 to gain further insights into its
function. GFP-Cts1 localizes to the mother bud neck as a
single ring in large budded cells. The constriction of this ring
closely follows that of the actomyosin ring component Myo1.
While the Cts1 dynamics do not completely match those of the
actomyosin ring, it is still possible that at some point during the
cell cycle, Cts1 associates with the actomyosin ring. The double
ring of GFP-Cts1 after it constricts suggests a possible associ-
ation with the septin double ring, but testing this will require
further studies. Our localization studies support a potential
role for Cts1 in cytokinesis. A recent study in the fission yeast
Schizosaccharomyces pombe provides some clues to the poten-
tial role of Cts1 in cytokinesis (42). The S. pombe C2 domain
protein Fic1 was identified and shown to exhibit homology to
both Cts1 and the S. cerevisiae protein Inn1 (42). Fic1 adds
structural integrity to the contractile ring during cytokinesis
and binds to the SH3 domain of Cdc15, which participates in
cellular processes that bridge the plasma membrane and cyto-
skeleton. It is possible that Cts1 carries out similar functions
during cytokinesis in C. neoformans. Importantly, Cts1 is rich
in prolines, which is consistent with a possible interaction with
SH3 domains (33). Future studies should further explore the
specific role of Cts1 in cytokinesis.
GFP-Cts1 also localizes to cytoplasmic puncta that colocal-
ize with the endocytic tracker FM4-64 at late endosomes. Strik-
ingly, compared to the wild type, the cts1? strain showed a
higher percentage of cells with vacuolar FM4-64 staining dur-
ing progression through endocytosis, suggesting that Cts1 may
play a role in vacuolar membrane trafficking. This is consistent
with previous studies that have reported involvement of C2
domain proteins, including synaptogamin I and rabphilin-3A,
in membrane trafficking (4, 46).
It is also unknown whether there are connections between
the roles of Cts1 in membrane trafficking and cytokinesis.
Schink and Bolker recently reported that the Ustilago maydis
Cdc42-specific guanine nucleotide exchange factor Don1 local-
izes to fast-moving endosomal vesicles that accumulate at the
site of septation during cytokinesis (44). Cts1 may have a sim-
ilar function in the cytoplasmic puncta and at the mother bud
neck, but further studies are necessary to investigate this hy-
In addition to potential roles in endocytic trafficking and
cytokinesis, Cts1 may also be involved in mRNA processing
and regulation of protein translation during high-temperature
stress. At 37°C, Cts1 colocalizes with the calcineurin catalytic
subunit Cna1, the P-body constituent Dcp1, and the stress
granule component Pub1. Colocalization of Cts1 and Cna1 at
sites of mRNA processing suggests that Cts1 may also play a
role in regulation of translation. Future studies should inves-
tigate this potential role.
This study was supported by NIH/NIAID R01 grants AI42159 and
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