Candida infections are a major cause of death in immuno-compromised
patients, such as HIV and transplant patients and patients receiving cancer
chemotherapy (White et al., 1998). Candida
albicans infections are typically treated with azole drugs that inhibit
the enzyme Erg11p/Cyp51p/lanosterol demethylase (Lepesheva
and Waterman, 2007; Waterman and Lepesheva, 2005)
or with drugs that the ergosterol biosynthetic pathway. Erg11p is a cytochrome
P450 protein (Nebert and Russell, 2002), a class of
proteins that was named for its unusual absorption spectrum that arises from
bound heme (Werck-Reichhart and Feyereisen, 2000). Erg11p
requires a reductase partner and Erg11p activity is elevated by cytochrome b5,
Cyb5p (Lamb et al., 1999; Rogers
et al., 2004; Schenkman and Jansson, 2003).
Disrupted ergosterol synthesis triggers alterations in the expression patterns
of numerous genes (Agarwal et al., 2003; Bammert
and Fostel, 2000; Hughes et al., 2000),
including 11 ERG (ergosterol synthetic) genes, CYB5/cytochrome b5
(Agarwal et al., 2003), heme synthetic genes
and DAP1 (damage resistance protein 1). Dap1p is composed largely of a region
of limited homology with cytochrome b5 called a heme-1 domain (Hand
et al., 2003; Mifsud and Bateman, 2002).
Cells deleted for DAP1 are defective in ergosterol biosynthesis at the step
catalyzed by Erg11p (Hand et al., 2003) and
as a result, dap1Δ cells are sensitive to azole antifungal drugs (Hand
et al., 2003; Mallory et al., 2005a),
which inhibit Erg11p. While Dap1p was originally characterized in S. cerevisiae,
Dap1p has a similar role in the phylogenetically distant yeast Schizosaccharomyces
pombe (Hughes et al., 2007). Dap1p has been
included as part of the global antifungal drug resistance network (Parsons
et al., 2004) and likened to a helping hand for P450
proteins (Debose-Boyd, 2007).
ERG11 over-expression suppresses itraconazole sensitivity in dap1Δ cells
and restores normal ergosterol synthesis to dap1Δ cells (Mallory
et al., 2005a) and in some strain backgrounds, Dap1p regulates the
stability of Erg11p (Mallory et al., 2005a).
Dap1p binds to heme through a penta-coordinate mechanism that utilizes a carboxy-terminal
tyrosine (Ghosh et al., 2005) and heme binding
is required for sterol synthesis and for stabilizing Erg11p (Mallory
et al., 2005a). In addition to regulating sterol synthesis, Dap1p
directs resistance to the methylating agent, methyl methanesulfonate (Hand
et al., 2003). MMS sensitivity is also due to defective Erg11p function
(Mallory et al., 2005a), suggesting that sterol
synthesis is needed for the repair of chemically induced damage, a conclusion
that is supported by genome-wide studies (Bennett et
al., 2001). Dap1p is also required for the uptake and storage of iron
and like the other Dap1p phenotypes, Dap1p-mediated iron metabolism is mediated
by Erg11p (Craven et al., 2007).
In S. cerevisiae, Dap1p lacks a putative membrane-spanning sequence
and localizes to punctuate cytoplasmic sites that co-fractionate with endosomes
(Craven et al., 2007). The localization of S.
cerevisiae Dap1p overlaps partially with that of Erg11p (Craven
et al., 2007) but no direct interaction for the two proteins has
been reported. In contrast, S. pombe Dap1p has a putative amino-terminal
membrane spanning sequence and co-precipitates with Erg11p (Hughes
et al., 2007), as does the mammalian Dap1p homologue, PGRMC1/Hpr6
(Hughes et al., 2007). PGRMC1 and the related
rodent homologues localize to the endoplasmic reticulum (Crudden
et al., 2005; Nolte et al., 2000),
with a smaller fraction localizing to the cell membrane (Krebs
et al., 2000).
Because S. cerevisiae and S. pombe Dap1p regulate azole drug
resistance and iron metabolism, pathways that are important for the biology
of pathogenic yeast, we have tested the extent to which C. albicans Dap1p
performs analogous functions. We have found that, like its relatives in non-pathogenic
yeast, C. albicans Dap1p regulates ergosterol synthesis at the step catalyzed
by Erg11p and promotes azole drug resistance. Furthermore, C. albicans Dap1p
is required for filamentous growth and for resistance to the DNA damaging agent
methyl, methanesulfonate (MMS). Thus, C. albicans Dap1p activates an
essential pathway in pathogenic fungi that regulates drug susceptibility and
MATERIALS AND METHODS
Culture conditions and chemicals: Unless stated otherwise, cells
were maintained by culturing in Yeast Peptone-Dextrose (YPD) medium at 30°C.
Fluconazole (LKT laboratories), ketoconazole (Sigma), itraconazole (Sigma),
PD98059 (Calbiochem), methyl methanesulfonate (Sigma) and ferrozine (Acros)
were added at the concentrations indicated in the text.
Strain construction: All strain manipulations were performed in the
wild-type strain BWP17 (Wilson et al., 1999).
The first copy of DAP1 was deleted by targeted integration of the plasmid pJM72
digested with Hind III and Apa I. In all cases, cells were transformed using
the standard lithium acetate transformation procedure, except that cells were
incubated for 1 h at 30°C in YPD media before plating. We used this approach
because we identified unusual recombination events at the 3 end of the
DAP1 gene when smaller regions of homology were used. We did not detect these
events during the deletion of the second copy of DAP1 with the HIS3 gene. The
disruption of the DAP1 gene by URA3 was verified initially by PCR using the
primers CaDAP1- 1081F and CaURA3-154R for the 5 end and CaURA3+750F and
CaDAP1+1600R for the 3 end. Oligonucleotide sequences are shown in (Fig.
s1). The DAP1/dap1Δ:: URA3 strain was named JMCa3.
The second copy of DAP1 was deleted by transformation of JMCa3 with a PCR product
generated by amplification of the HIS1 gene from the plasmid pFA-HIS1 (Gola
et al., 2003) with the primers CaDAP1-54-KOF and CaDAP1+573-KOR.
The deletion of DAP1 was verified initially by PCR using the primers CaDAP1-1081F,
CaHIS1-280R, CaHIS1+983F and CaDAP1+1600R. The dap1Δ::URA3 dap1Δ::HIS1
strain was named JMCa5. All of the primers are outside the region of homology
targeted by the integration. To re-introduce the DAP1 gene to dap1Δ/dap1Δ
cells, the plasmid pRC65 was digested with BbsI and used to transform JMCa5
cells. Arg+ colonies were tested for re-integration using the primers
CaDAP1-1081F and CaDAP1+488R-Xho.
Plasmids: The CaDAP1 deletion plasmid pJM72 was prepared as follows.
(1) The URA3 gene was amplified from pFA-URA3 (Gola et
al., 2003) with the primers CaURA3-220F and CaURA3+940R and sub-cloned
into the plasmid pCR2.1 (InVitrogen), forming the plasmid pJM65. (2) The 1 kb
of genomic DNA flanking the 5 end of DAP1 was amplified from BWP17 genomic
DNA using the primers CaDAP1-1000F and CaDAP1-1R and sub-cloned into the Hind
III and Sac I sites of pJM65, forming the plasmid pJM70. (3) The 1 kb of genomic
DNA flanking the 3 end of the DAP1 gene was sub-cloned into pCR2.1 using
the primers CaDAP1+469F and CaDAP1+1468R, forming the plasmid pJM67. (4) The
Not I-Apa I fragment containing the DAP1 flanking sequence from pJM67 was sub-cloned
into the same sites of plasmid pJM70, forming the plasmid pJM72.
The CaDAP1 knock-in plasmid pRC65 was constructed as follows. The CaDAP1 open
reading frame, along with 1000 bp of upstream sequence, was PCR amplified using
the primers CaDAP1-1000F-Hind and CaDAP1+488R-Xho cloned into plasmid pCR2.1
(Invitrogen), resulting in the plasmid pRC63.
|Fig. s1: Oligonucleotide sequences used in this study
A NotI fragment containing the ARG4 gene from the plasmid pFA-ARG4 was then
cloned into the NotI site of pRC63, forming the plasmid pRC65.
Spotting assays and plate preparation: For all spotting assays, cells
were serially diluted 1:10 in water and spotted on plates containing 10 μM
fluconazole, 0.02% methyl methanesulfonate, or 100 μM bathophenanthroline.
Colonies were photographed after 48 h incubation at 30°C. The growth of
the dap1Δ/dap1Δ strain JMCa5 was compared with that of CNC44, an Arg-
Ura+ His+ SC5314 derivative (Negredo
et al., 1997). For halo formation assays, cells were grown to log
phase (approximately A600 = 1), whereupon the A600 was measured.
Cells were diluted to 0.3 A600 units in 3 mL of water containing
0.7% of melted Bacto-agar that was maintained at 48°C. The cell suspension
was immediately spread on YPD plates and allowed to harden. Paper disks were
placed on the agar and 10 μL of various drugs were spotted on the disks.
The plates were then incubated for 24-48 h at 30°C and photographed.
Sterol analysis: Sterol profiles were analyzed by the KOH/n-heptane
extraction procedure of Molzahn and Woods (Molzahn and
Woods, 1972) as previously described (Hand et al.,
2003). Cells were grown in liquid medium and harvested for sterol analysis
at an A600 of 0.5-1. Cells were centrifuged, washed once with dH2O
and resuspended in 4.5 M KOH/60% ethanol. Cells were then heated at 88-90°C
for 1 h in a round-bottom flask. Ethanol (95%) was then added and the cells
were heated for an additional hour and cooled to room temperature. The mixture
was then extracted with n-heptane and dH2O and the n-heptane layer
was analyzed by gas chromatography at the University of Kentucky GC-MS facility.
Morphological analysis: Approximately 1000 cells mL-1 were
plated on YPD containing 10% fetal bovine serum. The plates were incubated at
37°C for 7 days. For filamentous growth, log phase cells were suspended
in molten YPS agar (1% yeast extract, 2% bacto-peptone, 2% sucrose and 2% agar)
at a concentration of 100 cells/mL and plated. After 1-3 days at 37°C, the
colonies were photographed. In other experiments, cells were suspended in spider
media [1% nutrient broth, 0.2% K2HPO4, 1.35% agar and
1% mannitol (Lee et al., 1975)].
Expression analysis: RNA was isolated from log phase yeast cells using
the RNAeasy kit from Qiagen using the manufacturers instructions, except
that cells were spheroplasted in 1 M sorbitol, 100 mM Tris, pH 7.8 and 100 mM
EDTA containing 150 μg zymolase. Three micrograms of RNA was reverse transcribed
as described previously (Mallory et al., 2005a).
The ratios of DAP1:TUB1 (an internal control for cDNA loading) were determined
using the primers CaDAP1+2F, CaDAP1+250R, CaTUB1+421F and CaTUB1+600R in the
same reaction. PCR reactions were separated on a 1.5% agarose 1000 gel (InVitrogen).
The DAP1 gene conserved among fungi: Dap1p has homologues in virtually all
fungi, including pathogenic fungi such as the Candida species, Aspergillus
and Cryptococcus (Fig. 1). All of the key sequences
in ScDap1p are conserved in this protein family, including the strictly conserved
FYGPxGPYxNFAGxDASRGLA motif at the heart of the heme-binding domain (Fig.
1, center). The S. cerevisiae Dap1p Asp91 and Tyr 138 are required
for heme binding and both residues are conserved among all of the fungal Dap1p
homologues (Ghosh et al., 2005; Mallory
et al., 2005a). However, some Dap1p homologues lack a hydrophobic
membrane-spanning sequence at their amino-termini, while others, including CaDap1p,
contain this sequence. In this way, CaDap1p resembles its mammalian homologues
(Falkenstein et al., 1996; Gerdes
et al., 1998; Krebs et al., 2000;
Meyer et al., 1998; Nolte
et al., 2000; Selmin et al., 1996).
The entire open reading frames of each copy of DAP1 were replaced with the
URA3 and HIS1 genes, resulting in the dap1Δ::URA3/ dap1Δ::HIS1 strain
JMCa5 (Fig. s2).
|Fig. 1: Dap1p is part of a highly conserved family of fungal
proteins. Proteins are aligned in two groups based on the presence of an
amino-terminal putative trans-membrane sequence. Candida albicans Dap1p
is aligned with homologues from Aspergillus nidulans, Aspergillus fumigatus
and Cryptococcus neoformans, while the Saccharomyces cerevisiae
homologue is aligned with Candida glabrata Dap1p. Residues that
are shared by both groups are shown in dark gray, while residues shared
within either group are highlighted in light gray. The amino-terminal putative
trans-membrane sequences and the central heme-1 domain homologies are boxed
and asterisks mark residues that are required for heme binding
|Fig. s2: Deletion of both copies of the C. alicans
DAP1 gene. One copy of DAP1 (a) was initially deleted by integrating the
plasmid pJM72, containing 1 kb of DNA flan the DAP1 open reading frame adjacent
to URA3 (b) resuling in strain JMCa3 (c) the second copy of DAP1 was then
deleted using a PCR product consisting of the HlS1 gene adjacent to 100
bp of DNA adjacent to the 5′ and 3′ ends of the DAP1 open reading
frame. The dap1Δ/dap1Δ strain is called JMCa5 (d) the deletion
of DAP1 was tested by PCR using primers within the inserted auxotrophic
marker (H-F and H-R for HlS1 and U-F and U-R for URA3) and outside the region
of homology (grey stippled line) used to target the integation event (primers
D-F and D-R). The panels in (f) show PCR products using the primers shown
in part (e) with wild-type (wt) DNA from the strain BWP17 and dap1Δ/dapΔ
DNA from the strain JMCa5 as template. The JMCa5 strain was subsequently
used for analying azole drug sensitivity, ergosterol synthesis and invasive
Fig. 2 (a-c): Mutants lacking Dap1p have
altered levels of sterol metabolites. (a) Depiction of the sterol biosynthetic
pathway showing the relevant intermediates. (b) The sterol profiles of
wild- type CNC44 or dap1Δ/dap1Δ JMCa5 and (c)
cells were analyzed by gas chromatography, showing increased peaks for
lanosterol, 24-methylene lanosterol, ergosta-5,7- dienol and episterol
in the dap1Δ/dap1Δ strain. For b and c, the X
axis represents retention time and the Y-axis represents relative abundance
The dap1Δ/dap1Δ strain grew at a wild-type rate under normal growth
conditions and appeared morphologically normal microscopically, similar to analogous
strains in other yeast species. JMCa5 did not exhibit temperature sensitivity
or defective growth on synthetic media.
The C. albicans dap1Δ/dap1Δ mutant has increased azole susceptibility: The dap1Δ/dap1Δ strain JMCa5 were measured using gas chromatography. Ergosterol is synthesized via a multi-step pathway that includes the first sterol intermediate, lanosterol (Fig. 2a). Compared to the wild-type strain (Fig. 2b), JMCa5 exhibited a marked increase in lanosterol and a smaller increase in 24-methylene lanosterol (Fig. 2c), suggesting a partial arrest at the step of ergosterol synthesis catalyzed by Erg11p. In addition, dap1Δ/dap1Δ cells accumulated the Erg5p and Erg3p substrates ergosta-5,7-dienol and episterol, respectively (Fig. 2c).
The C. albicans dap1Δ/dap1Δ strain JMCa5 grew poorly on plates
containing 2-20 μM fluconazole compared to the strain CNC44 (Fig.
3a) which was used because its auxotrophy is identical to JMCa5 (Negredo
et al., 1997). In addition, dap1Δ/dap1Δcells were hyper-sensitive
to the Erg11p inhibitors itraconazole and ketoconazole in spotting assays (Fig.
3a, respectively) and in halo-forming assays for fluconazole (Fig.
s3). In a liquid growth assay, the dap1Δ/dap1Δ strain had a 9-fold
lower MIC80 for fluconazole than the wild- type strain (2.0 versus
18.2 μM) which was highly significant (P = 6x10-5, two-tailed
t-test). Microscopic analysis revealed enlarged, elongated buds after fluconazole
treatment in the dap1Δ/dap1Δ strain that were not detected in wild-type
cells (Fig. s3). The fluconazole sensitivity of the dap1Δ/dap1Δ
strain was complemented by the insertion of the wild-type DAP1 1gene (Fig.
3b). Thus, like its S. cerevisiae and S. pombe analogues,
C. albicans strains lacking DAP1 are sensitive to azole drugs due to
aberrant ergosterol synthesis (Hand et al., 2003;
Hughes et al., 2007).
We identified the role for S. cerevisiae DAP1 in ergosterol synthesis,
in part, through its transcriptional induction by azole antifungal drugs. We
used PCR to test the expression of C. albicans DAP1 after treatment with
various azole drugs for 3 h and detected a 1.7-2.0-fold induction in the DAP1
transcript after treatment with fluconazole, itraconazole and ketoconazole (Fig.
4a). No DAP1 band was detectable in cDNA from the dap1Δ/dap1Δ
strain JMCa5 (Fig. 4a) and primers for the C. albicans
tubulin gene TUB1 were included in each reaction to control for sample loading
|Fig. 3 (a-b): C. albicans dap1Δ/dap1Δ strains
have increased azole drug susceptibility. (a) The wild- type CNC44 strain
or the dap1Δ/dap1Δ strain JMCa5 were plated on YPD plates without
(rows 1-2) or with 10 μM fluconazole (rows 3-4), 10 μM itraconazole
(rows 5-6) or 10 μM ketoconazole (rows 7-8) and (b) Fluconazole sensitivity
in the dap1Δ/dap1Δ strain JMCa5 (row 5) was complemented by an
inserted copy of DAP1 (row 6)
DAP1 regulation has been reported to be under the control of the MAP kinase
Hog1p and the addition of a MAP kinase inhibitor, PD98059, inhibited the induction
of DAP1 from 1.9-fold with fluconazole to baseline levels (Fig.
4b). These results suggest that MAP kinases may contribute to the regulation
of DAP1 by azole drugs.
Dap1p regulates filamentous growth: Because Dap1p regulates sterol synthesis
in C. albicans, we tested the ability of dap1Δ/dap1Δ mutants
to undergo filamentous growth.
|Fig. s3 (a-f): Dap1p-associated azole susceptibility phenotypes.
Wild-type (a) and dap1A (b) were tested for azole susceptibility
by halo formation assay in which paper disks were saturated with 5 I of
10 M fluconazole. The dap11l strain had decreased residual growth
after treatment. The morphology of the wild-type CNC44 strain changed little
after 3 h of fluconazole treatment (c and d), while the dap1A strain
JMCa5 developed an increased number of elongated cells (e and f)
The wild-type CNC44 and the dap1Δ/dap1Δ JMCa5 strains were plated
on media containing 10% serum and incubated at 30°C for 7 days. The wild-type
strain formed colonies with a ruffled appearance (Fig. 5a),
while the dap1Δ/dap1Δ strain formed a larger proportion of smooth
colonies (Fig. 5b). When grown in suspension in YPS medium,
wild-type cells formed foci with prominent filaments (Fig. 5c),
while foci of the dap1Δ/dap1Δ strain were generally smooth, with a
few single filaments detectable (Fig. 5d). Wild-type and dap1Δ/dap1Δ
strains formed similar structures when suspended in Spider media (Fig.
5e, f). However, when the same strains were grown in liquid spider media
or RPMI-1640, there was a modest difference in morphologies (data not shown),
suggesting that cells lacking DAP1 switch to a filamentous morphology under
specific growth conditions.
|Fig. 4(a-b): DAP1 transcription is induced by azole drugs. (a) DAP1 expression
in the wild-type SC5314 strain after 3 h of 10 μM fluconazole (lane
2), 10 μM itraconazole (lane 3) or 10 μM ketoconazole (lane 4)
treatment. Expression was measured by reverse-transcriptase PCR using primers
to the TUB1 gene in the same PCR reaction as controls for cDNA loading (lower
bands). For lanes 1-4, the DAP1:TUB1 ratios were 0.21, 0.36, 0.37 and 0.41,
respectively. The dap1Δ/dap1Δ strain JMCa5 was used to test the
identity of the DAP1 band (lanes 5 and 6), before or after the same treatments
as were used in lane 2. (b) DAP1 induction by 10 μM fluconazole after
3 h treatment (lane 2) was reversed by the addition of the MAP kinase inhibitor
PD98059 at 10 μM (lane 4), while 10 μM PD98059 had no effect by
itself (lane 3). For lanes 1-4, the DAP1:TUB1 ratios were 1.11, 2.13, 1.47
and 1.40, respectively. The analysis was performed using the same conditions
described in (a)
MMS sensitivity and low iron growth: Dap1p mediates resistance to the
DNA damaging agent MMS in S. cerevisiae. The dap1Δ/dap1Δ strain
JMCa5 was markedly sensitive to MMS compared to the wild-type CNC44 strain (Fig.
6a). On the iron chelating agent ferrozine, wild-type strains grew normally
and retained their white color, while dap1Δ/dap1Δ strains became dark
red, although their growth was not affected (Fig. 6b).
|Fig. 5 (a-f): C albicans cells lacking DAP1 have an altered morphology.
The wild-type strain CNC44 (a, c and e) or the dap1Δ/dap1Δ strain
JMCa5 (b, d and f) were plated on media containing 10% serum (a and b) or
were resuspended in YPS (yeast-peptone- sucrose, c and d) or spider media
(e and f)
|Fig. 6 (a-b): MMS sensitivity in cells lacking Dap1p. (a) The wild-type
CNC44 strain or the dap1Δ/dap1Δ strain JMCa5 were plated on YPD
plates without (rows 1-2) or with 0.015% MMS (rows 3-4) and (b) The same
strains were plated on YPD without (rows 5-6) or with 800 μM ferrozine
C. albicans Dap1p is the first member of the Dap1p/PGRMC1 family
to be characterized in pathogenic fungi. C. albicans cells lacking Dap1p
accumulate lanosterol, suggesting that Dap1p is required for wild-type Erg11p
function and are hyper-sensitive to inhibitors of the ergosterol biosynthetic
pathway, like comparable strains in S. cerevisiae and S. pombe (Hand
et al., 2003; Hughes et al., 2007;
Mallory et al., 2005a). It is possible that
azole susceptibility is due, in part, to the elevated levels of lanosterol in
dap1Δ strains, rather than decreased ergosterol synthesis. However, the
targets of elevated lanosterol in C. albicans are largely unknown. In
addition to lanosterol, C. albicans dap1Δ mutants accumulate ergosta-5,
7-dienol, the Erg5p/sterol C-22 desaturase substrate and episterol, the substrate
of Erg3p/sterol C-5 desaturase. This effect was detected in S. cerevisiae
(Hand et al., 2003; Mallory
et al., 2005a), suggesting that this is a conserved function of Dap1p.
Both the sterol C-5 desaturases and sterol C-22 desaturases are activated by
cytochrome b5 with which Dap1p shares homology (Mifsud
and Bateman, 2002). Like Erg11p, Erg5p is a P450 protein while Erg3p contains
sequences for iron binding and Dap1p has been implicated in the transport or
storage of iron (Craven et al., 2007).
In addition to sterol defects and increased azole susceptibility, one of the
most pronounced phenotypes associated with Dap1p was diminished filamentous
growth. In the early stages of the assay, filaments were notably absent (Fig.
4) and although dap1Δ colonies were ultimately able to form mycelae,
the filaments were smaller than the wild-type strain and contained clusters
of unbranched cells. Ergosterol synthesis is important in hyphal formation (Martin
and Konopka, 2004). The association between Dap1p and invasiveness may be
important for mammalian cells, because the Dap1p homologue, PGRMC1, is over-expressed
in clinical tumor samples (Crudden et al., 2005;
Irby et al., 2005) and invasiveness is critical
in cancer formation. Furthermore, PGRMC1 is expressed in neuronal cells following
damage (Guennoun et al., 2007; Labombarda
et al., 2003) and may contribute to the migration and morphology
of those cells.
The best characterized function of the Dap1p proteins is heme binding. The
Dap1p/PGRMC1 proteins were originally identified as progesterone binding proteins
but progesterone binding was not detected for recombinant forms these proteins
(Min et al., 2005). In contrast, multiple labs
have reported heme binding for Dap1p/PGRMC1 and in a particularly elegant series
of studies, S. cerevisiae Dap1p was shown to bind to heme through a
5-coordinate mechanism that utilizes a conserved carboxy-terminal tyrosine residue
(Ghosh et al., 2005). Heme binding has also
been reported for the S. pombe (Hughes et al.,
2007), rodent (Min et al., 2005) and human
(Crudden et al., 2006; Ghosh
et al., 2005) homologues. Nonetheless, there is some evidence that
the human Dap1p homologue has a role in progesterone signaling, perhaps through
a co-precipitating protein (Peluso et al., 2007).
We were unable to detect any difference in proliferation or morphology following
treatment of wild-type and dap1Δ strains with progesterone.
S. cerevisiae and S. pombe Dap1p regulate MMS susceptibility
and we have shown that C. albicans Dap1p shares this activity. In S.
cerevisiae, MMS resistance can be restored to dap1Δ mutants through
over-expression of the heme biosynthetic proteins Hem1p and Hem2p and by adding
exogenous heme (Craven et al., 2007; Mallory
et al., 2005a), suggesting that MMS may target the heme biosynthetic
pathway. Because Erg11p binds heme, one of the ultimate targets of MMS may be
Erg11p and we previously showed that Erg11p over-expression suppresses MMS sensitivity
in dap1Δ mutants (Mallory et al., 2005a).
This activity is likely conserved between yeast and humans, because the human
Dap1p homologue, Hpr6/PGRMC1, is induced by DNA damaging agents (Mallory
et al., 2005b) and promotes survival from DNA damage (Crudden
et al., 2006).
The mechanism through which Dap1p or its homologues activate Erg11p is under
investigation. The S. pombe and human homologues bind directly to P450
proteins (Hughes et al., 2007), although this
has not been demonstrated in other organisms and the human homologue activates
P450 activity in an over-expression system (Min et al.,
2005). Thompson et al. recently reported that S. cerevisiae Dap1p
has a reducing activity similar to that of P450 reductases (Thompson
et al., 2007). However, Dap1p has a poor affinity for ferrous heme,
which is inconsistent with a direct role in redox cycling (Thompson
et al., 2007). These results, together with the findings of the current
study, suggest that Dap1p represents a novel regulatory mechanism for P450 proteins
such as Erg11p. Thus, Dap1p may represent a novel approach to targeting the
most tractable pathway in pathogenic fungi.
We thank Dr. Aaron Mitchell, Jesus Pla and Jurgen Wedlund for the kind gift
of strains and plasmids for the analysis and Ikhlas Ahmed for reading the manuscript.
We thank Dr. Jack Goodman for the sterol analysis, Drs. Martin Bard and Robert
J. Barbush for advice in interpreting the sterol profiles and Dr. Martin Bard
for strains and many helpful suggestions for deleting DAP1. This study was supported,
in part, by the NIH grants COBRE P20 RR 15592 and BIRCWH K12 DA14040.
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