The PbMDJ1 Gene Belongs to a Conserved MDJ1/LON Locus in Thermodimorphic Pathogenic Fungi and Encodes a Heat Shock Protein That Localizes to both the Mitochondria and Cell Wall of Paracoccidioides brasiliensis

Abstract

J-domain (DnaJ) proteins, of the Hsp40 family, are essential cofactors of their cognate Hsp70 chaperones, besides acting as independent chaperones. In the present study, we have demonstrated the presence of Mdj1, a mitochondrial DnaJ member, not only in the mitochondria, where it is apparently sorted, but also in the cell wall of Paracoccidioides brasiliensis, a thermodimorphic pathogenic fungus. The molecule (PbMdj1) was localized to fungal yeast cells using both confocal and electron microscopy and also flow cytometry. The anti-recombinant PbMdj1 antibodies used in the reactions specifically recognized a single 55-kDa mitochondrial and cell wall (alkaline β-mercaptoethanol extract) component, compatible with the predicted size of the protein devoid of its matrix peptide-targeting signal. Labeling was abundant throughout the cell wall and especially in the budding regions; however, anti-PbMdj1 did not affect fungal growth in the concentrations tested in vitro, possibly due to the poor access of the antibodies to their target in growing cells. Labeled mitochondria stood preferentially close to the plasma membrane, and gold particles were detected in the thin space between them, toward the cell surface. We show that Mdj1 and the mitochondrial proteinase Lon homologues are heat shock proteins in P. brasiliensis and that their gene organizations are conserved among thermodimorphic fungi and Aspergillus, where the genes are adjacent and have a common 5′ region. This is the first time a DnaJ member has been observed on the cell surface, where its function is speculative.

Paracoccidioides brasiliensis is the fungal species responsible for paracoccidioidomycosis (PCM), which is endemic in certain areas of Latin America. PCM is one the four deep-seated granulomatous mycoses caused by thermodimorphic fungi that also include Histoplasma capsulatum, Blastomyces dematitidis, and Coccidioides immitis. These fungal species are phylogenetically related, according to sequence analysis of the ribosomal RNA gene locus (3, 36), and have been classified as Ascomycetes of the Onigenaceae family. P. brasiliensis grows as a multibudding yeast when in parasitism or cultivated at 37°C and as a mycelium when incubated at temperatures below 28°C. The fungus is multinucleated, and its sexual form is still unknown. Genetic manipulation in this species is only starting to be attempted (23).

The human host most frequently acquires the fungus by inhalation of mycelial particles, but infection can be established only after transition to the yeast parasitic phase takes place (28). A number of biochemical processes are associated with fungal adaptation to the host's environment and temperature, and many of them are probably responsible for phase transition and/or phase maintenance. Different groups have recently addressed the overall scenario of gene expression in P. brasiliensis yeast versus mycelium, or undergoing phase transition in vitro, making use of microarrays, suppression subtraction hybridization, real-time reverse transcriptase (RT) PCR, and in silico analyses (14, 15, 27, 33). Considering that these analyses have been undertaken with P. brasiliensis transforming to the yeast phase upon temperature increase, differentially regulated heat shock protein homologues have indeed been detected, especially in the initial hours of temperature change (15, 33). Some of them, however, seem to be necessary for growth in the yeast phase, e.g., the HSP70 and HSP60 genes in P. brasiliensis that have been previously characterized (18, 41).

We have characterized a LON gene homologue from P. brasiliensis (PbLON) (2). Lon proteins are conserved ATP-binding, heat-inducible serine proteinases. They form high-molecular-mass complexes of homo-oligomers, which in Saccharomyces cerevisiae contain the stoichiometry of seven subunits of 117 kDa (46). The yeast Lon, also called PIM1, is synthesized as a pre-proenzyme precursor in the cytosol and then is sorted to the mitochondrial matrix where it is processed (54). It controls proteolysis by mediating cleavage of misfolded or unassembled matrix proteins and has an essential role in the maintenance of mitochondrial DNA integrity and mitochondrial homeostasis (51). Lon is also a DNA-binding protein (26) and is essential for cell survival in humans (4).

In P. brasiliensis, PbLon consists of 1,063 residues containing a mitochondrial import signal, a conserved ATP-binding site, and a serine catalytic motif (2). Its open reading frame (ORF) is within a 3,369-bp fragment interrupted by two introns located in the 3′ segment; an MDJ1-like gene was partially sequenced in the opposite direction but sharing with PbLON a common 5′ untranslated region (2). This chromosomal organization might be functionally relevant, since Mdj1p is a type I DnaJ molecule located in the yeast mitochondrial matrix and is essential for substrate degradation by Lon and other stress-inducible ATP-dependent proteinases (39, 53). In bacteria, DnaJ alone and/or the DnaJ/DnaK complex determine the fate of nonnative substrates to be cleaved by Lon (17).

DnaJ members (more recently referred to as J-domain proteins) belong to the Hsp40 family of molecular chaperones and localize to various cellular compartments, where their primary role is to regulate the activity of their cognate Hsp70 homologues (55). In the bacterial DnaK (Hsp70)/DnaJ/GrpE complex, a transient association of DnaJ stimulates the ATPase activity of DnaK, prompting substrate binding, while GrpE exchanges nucleotides (ADP/ATP), with the consequent substrate dissociation (12). This complex prevents aggregation of nonnative proteins, thus facilitating their folding by Hsp60 (22). In S. cerevisiae, Mdj1 is essential for the biogenesis of functional mitochondria, but it is not involved in protein translocation into the matrix (reviewed in reference 52). Typically, DnaJ members are constituted of a structurally conserved J domain located near the N-terminal end, followed by a glycine/phenylalanine-rich segment (G/F domain) and four repetitions of a zinc-binding CXXCXGXG motif (55). The J domain and the G/F segment are essential for interaction with DnaK, while the zinc finger-like motif contains elements for binding non-native substrates (49) and also displays thiol-disulfide oxidoreductase activity (50). In S. cerevisiae, 22 J-domain proteins (types I, II, and III) have been found in the genome; their subcellular localizations are attributed to the cytoplasm, nucleoplasm, mitochondria, and endoplasmic reticulum (55).

We have presently characterized the P. brasiliensis PbMDJ1 gene and verified that the PbLON/PbMDJ1 locus is conserved among the dimorphic pathogenic fungi besides Aspergillus nidulans and A. fumigatus. We show that PbLON and PbMDJ1 encode mitochondrial heat shock proteins; however, PbMdj1 has also been found in the cell wall of P. brasiliensis. The finding of a DnaJ member on the cell surface is a novel observation.

MATERIALS AND METHODS

Fungal strains and culture conditions.

We used P. brasiliensis isolates B-339 and 18, provided by Angela Restrepo (Colombia) and Zoilo P. Camargo (Brazil), respectively. More details about the fungal isolates can be found elsewhere (29). Cultures were maintained at 36°C (yeast phase) in solid modified YPD (0.5% casein peptone, 0.5% yeast extract, 1.5% glucose, pH 6.3).

Isolation of total DNA and RNA.

For DNA extraction, strain B-339 was expanded for 10 days at 36°C in liquid modified YPD under agitation. Total DNA was extracted from a 10-ml pellet of nitrogen-frozen cells that were disrupted in a mortar and then briefly in a pestle, as described previously (11). Total RNA was isolated from fresh cells mechanically disrupted by vortexing with glass beads for 10 min in the presence of TRIzol reagent (Invitrogen), according to the instructions provided by the manufacturer. Contaminating DNA in these preparations was digested with RNase-free DNase I (Promega), as described previously (15). The efficiency of hydrolysis was tested by PCR with primers of the PbGP43 gene (11). For analysis of gene expression in cells undergoing heat shock at 42°C, yeast cells growing logarithmically at 36°C under shaking in modified YPD were transferred to 42°C for 30 and 60 min.

Cloning of the 3′ end of PbMDJ1.

We followed the strategy of 3′ rapid amplification of cDNA ends to clone the 3′ end of the PbMDJ1 cDNA, using the SuperScript II RNase H RT kit (Invitrogen), because at that time the PbMDJ1 expressed sequence tag (EST) had not yet been found in the P. brasiliensis EST bank (15). The positions of the primers can be seen in Fig. 1A. The first strand of the total cDNA pool was obtained from total RNA (2.5 μg) extracted from strain B-339 yeast cells previously heat shocked for 60 min at 42°C. The template was incubated (10 μl, final volume) with a 1 μM concentration of a poly(T)-containing cassette primer (511, 5′-GACTCGAGTCGACATCGT₁₇-3′) at 65°C (5 min), cooled in ice, mixed with synthesis buffer (final concentrations of 0.05 M Tris-HCl [pH 8.3], 0.075 M KCl, 3 mM MgCl₂, 0.01 M DDT, 1 mM [each] deoxynucleoside triphosphate, 2 U/μl of RNAseOUT, and 10 U/μl of SuperScript II RT), and incubated at 55°C (50 min) and then at 85°C (5 min). Template RNA was removed with 0.1 U/μl of RNase for 20 min at 37°C. Negative controls were incubated in the absence of RT. The second strand was obtained from total cDNA (1 μ1 of the above reaction product) with an internal sense primer (B4, 5′-TGGTGGTGGGTTCTCTGC-3′) under standard PCR conditions at an annealing temperature of 52°C. We used Platinum Taq polymerase (Invitrogen) and an antisense cassette primer (512, 5′-GACTCGAGTCGACATCG-3′) for amplification of the double-stranded PbMDJ1 fragment. The final product was used as a template in a second round of PCR primed with antisense primer 512 and nested sense primer G5 (5′-TGCCTTTGCGGGTGGTTC-3′). Sequencing information from the resulting 1,161-bp amplicon was used to design primers located at the 3′ end (antisense 2A7, 5′-TACCATTTTTTGCCTGCC-3′), which enabled PCR amplification of the whole genomic PbMDJ1 gene using genomic DNA as a template. A cDNA segment of the 5′ region, used later for heterologous expression, was amplified by RT-PCR carried out with total RNA (1 μg) and the ThermoScript RT-PCR System (Gibco BRL). The first cDNA strand was amplified with the internal antisense primer E4 (5′-GCCGCACGAGGAACAGG-3′), according to the supplier's recommendations. A PCR product of 757 bp was then obtained with primers B3 (sense, 5′-CTCCCGGCTCGTCTCCTG-3′) and A7 (antisense, 5′-CCGCTTTGTACCCTGTTT-3′), with the first-strand reaction product as a template. Sequence comparison between DNA and cDNA fragments enabled the precise localization of two introns. Negative controls for 3′ rapid amplification of cDNA ends and RT-PCRs were incubated in the absence of RT. DNA fragments were recovered from agarose gels using either the Nucleiclean kit (Sigma) or the Concert gel extraction system (Gibco BRL). Purified amplicons were cloned into the pGEM-T easy vector (Promega) for maintenance and sequencing. Nucleotide sequencing was carried out in the facilities of the Center of Human Genome at the São Paulo University. Sequences were compared with those of the National Center for Biotechnology Information through the GenBank database and the BLAST network service and analyzed with the EditSeq, Seqman, Megalign, and Protean programs of the Lasergene System (DNAstar Inc.).

FIG. 1.

(A) Schematic representation of PbMDJ1, highlighting the localization of the primers and probes used in this work. Introns were localized from nt 205 to 313 and nt 1552 to 1683. (B) Schematic representation of PbMdj1 showing the localization of conserved domains. Kyte-Doolittle hydrophilicity plots (Protean module; DNAstar Inc.) of fungal Mdj1-like sequences are as follows: Pb, P. brasiliensis (AF334811); Bd, B. dermatitidis (http://genome.wustl.edu/BLAST/blasto_client.cgi); Hc, H. capsulatum (http://genome.wustl.edu/BLAST/histo_client.cgi); Ci, C. immitis (http://www.broad.mit.edu/annotation/fungi/coccidioides_immitis); An, A. nidulans (EAA57980); Cn, C. neoformans (EAL21819); Ca, C. albicans (EAK92195); Sc, S. cerevisiae (CAA82189); Ec, Escherichia coli (BA000007). The boxes indicate the predicted mitochondrial target sequence (MT) and the J domain. The dots localize the Zn²⁺ finger domains. Horizontal bars indicate the location of mouse T-cell epitopes with a minimum of 12 amino acids, as predicted by the Sette major histocompatibility complex motif (Protean module; DNAstar Inc). Percentages of identity with PbMdj1, as calculated with the MegAlign module of the DNAstar program, are given at right.

Northern blotting.

Standard conditions were used for electrophoresis and Northern blotting (38), carried out with 25 μg of total RNA per lane. Hybridizations with [α-³²P]dCTP-labeled probes were done in nylon Hybond-N membranes (Amersham) under high-stringency conditions with a B3/2A7 DNA fragment of PbMDJ1 (Fig. 1A) and a PbLON amplicon of 850 bp (from nucleotide [nt] 536 to nt 1386). The probes were labeled using the RadPrime DNA labeling system (Gibco BRL) as specified by the manufacturer and separated from free nucleotides in a molecular biology-grade Sephadex G-50 column (Pharmacia). The membranes were hybridized with the labeled probes (1 × 10⁶ cpm/ml) at 42°C overnight in 50% formamide, 6× SSC (1× SSC is 0.15 M NaCl, 0.015 M sodium citrate), 5× Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), and 100 μg/ml of salmon sperm DNA; washed one time with SSC-0.1% SDS (20 min, 42°C) and three times with 0.2× SSC-0.1% SDS (20 min, 68°C); covered with Vitafilm (Goodyear); and exposed to an X-ray film (X-Omat; Kodak) at −70°C with an intensifying screen.

Protein extraction.

Total cell extracts of P. brasiliensis were obtained through glass bead disruption (8). For isolation of total mitochondrial fractions, we adapted for P. brasiliensis the method described by Suzuki et al. (48) for S. cerevisiae. In order to accomplish cell lysis under mild conditions, nitrogen-frozen yeast cells were mechanically disrupted in a mortar until they formed a fine white powder, which was subsequently thawed in BB buffer (0.6 M sorbitol, 20 mM HEPES, pH 7.4) and sonicated for 5 min, alternating 15-s pulses with 15 s in ice. Cell debris were pelleted by centrifugation (1,500 × g, 5 min) and the ice-cooled supernatant was centrifuged at 1,200 × g for 10 min to precipitate the mitochondrial fraction (brownish pellet), which was then rinsed with BB and centrifuged (1,500 × g, 5 min). The supernatant was subjected to the same procedure two times, the resulting brownish pellets were pooled and washed two times with BB and suspended in 0.2 ml of BB, and the protein concentration was estimated by spectrometry in an aliquot diluted 100 times in 0.6% SDS (an A₂₈₀ of 0.2 corresponds to 10 mg/ml of mitochondrial proteins). Total mitochondrial protein preparations were kept at −70^o in 20% glycerol-1 mg/ml bovine serum albumin (BSA).

A β-mercaptoethanol cell wall extract was obtained from intact cells as described previously (9). P. brasiliensis yeast cells grown under shaking for 10 days (cell viability, >85%) in modified YPD were pelleted by centrifugation (1,500 × g, 15 min) and rinsed three times with an excess volume of phosphate-buffered saline (PBS). Washed cells (12 ml of wet pellet) were suspended in 30 ml of 20 mM ammonium carbonate (pH 8.6)-1% β-mercaptoethanol-1 mM phenylmethylsulfonyl fluoride and agitated for 1 h at 36°C. The released soluble components were separated from the cells by centrifugation, dialyzed in H₂O, and concentrated to 1.5 ml by lyophilization (about 8 mg/ml of total protein). The treatment did not affect cell integrity, as verified by microscopy of the treated cells. Protein contents were estimated using BSA as a standard (5).

Expression of PbMdj1 and PbLon in bacteria.

Expression of PbMdj1 and PbLon in bacteria was achieved with the pHIS1 and pHIS3 vectors (40). A 757-bp cDNA fragment of the 5′ region of PbMDJ1, obtained by RT-PCR with primers B3/A7 (Fig. 1A; see description above), was excised from pGEM-T with EcoRI/SpeI and subcloned in the same sites of pHIS3 in frame with the sequence encoding a His₆ tag. The resulting construct was called pHISMdj1. For expression of PbLon, an 845-bp BamHI/HindIII intron-free 5′ gene fragment was excised from a pUCSmaI plasmid containing the entire PbLON genomic sequence (2) and subcloned into the same sites of pHIS1 to generate a pHISLon expression plasmid. Vector pHIS1 expresses heterologous proteins bearing His₆ in both the N and C termini. Plasmids pHISMdj1 and pHISLon were used to transform Escherichia coli DH5α for maintenance and amplification, where positive clones were selected for resistance to ampicillin. Correct in-frame ligation was checked by endonuclease restriction and sequencing. Selected plasmids were inserted into E. coli BL21/pLysS (Novagen) for IPTG (isopropyl-β-d-thiogalactopyranoside)-induced protein expression. For purification of recombinant PbMdj1 (rPbMdj) and rPbLon, individual bacterial clones were cultivated overnight at 37°C under shaking in LB medium containing 100 μg/ml of ampicillin and 37 μg/ml of chloramphenicol. An aliquot of this initial culture was diluted 1:100 in fresh medium and grown until it reached an A₆₀₀ of 0.6. At this point, protein expression was induced for 3 h with 0.5 M IPTG. Bacterial cell pellets obtained by centrifugation (10,000 × g, 5 min) were suspended in 50 mM Tris-HCl (pH 7.0)-0.2 M NaCl-10% sucrose-1 mM phenylmethylsulfonyl fluoride and disrupted by sonication for 5 min (15-s pulses alternated with 15 s in ice). Precipitates were pelleted by centrifugation (10,000 × g, 5 min), suspended in 100 mM NaH₂PO₄-10 mM Tris-HCl, pH 8.0, containing 8 M urea, and centrifuged, and the supernatant was chromatographed in Ni-nitrilotriacetic acid (NTA) columns (QIAGEN), according to the manufacturer's instructions. rPbMdj1 and rPbLon fractions eluted at pH 4.5 were pooled for further use. Fractions eluted at pH 6.3 and 5.9 were discarded.

Rabbit immunization, antibody purification, and Western immunoblotting.

Approximately 300 μg of Ni-NTA-eluted rPbMdj1 and rPbLon were fractionated in preparative SDS-polyacrylamide gel electrophoresis (PAGE) gels (21) and stained briefly with Coomassie blue. The correspondent bands were cut off the polyacrylamide gels, homogenized in PBS, and injected subcutaneously at several spots on the shaved backs of rabbits. The same procedure was followed 40 days later; 25 days after this booster, the rabbits were bled and the serum was separated, tested, and aliquoted at −20°C. The rabbits were also bled before immunization to obtain preimmune sera. Polyclonal rabbit immunoglobulin G (IgG) anti-rPbMdj1 and anti-rPbLon were purified from both preimmune and immune sera in protein A-Sepharose (Pharmacia), checked for purity in SDS-PAGE gels, pooled, dialyzed in PBS, aliquoted, and frozen at −20°C. The IgG concentration was estimated by spectrometry at A₂₈₀. For purification of affinity-purified antibodies, rPbMdj or rPbLon eluted from Ni-NTA columns (150 μg, as estimated in SDS-PAGE gels) were immobilized onto nitrocellulose membranes (0.45 μm) (Amersham) over an area of 12 cm², which was then quenched with 5% skim milk in PBS (overnight at 4°C) and rinsed with PBS for 10 min under shaking. The membranes were incubated for 1 h in ice with total immune sera (1ml) and/or immune IgG diluted 1:1 (vol/vol) in PBS. The membranes were thoroughly rinsed with PBS-0.5 M NaCl, and the bound antibodies were eluted with 50 mM glycine, pH 3.0 (500 μl), which was immediately neutralized with 1 M Tris-HCl, pH 9.0. The protein profile of the eluted material in silver-stained SDS-PAGE gels indicated the presence of a major IgG band. Preimmune sera and/or IgG fractions were processed similarly. IgG and affinity purified aliquots were kept at −20°C.

Western immunoblot reactions with sera from hyperimmune rabbits or patients with PCM were carried out for 3 h at room temperature under shaking, after which the membranes were washed six times with 0.1% Tween 20 in PBS and incubated for 1 h at room temperature with goat anti-rabbit or horse anti-human Ig conjugated to peroxidase (Sigma). The reactions were developed with an enhanced chemiluminescence kit (Amersham Pharmacia).

Confocal analysis.

P. brasiliensis yeast cells growing in aerated liquid cultures in logarithmic phase (viability, >90%) were collected, washed twice in modified YPD, and adjusted to a concentration of 2 × 10⁶ to 3 × 10⁶ viable cells/ml. Washed cells were initially incubated with a 20 nM concentration of MitoTracker Red CMXRos probe (Molecular Probes) for 20 min at 36°C, in order to stain for active mitochondria, and then rinsed three times with PBS and fixed in cold methanol for 30 min in the dark. The MitoTracker Red-labeled cells were incubated with 3% (wt/vol) BSA in PBS (blocking buffer) for 16 h at 4°C, washed three times with PBS, and then incubated with either 100 μg/ml of IgG or 60 μg/ml of affinity-purified antibodies from polyclonal rabbit anti-rPbMdj and anti-rPbLon for 4 h at room temperature. Control reactions used preimmune IgG. The cells were then incubated for 1 h in the dark with 7.5 μg/ml of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) in blocking buffer. Between each incubation step with antibodies, the fungal cells were washed six times with PBS. A drop of 10 μl of each suspension was mounted on glass slides with Vectashield mounting medium (VECTOR Laboratories, Inc., Burlingame, CA), and the slides were sealed with nail polish. Labeling was analyzed using a laser scanning confocal microscope, LSM-510 NLO (Carl Zeiss, Jena, Germany).

Electron microscopy.

P. brasiliensis yeast cells growing in logarithmic phase (viability, >90%) under aerated conditions were collected by centrifugation, washed twice with 0.1 M sodium cacodylate buffer, pH 7.2, and fixed with 2% (wt/vol) formaldehyde and 2.5% (vol/vol) glutaraldehyde in cacodylate buffer for 3.5 h at room temperature under agitation (47). Yeast cells were stored in fixative at 4°C until use. Cell pellets were solidified in 2.5% (wt/vol) agarose and sliced at a thickness of approximately 1 to 2 mm. Cell slices were postfixed with 1% potassium ferrocyanide-reduced osmium tetroxide (1%) in cacodylate buffer for 1 h at room temperature under shaking (56). After two washes with ultrapure water, the cell slices were transferred to 1% aqueous uranyl acetate for 1 h in the dark. Finally, the cell pellets were washed twice in water, dehydrated, and embedded in Spurr resin (Electron Microscopy Sciences). Ultrathin sections (70 to 120 nm) were collected on Formvar/carbon-coated nickel grids, treated for 15 min with saturated sodium meta-periodate, freshly prepared, and washed five times with ultrapure water. The preparation was quenched with 5% (vol/vol) acetylated BSA (cBSA) (Aurion) in PBS for 1 h and incubated for 30 min with a drop of 0.1 M glycine in PBS. The grids were washed three times with PBS containing 1% cBSA and 0.5% Tween 20 (buffer A) and incubated overnight with 60 μg/ml of rabbit anti-rPbMdj1 IgG in 1% cBSA-PBS (buffer B) in a moist chamber at 4°C. After five washes with buffer A, the grids were incubated with12-nm colloidal gold-AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch) at 1:30 in buffer B for 1 h. The washing step was repeated, followed by three rinses in PBS and a fixing step with 2.5% glutaraldehyde in PBS for 5min. The grids were washed five times with ultrapure water and dried. The ultrathin sections were then contrasted with 2% uranyl acetate and lead citrate. Control grids were incubated with rabbit preimmune IgG (60 μg/ml). The sections were examined in a JEOL 1200 EX-II electron microscope.

Flow cytometry (FACS) analysis.

For fluorescence-activated cell sorter (FACS) analysis, P. brasiliensis yeast cells were prepared as described by Soares et al. (43), with modifications. Fungal cells were cultivated for 5 days at 36°C in modified YPD under shaking (viability, >90%), collected by centrifugation, rinsed, suspended in PBS, and left to rest for 3 min, when clumped cells tend to sediment by gravity. The supernatant was collected, adjusted to 1 × 10⁶ cells/ml, and fixed for 1 h at room temperature with an equal volume of 4% (vol/vol) paraformaldehyde in PBS, pH 7.2. Fixed cells were precipitated by centrifugation (1 min at 5,600 × g), rinsed three times with filtered PBS, and incubated for 30 min in 150 mM NH₄Cl to block free amine groups. Quenching was carried out for 1 h at room temperature, with shaking, in PBS containing 1% BSA (blocking buffer). The cells were then incubated overnight at 4°C with rabbit IgG anti-rPbMdj1 (200 μg/ml in blocking buffer) or with the same concentration of rabbit preimmune IgG. Following five rinses with PBS, the fungal cells were incubated in the dark for 1 h at room temperature in FITC-polyclonal anti-rabbit IgG (Jackson ImmunoResearch) at 15 μg/ml (1:100) in blocking buffer. Labeled cells were rinsed five times in PBS, suspended in the same buffer (1 ml), and analyzed in a Calibur FACS (Becton Dickinson, Mountain View, CA). A total of 10,000 cells were analyzed for fluorescence at 492 to 520 nm. Unlabeled control cells were previously analyzed for autofluorescence, relative cell size, and granularity.

Effect of anti-rPbMdj1 on in vitro growth of P. brasiliensis.

The effect of polyclonal anti-rPbMdj1 IgG on in vitro growth of P. brasiliensis yeasts was tested in strain 18 cells growing at 36°C for 5 days (viability, >90%) in modified YPD under agitation. The cells were collected, rinsed in culture medium, and individuated by being passed through a 25-mm needle. The assay was performed as described by Rodrigues et al. (37), with modifications, in a 96-well culture plate using 100 to 500 viable yeast cells per well in modified YPD containing 0.5 mg/ml of ampicillin. The cells were incubated for 48 to 72 h at 36°C in the presence or absence of different concentrations (25 to 200 μg/ml) of either anti-rPbMdj1 or preimmune IgG. After this incubation period, the same concentration of antibodies was added again and the cultures were further incubated for 3 to 4 days. The number of CFU and the cell morphology were observed in an inverted microscope (Olympus CK40).

RESULTS

The PbMDJ1 ORF lies within a 1,897-bp fragment (GenBank accession number AF334811), where two introns were localized (Fig. 1A). The deduced PbMdj1 protein contains 551 amino acids and has a deduced molecular mass of 58.7 kDa and a basic isoelectric point of 8.9. In the sequence we can recognize the J domain and the glycine/phenylalanine-rich and zinc finger domains (Fig. 1B), which are characteristic of type I J-domain proteins. Although we found six potential glycosylation sites (Asp-X-Ser/Thr) in PbMdj1, apparently none is substituted with endoglycosidase H-sensitive oligosaccharide chains (not shown), suggesting that the molecule does not gothrough the secretory pathway. The computer program TargetP (http://www.cbs.dtu.dk/services/TargetP/ [30]) predicted a mitochondrial targeting peptide within the 28 first amino acid residues (2.94 kDa). A comparison among fungal Mdj1 homologues indicated a high percentage of identity (85%) with sequences from the dimorphs B. dermatitidis and H. capsulatum, which can be visualized by the similarities in the hydrophilicity profiles (Fig. 1B). The J domain is the most conserved region among the Mdj1 homologues analyzed. It contains four characteristic helixes (reviewed in reference 20) and a highly conserved HPD tripeptide between helixes II and III, which is essential for the DnaJ/Hsp interaction (35). In the C-terminal half of PbMdj1 there is one predicted T-cell mouse epitope with a minimum of 12 residues (LYTAQIPLTTALL) between amino acids 379 and 391 (Fig. 1B), which is conserved in the homologues from B. dermatitidis and H. capsulatum.

We observed an extremely conserved gene organization of MDJ1 and LON among members of the Eurotiomycetes family, i.e., P. brasiliensis, B. dermatitidis, H. capsulatum, C. immitis, A. nidulans, and A. fumigatus. The number and position of the introns are conserved, although their sizes and sequences may differ (Fig. 2). MDJ1 from Neurospora crassa and Fusarium graminearum have the same intron numbers and positions as in P. brasiliensis, while their LON gene has only one 3′ intron. In Cryptococcus neoformans, both genes bear several small introns. The most interesting finding of our comparative analysis was the fact that the whole MDJ1/LON locus is conserved in Eurotiomycetes species, where the genes are adjacent, inversely oriented, and separated by a common 5′ region ranging between 400 and 485 nt (Fig. 2). In these species, a BroA (Bro1) gene homologue is found adjacent to MDJ1, in the same direction. In S. cerevisiae, BroA encodes a cytoplasmic protein involved in the sorting of membrane proteins into the lumenal vesicles of endosomal multivesicular bodies (34).

FIG. 2.

Phylogenetic tree of fungal Mdj1 sequences according to the Clustal W program. Besides the fungal sequences indicated in the legend to Fig. 1, we included those of A. fumigatus (EAL93469), N. crassa (EAA32959), F. graminearum (EAA76137), S. pombe (CAB09769), C. glabrata (CAG60838), and Homo sapiens (AF061749). A phylogenetic tree of the correspondent Lon sequences showed similar distributions. On the right, schematic representations of the chromosomal organization and gene direction in the MDJ1/LON locus of the species that bear these genes in the same chromosome. The distances separating the genes (interrupted line) in N. crassa, F. graminearum, and C. glabrata are, respectively, 236,960 kb, 1,982,719 kb, and 19,091 kb.

We expressed a His-tagged N-terminal region of PbMdj1 (252 amino acids, 25.8 kDa), which included the entire J domain, the Gly/Phe-rich region, and two zinc finger domains. This fragment contains a number of hydrophilic regions (Fig. 1B) and antibody epitopes, as predicted by the Jameson-Wolf antigenic index of the Protean module of the Lasergene program (DNAstar Inc.). We additionally expressed a 282-amino-acid His-tagged N-terminal fragment of PbLon (31.7 kDa, between residues 179 and 463), which also contains several peaks of predicted antibody epitopes. Both truncated recombinant proteins (rPbMdj1 and rPbLon) were expressed as major insoluble cytoplasmic proteins of calculated molecular masses of approximately 31 kDa for rPbMdj1 and 36 kDa for PbLon, including their vector sequences. These values are compatible with their SDS-PAGE mobilities (Fig. 3A). The correspondent pH 4.5-eluted SDS-PAGE bands were cut off the gels to immunize rabbits. Anti-rPbMdj1 and anti-rPbLon rabbit immune sera had high immunoblot titers, of 1:8,000, when tested with small amounts of the correspondent recombinant proteins, as seen in Fig. 3B. We therefore used these antibodies in subcellular localization experiments.

FIG. 3.

(A) Coomassie blue-stained 10% SDS-PAGE gels showing insoluble total bacterial extracts from recombinant bacteria expressing PbMdj1 or PbLon (extract) and the respective proteins eluted from Ni-NTA columns with pH 4.5 buffer. (B) Titration of anti-rPbMdj1 and anti-rPbLon rabbit immune sera (1:500 to 1:16,000) in immunoblotting. As a substrate, 100 ng of rPbMdj1 (left) or rPbLon (right), as shown in panel A, was used. NC, negative control with preimmune serum. The same lack of reactivity was observed when testing insoluble bacterial components from recombinant bacteria with vector alone eluted at pH 4.5 from a Ni-NTA column. (C) Coomassie blue-stained 10% SDS-PAGE gel profiles of total cell (T) and mitochondrial (M) extracts (20 μg per lane) from a P. brasiliensis yeast culture growing at 36°C before and after heat shock at 42°C for 60 min. (D) Western blot of the gels shown in panel B, probed with the indicated rabbit sera at a concentration of 1:1,500. SDS-PAGE molecular markers are indicated (in kilodaltons) in panels A through D. (E) Northern blot analysis of total RNA isolated from P. brasiliensis cells growing at 36°C before and after heat shock at 42°C for 30 and 60 min, using PbMDJ1 and PbLON probes (upper panels). The band sizes are indicated (in kilobases). Lower panels show ethidium bromide-stained agarose gel replicas of the blotted gels in which the 18S and 28S rRNA components are visible.

Immunoblot reactions were carried out with both cell lysates and total mitochondrial proteins from P. brasiliensis yeast cells growing at 36°C or heat shocked for 60 min at 42°C (Fig. 3C). Anti-rPbMdj1 immune serum specifically reacted with a single mitochondrial component of approximately 55 kDa (Fig. 3D), which is comparable to the molecular mass predicted for the processed protein lacking the mitochondrial targeting region. Expression of this component increased in heat-shocked cells, demonstrating that the PbMDJ1 gene encodes a heat shock protein in P. brasiliensis. A single 55-kDa band was also seen when total mitochondrial extracts were captured with immobilized anti-PbMdj1 IgG and then tested by immunoblotting (not shown). When the same strategy was applied with whole- cell extracts, the reaction was negative (not shown), showing that these antibodies were not cross-reacting with other intracellular components. The anti-rPbLon immune serum recognized a mitochondrial component of approximately 110 kDa, whose expression also increased in heat-shocked cells, suggesting that PbLON encodes a high-molecular-weight heat shock mitochondrial proteinase. The estimated molecular mass corresponds well to the PbLon monomer (117.6 kDa) deprived of the 6.1-kDa mitochondrial targeting region of 54 amino acids. In nonreducing SDS-PAGE gels (not shown), anti-rPbLon reacted with a mitochondrial component migrating slowly near the gel top, which is in accordance with the polymeric nature of the Lon-like proteases. The migration of the component recognized by anti-rPbMdj1 was not changed in the absence of a reducing agent (not shown). Neither PbLon nor PbMdj1 could be detected when 20 mg of total cell extracts was tested, possibly because of their small relative concentration in the cytoplasm. The heat shock nature of the mitochondrial proteins was confirmed at the transcription level. PbMDJ1 and PbLON RNA bands could be seen in Northern blots with total RNA extracted from P. brasiliensis yeast cells only after heat shock of 30 or 60 min at 42°C, but not with genetic material isolated from the starting culture growing at 36°C (Fig. 3E). The estimated sizes of the transcripts (2.05 kb for PbMDJ1 and 3.4 kb for PbLON) are in agreement with the correspondent genes and protein monomers labeled with anti-PbMdj1 and anti-PbLon (Fig. 3D).

Using confocal microscopy of P. brasiliensis yeast cells growing under aerobic conditions, we observed that anti-rPbMdj1 and anti-rPbLon antibodies reacted with the correspondent proteins in the mitochondria, as suggested by colocalization with MitoTracker Red (Fig. 4). Both antibodies (green) and MitoTracker (red) stained the cytoplasmic compartment with a punctuated pattern, which merged in tones of yellow and orange. Surprisingly, however, anti-rPbMdj1 antibodies also labeled the whole cell surface, where patches of stronger green staining could be seen at the budding sites (Fig. 4A). This is particularly evident in the images of Fig. 4D, where the bud neck is intensely bright. The longitudinal slice of this image facilitated a view of a large fluorescent green surface on the daughter cell. Control images obtained with preimmune antibodies showed only background fluorescence (Fig. 4C). We tried to label S. cerevisiae, C. albicans, and C. neoformans with anti-PbMdj1 immune serum, but the reactions were negative, as were immunoblotting reactions with S. cerevisiae cell extracts (not shown).

FIG. 4.

Localization of PbMdj1 and PbLon by confocal microscopy of P. brasiliensis yeast cells. Red images are active mitochondria stained with MitoTracker Red. Green images are reactions with affinity-purified anti-rPbMdj1 or anti-rPbLon conjugated with FITC. Control reactions were carried out with preimmune IgG (panel C, same result for both preimmune sera). Merged images show colocalization of the labels (yellow-orange). Panel D shows two inner sections of budding cells reacting only with anti-rPbMdj1 (lower panel) or as a merged image (upper panel). Similar results were obtained with anti-rPbMdj1 IgG or affinity-purified antibodies, while only affinity-purified anti-PbLon showed interpretable images.

Details of the cellular localization of PbMdj1 were further exploited by transmission electron microscopy using immunogold labeling (Fig. 5). Our preparations had preserved cell surface and organelle structures (Fig. 5A), and the images show intense labeling with anti-PbMdj1 antibodies of the double-layered cell wall. A large number of mitochondria are seen surrounding the cell membrane (Fig. 5A) yet are not in contact with it (Fig. 5B and C). The immunogold particles seem to be clustered inside the mitochondria, but in Fig. 5C they can also be seen near/at the mitochondrial surface and within the thin space between the mitochondrial surface and the cell membrane. Mitochondria that were not near the cell wall were less labeled, and the gold particles in the cytoplasm (far from the cell wall) were scarce. The bud neck in Fig. 5D has a great concentration of gold particles, in accordance with the intense fluorescence of this region seen in Fig. 4D. Nonspecific labeling with preimmune rabbit antibodies was reduced to scattered particles in the cytoplasm and the cell wall (Fig. 5E).

FIG. 5.

Ultrastructural localization of PbMdj1 in P. brasiliensis yeast cells incubated with IgG fractions (60 μg/ml) of either anti-rPbMdj1 (Athrough D) or preimmune control (E). Each panel shows a different cell. (A) Panoramic photomicrograph. (B and C) Details. Note labeling inside the mitochondria (M, arrows) and in the cell wall (CW). In panel C, labeling is also visible in the region between the cell membrane and the mitochondrion. (D) The bud neck is intensely labeled.

Surface labeling of P. brasiliensis yeast cells was also suggested by FACS. We observed a typical dose-response curve upon incubation of paraformaldehyde-fixed cells with 50, 100, or 200 μg/ml of anti-rPbMdj1 IgG. At a concentration of 200 μg/ml, over 50% of the cells were labeled, against 7% of preimmune IgG (Fig. 6A). In order to characterize the cell wall component that was reacting with anti-rPbMdj1 antibodies, cell wall components were released under mild conditions with β-mercaptoethanol at an alkaline pH (Fig. 6B). When this extract was assayed by immunoblotting with anti-rPbMdj1, a 55-kDa component was revealed, which was comparable in size to the mitochondrial band recognized by the same serum (Fig. 6B). This result adds evidence to the dual mitochondrial and cell wall localization of PbMdj1 in P. brasiliensis. It is of note that we have not detected measurable amounts of PbMdj1 in culture supernatants.

FIG. 6.

(A) FACS analysis of P. brasiliensis yeast cells incubated with 200 μg/ml of either anti-rPbMdj1 or preimmune IgG. (B) Coomassie blue-stained SDS-PAGE profile of an alkaline β-mercaptoethanol cell wall extract (β-ME, 40 μg), which was also Western blotted and assayed with anti-rPbMdj1, anti-PbLon, and preimmune (PI) IgG at a dilution of 1:1,500. A mitochondrial extract (Mito) was run in parallel and assayed with anti-rPbMdj1. Molecular masses are indicated (in kilodaltons).

Since the structure of PbMdj1 predicts several antibody epitopes and one potential T-cell epitope, we questioned whether paracoccidioidomycosis patients produced antibodies recognizing the molecule and whether anti-rPbMdj1 would be able to interfere with fungal growth. We addressed the first question by testing the reactivity of rPbMdj1 with three patients' sera by immunoblotting. As seen in Fig. 7, two of them recognized rPbMdj1 at a 1:200 dilution. The effect of polyclonal anti-rPbMdj1 IgG on in vitro growth of P. brasiliensis yeasts was tested in strain 18 cells, as described in Materials and Methods. Under these conditions, there was no inhibitory effect on the growth pattern or CFU counts compared with controls (Table 1).

FIG. 7.

Immunoblot reactivity of three PCM patients' sera with rPbMdj1 (note visible bands for patients 1 and 2). Controls were serum from a healthy individual (−) and anti-rPbMdj1 (+ [at 1:16,000]). Human sera were tested at a 1:200 dilution. The PCM sera reacted with gp43 with immunodiffusion titers of 1:64 and 1:32.

TABLE 1.

Lack of inhibition of P. brasiliensis growth by anti-rPbMdj1 IgG

Exptl condition (μg/ml)a	No. of CFUb
Culture medium	37 ± 5.2
Anti-rPbMdj1
200	38.33 ± 4.7
100	46.75 ± 9.0
50	37.75 ± 2.7
25	38 ± 0.8
12.5	39 ± 0.8
Preimmune serum
200	46.5 ± 3.5
100	41.75 ± 4.9
50	42.25 ± 5.7
25	43.25 ± 3.1
12.5	41.6 ± 5.8

^aYeast cells (10²) were cultivated in the presence of 200, 100, 50, 25, or 12.5 μg/ml of IgG. After incubation for 48 or 72 h at 36°C, the cultures were supplemented with the same amount of IgG.

^bNumbers of CFU were determined on day 6 of culture. Differences in numbers of CFU were not statistically significant (P > 0.05).

DISCUSSION

The present work demonstrates the presence of a mitochondrial member of the J-domain protein family, Mdj1, not only in the mitochondria but also in the cell wall of the pathogenic dimorphic fungus P. brasiliensis. To our knowledge, this is the first description of a DnaJ member on the cell surface. The unexpected cell wall localization of PbMdj1 was visualized in the fungal yeast cells using both confocal and electron microscopy and was confirmed by FACS analyses. The protein was labeled with rabbit polyclonal antibodies (IgG and affinity-purified fractions) raised against a recombinant PbMdj1 containing the N-terminal half of the protein. Evidence for the specificity of the immunological reactions with PbMdj1 comes from the following observations: (i) the anti-rPbMdj1 antibodies specifically recognized in immunoblotting, before or after antibody capture of soluble proteins, a single 55-kDa component from total P. brasiliensis mitochondrial extracts; (ii) the anti-rPbMdj1 antibodies specifically recognized a single 55-kDa immunoblotted component from a cell wall extract obtained from yeast cells of P. brasiliensis with mildly alkaline β-mercaptoethanol treatment; and (iii) confocal reactions carried out with affinity-purified antibodies resulted in a labeling pattern of both the cell wall and mitochondria.

Although the presence of surface J-domain proteins has not been reported before, the occurrence of extramitochondrial DnaJ homologues had been predicted by Soltys and Gupta (45). The presence of discrete amounts of mitochondrially sorted Hsp70 and Hsp60 on the plasma membrane, cytoplasmic vesicles, and cytoplasmic granules of mammalian cells has been well documented (42, 44; reviewed in reference 45). Hsp60 has been detected in small clusters at discrete points on the H. capsulatum cell wall, and it has been shown to mediate attachment of the fungus to macrophages via CD11/CD18 receptors (24). Both Hsp70 and Hsp60 need specific cochaperones for optimum chaperone function, which is thus likely to occur extramitochondrially. Therefore, outside the mitochondria Mdj1 could be assisting Ssc1 (mitochondrial Hsp70), but it could also be functioning as an independent chaperone. Although an Ssc1 homologue has not been described in fungal cell walls, an Hsp70-like protein has been detected at the outer surface of the plasma membrane, throughout the cell wall, and at the cell surface of C. albicans (25). It will be interesting to find out if the P. brasiliensis Ssc1, whose gene homologue has been identified in the database, will also be found in the cell wall and if it colocalizes with Mdj1.

The fungal cell wall is a dynamic compartment that determines and maintains cell shape but is also actively involved in many other biological events. Although glucans and chitin are the main scaffold components of the cell wall, it has a fascinating, complex structure with a number of other constituents (glycoproteins, proteins, and lipids), such as enzymes, adhesins, and structural and heat shock proteins, whose features depend on multiple intrinsic and external factors (reviewed for C. albicans in reference 10). Some of these components from pathogenic fungi are promising targets for drugs and immunotherapy (31). The interactions among these molecules seem to involve not only hydrogen and hydrophobic bonds but also covalent and phosphodiester (mediated by glycosyl phosphatidylinositol) linkages with polysaccharides. In the present study, we detected abundant labeling of the cell wall with anti-rPbMdj1 antibodies; however, low yields of the reactive 55-kDa protein were obtained upon alkaline extraction with β-mercaptoethanol, suggesting that the molecule is not loosely bound to this compartment. An additional observation was that when yeast cells were first treated with β-mercaptoethanol and then labeled for FACS analysis, the percentage of fluorescent cells increased instead of going down to background levels, as would be expected if PbMdj1 had been totally extracted (not shown). Therefore, it is likely that the initial peeling caused by mild alkaline and β-mercaptoethanol treatment improved the access of anti-rPbMdj1 antibodies to formerly inaccessible PbMdj1 target molecules. That might also explain why, although PbMdj1 seems to be actively participating in yeast cell budding, anti-rPbMdj1 antibodies could not interfere with fungal growth in vitro within the range of concentrations tested: other cell components might be blocking antibody access to their target molecules. An example of such a blockage mechanism has been recently detected in Fonsecaea pedrosoi, for which anti-monohexosylceramide can react with the cell wall only when melanin is absent (32). Another possible explanation is that the antibodies cannot recognize PbMdj1 well in growing cells. On the other hand, genetic manipulation in P. brasiliensis is still an obstacle to research of P. brasiliensis; until we have mutants for both Lon and Mdj1, one can only speculate about their role in the fungal cell wall. Another aspect to be investigated is the functionality of the potential T-cell receptor that is conserved among dimorphs.

A Southern blot of P. brasiliensis DNA restricted with endonucleases and assayed with an E5/E4 PbMDJ1 probe (Fig. 1A) resulted in a pattern of single bands (not shown), which suggests the presence of a single copy of the PbMDJ1 gene in the genome, as seen before with the adjacent PbLON gene (2). This is supported by the previous observations that PbMDJ1 and PbLON were mapped to a unique chromosomal band in 12 individual P. brasiliensis isolates (13). In other fungi analyzed here for which the genome is completely sequenced (Aspergillus and Candida), both LON and MDJ1 are present in single copies, reinforcing those observations. We have not found any evidence for the occurrence of alternative splicing in PbMDJ1: we visualized only one PbMDJ1 RNA band in Northern blots. Besides, RT-PCRs using primers designed to elongate the full-length ORF consistently resulted in a single band of the samesize when we tested different RNA preparations of yeast cellseither growing at 36°C or heat shocked at 42°C (not shown).Therefore, the components recognized by anti-rPbMDJ1 bothin mitochondria and in the cell wall apparently derive fromthesame gene and are not a product of differentially sortedmoleculesbearing particular targeting sequences due to alternative splicing. The gene encoding alanine-glyoxylate aminotransferase from amphibian livers has two alternative transcription start sites at either side of the 24-nt-long mitochondrial targeting signal, so that the protein encoded by the long transcript localizes to the mitochondria, while the product of the shorter transcript is cytosolic (16). We have not been able to determine the transcription initiation site of PbMDJ1; however, computer analysis does not predict any internal translation start site that would result in an ORF lacking the mitochondrial matrix-targeting signal. Moreover, the similar SDS-PAGE gel migration of the protein labeled in mitochondria and cell wall extracts with anti-rPbMdj1 suggests that the molecule was first sorted to that organelle, where it was probably processed by a matrix-processing peptidase into a mature 55-kDa form and then migrated to the cell wall. A matrix-processing peptidase homologue has been found in the P. brasiliensis database.

Recent years have seen a growing number of examples of proteins that are sorted to the mitochondrial matrix and then apparently exported to an additional compartment(s) of the cell (extensively reviewed in reference 45). The extramitochondrial destination varies with the molecules, which include antigens, enzymes, receptors, hormones, and chaperones, and with their specific roles in the destination sites. The mechanism(s) involved in mitochondrial export and its control are so far speculative, but the endosymbiotic origin of mitochondria suggests that some ancient bacterial secretory pathways must have been retained and modified by the organelle (45). It is noticeable that in a recent proteomics study of plasma membrane lipid rafts, 24% of the 196 identified proteins were mitochondrial; however, rafts have not been found in that compartment (1). In our electron microscopic images, mitochondria formed a ring near the cell surface. This pattern has been mentioned previously for P. brasiliensis yeast cells (19), and they can be seen in electron microscopic pictures of recently transformed yeasts (7), although in hyphae that pattern was not evident (6). The image shown in Fig. 5C suggests that some gold particles, in small clusters, are leaving the mitochondrion and moving toward the cell surface, but it is not clear whether these clusters are inside vesicles. Thus, the peripheral localization of mitochondria might not be fortuitous but rather may suggest that active trafficking of molecules to the cell surface might be taking place.

We have previously characterized the P. brasiliensis LON homologue (2). Here we show that PbLON is a heat shock gene that encodes a 110-kDa mitochondrial heat shock protein. In our confocal analyses, PbLon localized to the mitochondria, showing that the presence of PbMdj1 in the cell wall does not include helping protein degradation by PbLon. We observed that the deduced Lon and Mdj1 sequences are remarkably homologous among dimorphic fungi, especially P. brasiliensis, H. capsulatum, and B. dermatitidis. It will be interesting to find out if Mdj1 localizes to the cell wall of these microorganisms. We found that the LON/MDJ1 locus organization is comparable among Eurotiomycetes species. The entire homologous locus probably also includes Bro1, CreA, a little farther from BroA, and a hypothetical protein adjacent to LON so far found in A. nidulans, A. fumigatus, and H. capsulatum. In terms of comparative genomics, this is a relevant finding that might have evolutionary significance. This is the first conserved chromosomal organization reported for Ascomycetes, for which genome sequencing of several species will soon be finished. We are presently studying the regulatory elements contained in the 5′ untranslated region shared by LON and MDJ1, and we hope to find conserved mechanisms of regulation among fungal heat shock proteins.