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Expression of genes encoding cytosolic and endoplasmic reticulum HSP90 proteins in the aquatic fungus Blastocladiella emersonii
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Gene 411 (2008) 59 – 68 www.elsevier.com/locate/gene
Expression of genes encoding cytosolic and endoplasmic reticulum HSP90 proteins in the aquatic fungus Blastocladiella emersonii Luciana Pugliese a , Raphaela C. Georg a , Luciano G. Fietto a,b , Suely L. Gomes a,⁎ a
b
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Brazil
Received 21 September 2007; received in revised form 18 December 2007; accepted 6 January 2008 Available online 24 January 2008 Received by R. Britton
Abstract HSP90 proteins are important molecular chaperones involved in multiple cellular processes. This work reports the characterization of cDNAs encoding two distinct HSP90 proteins (named HSP90A and HSP90B) from the chytridiomycete Blastocladiella emersonii. Deduced amino acid sequences of HSP90A and HSP90B exhibit signatures of the cytosolic and endoplasmic reticulum (ER) HSP90 proteins, respectively. A genomic clone encoding HSP90A was also characterized indicating the presence of a single intron of 184 bp interrupting the coding region, located near the amino-terminus of the protein. Expression of both HSP90A and HSP90B genes increases significantly during heat shock at 38 °C, with highest induction ratios observed in cells stressed during germination of the fungus. Changes in the amount of HSP90A transcript were also evaluated during B. emersonii life cycle at physiological temperature (27 °C), and its levels were found to increase both during germination and sporulation of the fungus. HSP90A protein levels were analyzed during B. emersonii life cycle and significant changes were observed only during sporulation. Furthermore, during heat stress a large increase in the amount of HSP90A protein was observed. Induction of HSP90A and HSP90B genes during heat stress indicates the importance of both genes in the response to high temperature in B. emersonii. © 2008 Elsevier B.V. All rights reserved. Keywords: Heat shock; Endoplasmic reticulum HSP90; Cytosolic HSP90; Chytridiomycete
1. Introduction HSP90 proteins are essential molecular chaperones involved in folding, transport, maturation and degradation of a diverse number of proteins, many of them identified as signal transducers involved in cell cycle and developmental control. In most organisms and cell types these proteins are already abundant prior to cellular stress and are typically induced only a few-fold. It has been estimated that HSP90 accounts approximately for 1% of the Abbreviations: bp, base pairs; cDNA, DNA complementary to RNA; dCTP, 2′-deoxycytidine 5′-triphosphate; EDTA, ethylenediamine tetracetic acid; ER, endoplasmic reticulum; EST, expressed sequence tag; HS, heat shock; HSP, heat shock protein; kDa, kilodalton(s); SSC, 0.15 M NaCl/0.015 M Na3citrate pH 7.6. ⁎ Corresponding author. Av. Prof. Lineu Prestes 748, 05508-000, São Paulo, SP, Brazil. Tel.: +55 11 3091 3826; fax: +55 11 3091 2186. E-mail address: [email protected] (S.L. Gomes). 0378-1119/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2008.01.005
total soluble cytosolic protein in unstressed cells, making it one of the most abundant proteins in the cell (Lai et al., 1984). Hsp90 proteins are distributed ubiquitously among all living organisms and are indispensable for the survival of yeast cells even under non-stressful conditions (Borkovich et al., 1989). In vivo studies revealed that HSP90 functions as a molecular chaperone for specific substrate proteins, the classical examples being steroid hormone receptors and signaling protein kinases (Picard et al., 1990; Xu and Lindquist, 1993). Since HSP90 is essential for maintaining the activity of numerous signaling proteins, it plays a key role in cellular signal transduction networks. At a molecular level, HSP90 binds to substrate proteins, which are in a near native state and thus at a late stage of folding (Jakob et al., 1995) poised for activation by ligand binding or interaction with other factors. In fulfilling its role, HSP90 operates as part of multichaperone machineries in the cytosol, which
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includes HSP70, peptidyl-prolyl cis–trans isomerases and other co-chaperones (Bose et al., 1996; Freeman et al., 1996). Besides its cytosolic localization, HSP90 (named HSP90A when in the cytosol) is also found in mitochondria of Animalia and some Protista species (denominated TRAP), in the endoplasmic reticulum (denominated HSP90B) of all eukaryotic cells including some fungi, which until very recently were believed to lack HSP90B, and in the chloroplasts of plants (HSP90C). The nomenclature to name the HSP90 proteins located in different cellular compartments described above was recently proposed by Chen et al. (2006). Comparative genomic studies and evolutionary analyses across all kingdoms of organisms revealed that mitochondrial TRAP and endoplasmic reticulum HSP90B separately originated from the ancestors of the bacteria HSP90 homologue HtpG, whereas HSP90A and HSP90C originated from HSP90B by independent gene duplication events (Chen et al., 2006). These different paralogues possess specific amino acid signatures that characterize each type of HSP90 protein observed in the distinct cellular compartments (Chen et al., 2006). The present work describes the characterization of the gene encoding a cytosolic HSP90 from B. emersonii (HSP90A) and analysis of its mRNA and protein levels throughout the life cycle of the fungus at normal temperature as well as during heat shock. In addition, a cDNA encoding a putative endoplasmic reticulum (ER) HSP90B protein has been also isolated and sequenced, and its corresponding transcript levels shown to be induced during heat shock. This is the first expression study of an HSP90B gene in fungi. 2. Materials and methods 2.1. Cloning of the HSP90A gene and DNA sequence analysis An incomplete cDNA (1.7 kb) encoding the C-terminal two thirds of the B. emersonii HSP90A protein was obtained by immunoscreening of a cDNA expression library constructed in the λgt11 vector (Marques and Gomes 1992), using a monoclonal antibody anti-HSP90 (Sigma). The 1.7 kb cDNA was labeled with [γ-32P]-ATP by random-primed synthesis (Feinberg and Vogelstein, 1983) and used as probe in a Southern blot of total B. emersonii DNA digested with different combinations of restriction enzymes, and a single hybridization band was obtained for each restriction digest. To isolate a genomic clone for HSP90A, a partial library was constructed in the vector pUCBM20 (Boehringer-Manheim), with B. emersonii NotI/BamHI genomic DNA restriction fragments, isolated from the region of the gel that hybridized to the HSP90A cDNA clone. The library was analyzed by colony blot hybridization using the 1.7 kb cDNA probe, as previously described (de Oliveira et al., 1994). A positive clone, containing a 4.0 kb NotI/BamHI genomic fragment, was isolated and shown by DNA sequence analysis to contain the complete HSP90A gene. Nucleotide sequence determination was carried out on both strands using the Big Dye II kit (Applied Biosystem), after subcloning several restriction fragments. Analysis of sequence data and sequence comparisons were performed using the programs from the GCG package (Genetics Computer Group, Madison, Wisconsin).
2.2. Cloning and sequencing of the HSP90B cDNA The complete cDNA corresponding to the HSP90B gene from B. emersonii was isolated from a cDNA library obtained from cells submitted to heat shock (38 °C) during sporulation (Georg and Gomes, 2007a). The entire cDNA sequence was obtained using the Big Dye III kit (Applied Biosystem) and several synthetic oligonucleotides. The nucleotide sequences of the HSP90A gene and HSP90B cDNA, as well as their deduced amino acid sequences, were deposited in the GenBank/EMBL Data Bank and assigned accession numbers DQ787196 and EF559261, respectively. 2.3. Determination of transcription initiation sites Primer extension assays were performed using an 18nucleotide (nt) primer complementary to nt − 2 to + 16 of the coding region of the HSP90A gene which was 5′ end labeled with [γ-32P] ATP and T4 polynucleotide kinase, and then hybridized to 30 μg of total B. emersonii RNA isolated from cells at 0 h of sporulation incubated for 30 min either 27 °C or 38 °C. The annealing reaction was carried out in 25 μl of 100 mM piperazine-N, N′-bis (2-ethanesulfonic acid) buffer (pH 7.0) containing 1 M NaCl and 5 mM EDTA at 42 °C for 16 h. The nucleic acids were ethanol precipitated and resuspended in 100 μl of a solution containing 50 mM Tris–HCl buffer (pH 8.3); 3 mM MgCl2; 75 mM KCl; 20 mM dithiothreitol; 1 mM (each) dATP, dCTP, dTTP, and dGTP; and 40 U of RNase inhibitor (Boehringer Mannheim). The annealed primer was extended with 200 U of reverse transcriptase SuperScript RNase H− (GibcoBRL) and incubating at 42 °C for 90 min. The RNA was digested with 50 μg of RNase A (Pharmacia) and incubating at 37 °C for 30 min. The extension products were analyzed by denaturing polyacrylamide gel electrophoresis (7 M urea–7.5% polyacrylamide) followed by autoradiography. The extension fragments were sized by comparison to a sequencing ladder of M13mp18, using the universal primer forward − 40. 2.4. RNA isolation and Northern blot assays Total RNA, isolated from synchronized cells at different stages of B. emersonii development, was prepared by the Trizol method (Invitrogen). Possible DNA contamination of RNA samples used in qRT-PCR assays was eliminated by incubation with RNase–free DNase (Promega). For Northern blot assays RNA samples were resolved by electrophoresis on a 1% agarose–2.2 M formaldehyde gel and blotted onto Hybond N+ membranes (Amersham). The blots were pre-hybridized for 1 h at 42 °C in 120 mM sodium phosphate buffer (pH 7.2) containing 250 mM NaCl, 7% SDS, and 1 mM EDTA and hybridized for 16 h in the same solution with the 1.7 kb hsp90A cDNA fragment as probe (1 × 106 cpm/ml). The membranes were sequentially washed under high-stringency conditions, as previously described (de Oliveira et al., 1994). The membrane was air dried and exposed to X-ray film at − 80 °C in the presence of an intensifying screen.
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2.5. Quantitative real-time RT-PCR All quantitative real-time RT-PCR experiments (qRT-PCR) were performed using the GeneAmp 5700 Sequence Detection System (Applied Biosystems) equipment and the Platinum SYBR Green qPCR SuperMix UDG kit (Invitrogen). The thermocycling conditions comprised an initial step at 50 °C for 2 min, followed by 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. For each gene analyzed, two independent RNA samples were used. The gene encoding the mitochondrial RNA helicase-like protein was used as the calibrator gene in all experiments. The determination of the expression ratios was carried out using the method 2− ΔΔCT, described by Livak and Schmittgen (2001). 2.6. Western blot analysis Western blots were performed according to the method of Towbin et al. (1979) with modifications as follows. Cells from synchronized liquid cultures at different stages of B. emersonii development were collected by filtration through nytex cloth, suspended in cold 10% trichloroacetic acid, and incubated for 30 min at 4 °C. After centrifugation at 1500 ×g for 15 min, the precipitated proteins were resuspended by sonication, washed with cold chloroform and ethanol (1:1), dried, and dissolved in Laemmli buffer (Laemmli, 1970) for SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane, and the blot was incubated for 30 min in blocking buffer (10 mM Tris–HCl, pH 7.5 containing 150 mM NaCl, 5% non-fat dried milk, and 0.05% sodium azide). A monoclonal antibody against Hsp90 was diluted 1:400 in blocking buffer and the blot was incubated for 16 h at 4 °C. The membrane was then washed with TBS (10 mM Tris–HCl, pH 7.5; 150 mM NaCl) containing 0.05% Tween-20 followed by TBS alone and was incubated with anti-mouse immunoglobulin G antiserum conjugated with alkaline phosphatase (Sigma). Nitrobluetetrazolium and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside were used as substrates to visualize the reaction. 2.7. Immunofluorescence assay Immunofluorescence assays were carried out with a polyclonal antiserum anti-HSP90A from B. emersonii obtained from a rabbit immunized with a fusion protein expressed in Escherichia coli, corresponding to about 26 kDa of the central portion of B. emersonii HSP90A protein, and containing an Nterminal histidine tag encoded by the expression vector pPROEX-HTa (Invitrogen). For this experiment, 2 × 108 freshly collected zoospores were washed in sporulation solution (1 mM Tris–maleate, pH 6.8; 1 mM CaCl2) and fixed by incubation with one volume of 4% p-formaldehyde during 30 min at room temperature. After fixation, the cells were washed two times with PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.4 mM KH2PO4) and allowed to attach to glass slides at 37 °C, until dry. The slides were washed with PBS, then with double distilled H2O and dried at room temperature. The slides were incubated with PBS containing
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anti-HSP90A antiserum (1v/20v) for 30 min at room temperature. After washing 4 times for 5 min with PBS, the slides were incubated for 1 h with a secondary antibody against rabbit IgG conjugated with fluorescein isothiocyanate (1v/50v). The slides were then washed again with PBS, dried at room temperature and mounted with PBS/glycerol 1:1 pH 8.0. Immunostained cells were examined using a Nikon Microphot-FX microscope. 3. Results and discussion 3.1. Isolation and sequence analysis of cDNA and a genomic clone encoding the HSP90A from B. emersonii To isolate a cDNA encoding the HSP90A, a λgt11 cDNA library constructed with mRNA isolated from B. emersonii cells exposed to heat shock as previously described (Stefani and Gomes, 1995) was screened with using a monoclonal antibody against the human Hsp90. Sequence analysis indicated that the 1658 bp cDNA isolated was incomplete, corresponding to the C-terminal two thirds of the protein. Southern blot analysis using the HSP90A cDNA as a probe resulted in a single strong hybridization band for all different sets of endonuclease restriction digestions tested, indicating the presence of a single HSP90A gene in this fungus. To obtain the complete HSP90A gene, a partial genomic library was constructed based on the different restriction fragments observed during Southern blot analysis. A genomic clone containing a 4.0 kb BamHI/NotI fragment was isolated, and nucleotide sequence analysis demonstrated the presence of the entire coding region of the HSP90A gene including 5′ and 3′ flanking sequences. The coding region of the HSP90A gene is interrupted by a single intron of 184 bp in length, located near the aminoterminus of the protein (Fig. 1A). The position of the intron was confirmed by RACE-PCR, carried out using total RNA from sporulation cells exposed to 38 °C (data not shown). Although very few heat-inducible genes have been shown to contain intervening sequences, this is the second heat shock gene characterized in this fungus shown to present an intron. The other one was the HSP70 gene, and previous studies have indicated that the splicing machinery in B. emersonii seems to be quite heat stable (Stefani and Gomes, 1995). The putative translation start site for HSP90A deduced protein was inferred by translation in all three reading frames and by comparison with amino acid sequences of other eukaryotic HSP90 family members. The deduced protein sequence encompasses 710 amino acid residues with a calculated molecular mass of 80,792 Da and a pI of 4.85. We also searched for conserved domains in HSP90A putative amino acid sequence using Pfam program (http://pfam.sanger.ac.uk). We observed that B. emersonii HSP90A possesses an ATPase domain as well as an HSP90 domain, which is the putative region responsible by peptide binding (Fig. 1B). B. emersonii HSP90A protein contains all the amino acid signature sequences of cytosolic HSP90 proteins (Chen et al., 2006) as the motifs GLIINT (residues 18 to 23), NNLGTIA (92 to 98), SMIGQFGVGFYA (117 to 128), AGG (154 to 156), RGT (171 to 173), KHFSVEGQLEF (304 to 314),
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Fig. 1. A — Schematic representation of the HSP90A gene. The figure shows a partial restriction map of the 4.0 kb genomic fragment NotI/BamHI containing B. emersonii HSP90A gene. Some restriction sites are shown. Grey rectangles represent the coding region and the black rectangle represents the unique intron. The lines represent 5′ and 3′ non-coding regions of the gene. The position of the cDNA isolated from the λgt11 library is indicated. B — Scheme of the structural domains of B. emersonii HSP90A and HSP90B proteins. The domains shown were detected using Pfam program (http://pfam.sanger.ac.uk). The S box corresponds to the putative N-terminal signal sequence for the secretory pathway. C — Alignment between the deduced amino acid sequences of HSP90A and HSP90B from B. emersonii. The amino acid signature sequences characteristic of cytoplasmatic HSP90 proteins are underlined, and those from endoplasmic reticulum HSP90 proteins are in bold and italicized. The HDEL motif is shaded. The alignment was performed using ClustalW program (www.ebi.ac.uk/Tools/clustalw/).
VKK (394 to 396), HED (427 to 429), IDEY (502 to 505), QALRD (594 to 598) and MEEVD (706 to 710) (Fig. 1B and 1C). The MEEVD region is responsible for the cytosolic interaction of HSP90 with tetratricopeptide repeat (TPR) domains present in some of its co-chaperones (Pratt and Toft, 1997).
To confirm the subcellular localization of B. emersonii HSP90A, zoospores were observed by immunofluorescence using a confocal laser-scanning microscope. After incubation of the zoospores with anti-HSP90A antiserum followed by incubation with a secondary antibody against rabbit IgG labeled with
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fluorescein, a homogenous signal was found diffusely spread throughout the zoospore indicating that HSP90A is localized in the cytosol in B. emersonii (Fig. 2A). No fluorescence was observed when zoospores were incubated only with the secondary antibody, as a negative control (Fig. 2C). 3.2. Transcription start site of the HSP90A gene and its regulatory region The 5′ non-coding region of gene HSP90A was analyzed for potential basal promoter consensus elements, since HSP90 has important roles at physiological conditions, as well as for potential transcription factor binding sites involved in stress responses. For that we initially determined the transcription start site of the HSP90A gene by primer extension assay, using an 18-nucleotide (nt) primer complementary to the 5′ end of the coding region of the gene and RNA from sporulation cells at normal temperature or exposed to 38 °C for 30 min. As depicted in Fig. 3, a single start site
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was localized 65 nt upstream from the translation initiation codon both in heat shocked or non-heat shocked cells. As expected, the extension product obtained from heat shocked RNA was more abundant. The sequence upstream of the transcription initiation site was analyzed concerning the presence of putative transcription factor binding sites. A putative CAAT-box (CAAAT) was observed in the region between nucleotides -112 and -108, about 42 nt upstream from the transcription start site (TSS), but no consensus sequence for a TATA-box was identified (Fig. 3). The presence of DNA sequences known as binding sites for stress-related transcriptional activators was also found in the upstream region of the gene, including many repeats of the pentanucleotide 5′nGAAn-3′, known as the heat shock element (HSE), the binding site of the heat shock transcription factor (HSF), but none of them conforming to the canonical HSE (nnGAAnnTTCnnGAAnn). The 5′-CCCCT-3′ sequence motif, which is known as the stress response element (STRE) was also found about 29 nt from the TSS, as well as STRE-like motifs (5′-CCCT-3′ and 5′-AGGG-3′)
Fig. 2. Immunolocalization of HSP90A by indirect immunofluorescence using confocal laser-scanning microscopy. B. emersonii zoospores were fixed and incubated either in the presence (A and B) or in the absence (C and D) of anti-HSP90A antiserum followed by incubation with secondary antibody conjugated with fluorescein. Intense fluorescence is observed in the zoospore (A) and no fluorescence is observed in the control experiment without the primary antibody (C). (B) and (D) correspond to DIC images. Total zoom is 200×. The zoospore size is approximately 8 μm.
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around nucleotides 38 and 90 from the TSS, respectively (Fig. 3). STRE and STRE-like motifs serve as binding sites for transcriptional activators containing zinc fingers and are activated in response to a broad range of stresses (Ruis and Schüller, 1995; Grably et al., 2002). Putative GC boxes (5′-GGGCGG-3′) which are binding sites for transcription factor Sp1 were also found in the regulatory region of the HSP90A gene. Non-canonical HSEs and putative Sp1 binding sites were also found in the regulatory region of B. emersonii HSP70 gene, however no STRE-sequences were present (Stefani and Gomes, 1995). 3.3. Expression of the HSP90A gene during B. emersonii development and in response to heat shock Northern blot analysis was carried out to investigate changes in the amount of HSP90A mRNA during B. emersonii life cycle and
in response to high temperature. Significant changes in HSP90A transcript levels were observed during B. emersonii life cycle. As shown in Fig. 4, a large increase in the amount of HSP90A mRNA occurs during B. emersonii germination and early growth at 27 °C, the amount of transcript rising from very low levels in the zoospore to 20-fold higher levels 120 min after induction of germination. Changes in HSP90A mRNA levels were also observed during sporulation at normal temperature, with maximum levels observed after 90 min of starvation, the amount of transcript decreasing drastically after that (Fig. 4). Comparison of the maximum amounts of HSP90A mRNA in cells during early vegetative growth and during sporulation revealed 2-fold higher levels in sporulation cells (not shown). The pattern of variation of HSP90A mRNA levels during the life cycle of the fungus is very similar to the one previously determined for the HSP70-1 gene of B. emersonii, which is the most highly heat induced gene of the
Fig. 3. A — Primer extension mapping of the transcription start site of B. emersonii HSP90A gene. An 18 nt primer complementary to nt +16 to − 2 was 5′ end labeled with [γ-32P] ATP and hybridized to 30 μg of total RNA from 30 min sporulation cells maintained at 27 °C (lane 1) or heat shocked at 38 °C for 30 min (lane 2). The hybrids were then extended with reverse transcriptase, and the extension products were resolved by denaturing polyacrylamide gel electrophoresis and autoradiography. The sequencing ladder was generated with M13mp18 and the universal primer forward − 40. B — Nucleotide sequence of the 5′ region of the HSP90A gene. Nucleotide +1 is the A of ATG of the initiator methionine. The sequence complementary to the oligonucleotide used in the primer extension experiment is underlined. The transcription start site is depicted with an arrow over the sequence. Putative core sequences representing transcription factor binding sites are indicated: CAAT-box (double underline), GC box (underline), HSE (rectangles) and STRE and STRE-like (bold).
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Fig. 4. HSP90A mRNA levels during B. emersonii life cycle at normal temperature and after heat shock. Total RNA was isolated from cells at different stages of B. emersonii life cycle and samples (8 μg) were analyzed by Northern blot using the 1.7 kb HSP90A cDNA as a probe. A1 — Autoradiogram of the blot during germination and early growth; lane 1: zoospores; lanes 2 to 5: germinating cells 30, 60, 90 and 120 min after inoculation in DM4 medium; lanes 6–8: 30 min germinating cells submitted to heat shock (HS) at 38 °C during 30, 60 and 90 min. B1 — Relative amount of HSP90A mRNA, determined by densitometry scanning of the autoradiogram in A1. A2 — Autoradiogram of the blot during sporulation; lanes 1–6: sporulating cells 0, 30, 60, 90, 120 and 150 min after starvation; lane 7: zoospores; lanes 8–10: time zero sporulating cells submitted to heat shock (HS) at 38 °C during 30, 60 and 120 min. B2 — Relative amount of the HSP90A mRNA, determined by densitometry scanning of the autoradiogram in A2. The results shown are representative of three independent experiments.
HSP70 family (Stefani and Gomes, 1995; Georg and Gomes, 2007b), indicating that both genes should be important in the two differentiation stages of the cycle. B. emersonii cells exposed to heat shock both during germination and sporulation produced a large increase in the amount of HSP90A mRNA, with maximum levels observed after 60 min at 38 °C in both developmental stages. Transcript levels decreased significantly after 90 min at high temperature, indicating that heat shock induction of the HSP90A gene is transient (Fig. 4).
3.4. HSP90A protein levels during B. emersonii life cycle and heat shock To investigate if the changes observed in HSP90A mRNA levels during sporulation and germination resulted in different amounts of HSP90A protein during these stages, Western blot analysis was carried out. As depicted in Fig. 5, no significant changes were observed in the amount of HSP90A during germination at normal temperatures. Thus, the changes in transcript
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Fig. 5. HSP90A protein levels during life cycle at normal temperature and after heat shock. Total protein extracts from B. emersonii cells were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and the protein blots were probed with anti-HSP90A monoclonal antibody, as described in Materials and methods. A1 — Western blot during germination and early growth; lanes 1–5: 0, 30, 60, 90 and 120 min after inoculation in DM4 medium; lanes 6–8: 30 min germination cells exposed to 38 °C for 30, 60 and 90 min. Equal amounts of protein were applied to each lane. B1 — Relative amounts of HSP90A determined by densitometry scanning of the membrane shown in A1. A2 — Western blot during sporulation; lanes 1–6: sporulating cells collected 30, 60, 90, 120 and 150 min after starvation, lane 7: zoospores and lanes 8–10: time zero sporulating cells submitted to heat shock (HS) at 38 °C during 30, 60 and 90 min. B2 — Relative amounts of HSP90A determined by densitometry scanning of the membrane shown in A2. The results shown are representative of three independent experiments.
levels detected during germination are not accompanied by changes in HSP90A protein levels. However, during sporulation at normal temperatures, the amount of HSP90A was observed to present a variation, with its levels increasing about 2-fold by 90 min of sporulation and decreasing after that to reach very low levels in the zoospores (Fig. 5). This pattern of variation is similar to the changes in HSP90A mRNA observed during this stage. Exposure to 38 °C during germination and sporulation produced large increases in the amount of HSP90A protein, with levels 5-
fold higher after 60 min of heat shock during germination and 2.5fold higher after 90 min of heat stress during sporulation. 3.5. Isolation and sequence analysis HSP90B cDNA During the sequencing of a new B. emersonii stress cDNA library, constructed using mRNAs extracted of cells exposed to heat shock (Georg and Gomes, 2007b), an EST encoding another HSP90 protein from this fungus was observed. The complete
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cDNA sequence determined comprises 2440 bp and encodes a putative protein of 785 amino acids (Fig. 1B and C) with a calculated molecular mass of 87,547 Da and pI of 5.12. As performed for HSP90A protein, we also searched for conserved domains in HSP90B putative amino acid sequence using Pfam program (Fig. 1B). We observed that B. emersonii HSP90B also possesses an ATPase domain as well as an HSP90 domain. The deduced amino acid sequence of the second B. emersonii HSP90 displayed the amino acid signature sequences of endoplasmic reticulum (ER) HSP90 proteins (FLREL — residues 71 to 75; IGQFGVGFYS — 159 to 168 and FPLNVSRE — 429 to 436) as described by Chen et al. (2006) (Fig. 1C). HSP90B proteins do not possess a completely conserved motif that alone can distinguish them from the other proteins of this family. However, the simultaneous presence of these three conserved motifs, can separate HSP90B proteins from other subfamily members. A fourth motif, the C-terminal KDEL, that was thought to be the ER retention signal, is not completely conserved across all HSP90B subfamily members and therefore is not considered a signature sequence of ER HSP90 proteins (Chen et al., 2006). In fact, B. emersonii HSP90B protein possesses this HDEL motif in its Cterminal, which could be responsible by the retention of this protein in the ER (Fig. 1C). In addition, we used the targetP program (Emanuelsson et al., 2000 — www.cbs.dtu.dk/services/ TargetP), which predicts the subcellular location of eukaryotic proteins, and we identified a putative signal peptide for the secretory pathway in the N-terminus of the B. emersonii HSP90B protein (Fig. 1B and C). We also investigated if the gene encoding HSP90B was induced by heat shock in quantitative real-time RT-PCR assays. To compare the induction levels of HSP90B gene relative to the HSP90A gene, we performed qRT-PCR experiments for both genes (Fig. 6). We observed that HSP90B gene is also highly induced in response to heat shock both in sporulation and germination cells, its induction levels being as high as those of the HSP90A gene. Interestingly, the highest mRNA levels for both B. emersonii HSP90 genes were observed during heat stress in germination cells (Fig. 6). It is important to notice that until very recently, it was believed that ER HSP90 proteins were not present in fungi.
Fig. 6. Heat shock induction of HSP90A and HSP90B genes from B. emersonii evaluated by real-time RT-PCR. A — Induction ratios of HSP90 genes during sporulation. Cells were exposed to heat shock (38 °C) from 30 to 60 min after induction of sporulation. B — Induction ratios of HSP90 genes during germination. Cells were exposed to heat shock (38 °C) from 30 to 60 min after induction of germination.
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However, Chen et al. (2006) found in C. neoformans genome database a sequence encoding a representative of this type of HSP90 protein. Taking into account this finding the authors suggested that ER HSP90 has been lost in many fungal lineages. 4. Final conclusions HSP90 is a ubiquitous, abundant and evolutionarily conserved molecular chaperone (Young et al., 2001). Two homologues of HSP90A, a heat inducible and a constitutive isoform, are found in Saccharomyces cerevisiae and one in Neurospora crassa, Schizosaccharomyces pombe, Candida albicans, and Podospora anserina (Borkovich et al., 1989; Farrelly and Finkelstein, 1984; Girvitz et al., 2000; Aligue et al., 1994; Swoboda et al., 1995; Loubradou et al., 1997). No HSP90B has been found in these late diverging fungi, except for Cryptococcus neoformans that recently has been shown to possess a putative HSP90B gene (Chen et al., 2006). B. emersonii belongs to the Chytridiomycete class, which is at the base of the fungal phylogenetic tree. As we show here, this fungus has a single gene encoding a cytoplasmic HSP90A and sequences encoding a putative ER HSP90 protein were found during analysis of a stress cDNA library. The expression of an HSP90B gene in B. emersonii led us to investigate if this type of gene is found in other early diverging fungi. We searched for sequences encoding putative HSP90B proteins in the genomes of the recently sequenced zygomycetes Rhizopus oryzae and Phycomyces blakesleeanus using C. neoformans HSP90B protein sequence (accession number XP_571124.1) and the BlastP tool against local databases (www.broad.mit.edu/ annotation/fgi and http://genome.jgi-psf.org). Interestingly, we observed in both genomes genes encoding a putative HSP90B protein (accession numbers: RO3G_08307.1 for R. Oryzae and Phybl1|17326|e_gw1.3.60.1 for P. blakesleeanus). These data suggest that ER HSP90 proteins could have been lost in most late diverging fungi. Analysis of B. emersonii HSP90A expression during the fungus life cycle revealed a developmental regulation of the gene. Transcript levels were observed to increase during germination and early growth at 27 °C, although without significant changes in protein levels. In contrast, a transient increase in mRNA levels was observed during the sporulation phase, which is accompanied by an increase in HSP90A protein levels. Comparison of HSP90A mRNA levels between the two phases of B. emersonii life cycle demonstrated a two-fold higher amount of transcript during sporulation when compared to germination. The same difference was observed in the amount of HSP90A protein in both phases (data not shown). The fact that B. emersonii sporulation is induced by starvation, which could be considered a stress condition, would explain why HSP90 mRNA and protein levels are higher at this stage. In addition, transient heat shock induction was observed both during germination and sporulation, with a large increase in the amount of the protein during heat stress. This differential pattern of expression of the HSP90A gene during fungal differentiation and after heat shock was also observed for N. crassa, with
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HSP90 mRNA levels being much higher in mycelium cells at normal temperature and more highly induced by heat shock when compared to mature conidia cells (Girvitz et al., 2000). The HSP90B gene was also induced by heat shock in B. emersonii both during germination and sporulation, and as observed for the HSP90A gene, its highest induction ratios were detected in heat shocked germination cells. These results indicate that both HSP90 genes are important for the response to heat shock in the fungus. Higher levels of induction in germination than in sporulation cells were also observed after heat stress for other heat shock genes, as for instance, in the B. emersonii HSP70 gene family (Georg and Gomes, 2007b). This observation suggests that during temperature stress heat shock gene induction could be more important in germination than in sporulation cells. Acknowledgements The authors would like to thank Sergio R. Matioli for helpful discussions, and Luci D. Navarro and Sandra M.F. Fernandes for technical assistance. This work was supported by a grant from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP). L.P., R.C.G. and L.G.F. were FAPESP predoctoral fellows. S.L.G. was partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References Aligue, R., Akhavan-Niak, H., Russell, P., 1994. A role for Hsp90 in cell cycle control: Wee1 tyrosine kinase activity requires interaction with Hsp90. EMBO J. 13, 6099–6106. Borkovich, K.A., Farrelly, F.W., Finkelstein, D.B., Taulien, J., Lindquist, S., 1989. Hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol. Cell. Biol. 9, 3919–3930. Bose, S., Weikl, T., Bugl, H., Buchner, J., 1996. Chaperone function of Hsp90associated proteins. Science 274, 1715–1717. Chen, B., Zhong, B., Monteiro, A., 2006. Comparative genomics and evolution of the Hsp90 family of genes across all kingdom of organisms. BMC Genomics 7, 156. de Oliveira, J.C., Borges, A.C., Marques, M.V., Gomes, S.L., 1994. Cloning and characterization of the gene for the catalytic subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Eur. J. Biochem. 219, 555–562. Emanuelsson, O., Nielsen, H., Brunak, S., von Heijne, G., 2000. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol. 21, 1005–1016. Farrelly, F., Finkelstein, D., 1984. Complete sequence of the heat-inducible HSP90 gene of Saccharomyces cerevisiae. J. Biol. Chem. 259, 5745–5751.
Feinberg, A.P., Vogelstein, B., 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13. Freeman, B.C., Toft, D.O., Morimoto, R.I., 1996. Molecular chaperonies machines: chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23. Science 274, 1718–1720. Georg, R.C., Gomes, S.L., 2007a. Transcriptome analysis in the aquatic fungus Blastocladiella emersonii in response to heat shock and cadmium. Eukaryot. Cell. 6, 1053–1062. Georg, R.C., Gomes, S.L., 2007b. Comparative expression analysis of members of the Hsp70 family in the Chytridiomycete Blastocladiella emersonii. Gene 386, 24–34. Girvitz, T.L., Ouimet, P.M., Kapoor, M., 2000. Heat shock protein 80 of Neurospora crassa: sequence analysis of the gene and expression during the asexual phase. Can. J. Microbiol. 46, 981–991. Grably, M.R., Stanhill, A., Tell, O., Engelberg, D., 2002. HSF and Msn2/4p can exclusively or cooperatively activate the yeast HSP104 gene. Mol. Microbiol. 44, 21–35. Jakob, U., Lilie, H., Meyer, I., Buchner, J., 1995. Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo. J. Biol. Chem. 270, 7288–7294. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 226, 680–685. Lai, B.T., Chin, N.W., Stanek, A.E., Keh, W., Lanks, K.W., 1984. Quantitation and intracellular localization of the 85 K heat shock protein by using monoclonal and polyclonal antibodies. Mol. Cell. Biol. 4, 2802–2810. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-deltadeltaCT method. Methods 25, 402–408. Loubradou, G., Begueret, J., Turcq, B., 1997. A mutation in an HSP90 gene affects the sexual cycle and suppresses vegetative incompatibility in the fungus Podospora anserina. Genetics 147, 581–588. Marques, M.V., Gomes, S.L., 1992. Cloning and structural analysis of the gene for the regulatory subunit of cAMP-dependent protein kinase in Blastocladiella emersonii. J. Biol. Chem. 267, 17201–17207. Picard, D., Khursheed, B., Garabedian, M.J., Fortin, M.G., Lindquist, S., Yamamoto, K.R., 1990. Reduced levels of Hsp90 compromise steroid receptor action in vivo. Nature 348, 166–168. Pratt, W.B., Toft, D.O., 1997. Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr. Rev. 8, 306–360. Ruis, H., Schüller, C., 1995. Stress signaling in yeast. BioEssays 17, 959–965. Stefani, R.M.P., Gomes, S.L., 1995. A unique intron-containing hsp70 gene induced by heat shock and during sporulation in the aquatic fungus Blastocladiella emersonii. Gene 152, 19–26. Swoboda, R.K., Bertram, G., Budge, S., Gooday, G.W., Gow, N.A., Brown, A.J., 1995. Structure and regulation of the HSP90 gene from the pathogenic fungus Candida albicans. Infect. Immun. 63, 4506–4514. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Proc. Natl. Acad. Sci. USA 76, 4350–4354. Xu, Y., Lindquist, S., 1993. Heat shock protein Hsp90 governs the activity of pp60v-src kinase. Proc. Natl. Acad. Sci. USA 90, 7074–7078. Young, J.C., Moarefi, I., Hartl, F.U., 2001. Hsp90: a specialized but essential protein-folding tool. J. Cell Biol. 154, 267–273.
Gene 411 (2008) 59 – 68 www.elsevier.com/locate/gene
Expression of genes encoding cytosolic and endoplasmic reticulum HSP90 proteins in the aquatic fungus Blastocladiella emersonii Luciana Pugliese a , Raphaela C. Georg a , Luciano G. Fietto a,b , Suely L. Gomes a,⁎ a
b
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Brazil
Received 21 September 2007; received in revised form 18 December 2007; accepted 6 January 2008 Available online 24 January 2008 Received by R. Britton
Abstract HSP90 proteins are important molecular chaperones involved in multiple cellular processes. This work reports the characterization of cDNAs encoding two distinct HSP90 proteins (named HSP90A and HSP90B) from the chytridiomycete Blastocladiella emersonii. Deduced amino acid sequences of HSP90A and HSP90B exhibit signatures of the cytosolic and endoplasmic reticulum (ER) HSP90 proteins, respectively. A genomic clone encoding HSP90A was also characterized indicating the presence of a single intron of 184 bp interrupting the coding region, located near the amino-terminus of the protein. Expression of both HSP90A and HSP90B genes increases significantly during heat shock at 38 °C, with highest induction ratios observed in cells stressed during germination of the fungus. Changes in the amount of HSP90A transcript were also evaluated during B. emersonii life cycle at physiological temperature (27 °C), and its levels were found to increase both during germination and sporulation of the fungus. HSP90A protein levels were analyzed during B. emersonii life cycle and significant changes were observed only during sporulation. Furthermore, during heat stress a large increase in the amount of HSP90A protein was observed. Induction of HSP90A and HSP90B genes during heat stress indicates the importance of both genes in the response to high temperature in B. emersonii. © 2008 Elsevier B.V. All rights reserved. Keywords: Heat shock; Endoplasmic reticulum HSP90; Cytosolic HSP90; Chytridiomycete
1. Introduction HSP90 proteins are essential molecular chaperones involved in folding, transport, maturation and degradation of a diverse number of proteins, many of them identified as signal transducers involved in cell cycle and developmental control. In most organisms and cell types these proteins are already abundant prior to cellular stress and are typically induced only a few-fold. It has been estimated that HSP90 accounts approximately for 1% of the Abbreviations: bp, base pairs; cDNA, DNA complementary to RNA; dCTP, 2′-deoxycytidine 5′-triphosphate; EDTA, ethylenediamine tetracetic acid; ER, endoplasmic reticulum; EST, expressed sequence tag; HS, heat shock; HSP, heat shock protein; kDa, kilodalton(s); SSC, 0.15 M NaCl/0.015 M Na3citrate pH 7.6. ⁎ Corresponding author. Av. Prof. Lineu Prestes 748, 05508-000, São Paulo, SP, Brazil. Tel.: +55 11 3091 3826; fax: +55 11 3091 2186. E-mail address: [email protected] (S.L. Gomes). 0378-1119/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2008.01.005
total soluble cytosolic protein in unstressed cells, making it one of the most abundant proteins in the cell (Lai et al., 1984). Hsp90 proteins are distributed ubiquitously among all living organisms and are indispensable for the survival of yeast cells even under non-stressful conditions (Borkovich et al., 1989). In vivo studies revealed that HSP90 functions as a molecular chaperone for specific substrate proteins, the classical examples being steroid hormone receptors and signaling protein kinases (Picard et al., 1990; Xu and Lindquist, 1993). Since HSP90 is essential for maintaining the activity of numerous signaling proteins, it plays a key role in cellular signal transduction networks. At a molecular level, HSP90 binds to substrate proteins, which are in a near native state and thus at a late stage of folding (Jakob et al., 1995) poised for activation by ligand binding or interaction with other factors. In fulfilling its role, HSP90 operates as part of multichaperone machineries in the cytosol, which
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includes HSP70, peptidyl-prolyl cis–trans isomerases and other co-chaperones (Bose et al., 1996; Freeman et al., 1996). Besides its cytosolic localization, HSP90 (named HSP90A when in the cytosol) is also found in mitochondria of Animalia and some Protista species (denominated TRAP), in the endoplasmic reticulum (denominated HSP90B) of all eukaryotic cells including some fungi, which until very recently were believed to lack HSP90B, and in the chloroplasts of plants (HSP90C). The nomenclature to name the HSP90 proteins located in different cellular compartments described above was recently proposed by Chen et al. (2006). Comparative genomic studies and evolutionary analyses across all kingdoms of organisms revealed that mitochondrial TRAP and endoplasmic reticulum HSP90B separately originated from the ancestors of the bacteria HSP90 homologue HtpG, whereas HSP90A and HSP90C originated from HSP90B by independent gene duplication events (Chen et al., 2006). These different paralogues possess specific amino acid signatures that characterize each type of HSP90 protein observed in the distinct cellular compartments (Chen et al., 2006). The present work describes the characterization of the gene encoding a cytosolic HSP90 from B. emersonii (HSP90A) and analysis of its mRNA and protein levels throughout the life cycle of the fungus at normal temperature as well as during heat shock. In addition, a cDNA encoding a putative endoplasmic reticulum (ER) HSP90B protein has been also isolated and sequenced, and its corresponding transcript levels shown to be induced during heat shock. This is the first expression study of an HSP90B gene in fungi. 2. Materials and methods 2.1. Cloning of the HSP90A gene and DNA sequence analysis An incomplete cDNA (1.7 kb) encoding the C-terminal two thirds of the B. emersonii HSP90A protein was obtained by immunoscreening of a cDNA expression library constructed in the λgt11 vector (Marques and Gomes 1992), using a monoclonal antibody anti-HSP90 (Sigma). The 1.7 kb cDNA was labeled with [γ-32P]-ATP by random-primed synthesis (Feinberg and Vogelstein, 1983) and used as probe in a Southern blot of total B. emersonii DNA digested with different combinations of restriction enzymes, and a single hybridization band was obtained for each restriction digest. To isolate a genomic clone for HSP90A, a partial library was constructed in the vector pUCBM20 (Boehringer-Manheim), with B. emersonii NotI/BamHI genomic DNA restriction fragments, isolated from the region of the gel that hybridized to the HSP90A cDNA clone. The library was analyzed by colony blot hybridization using the 1.7 kb cDNA probe, as previously described (de Oliveira et al., 1994). A positive clone, containing a 4.0 kb NotI/BamHI genomic fragment, was isolated and shown by DNA sequence analysis to contain the complete HSP90A gene. Nucleotide sequence determination was carried out on both strands using the Big Dye II kit (Applied Biosystem), after subcloning several restriction fragments. Analysis of sequence data and sequence comparisons were performed using the programs from the GCG package (Genetics Computer Group, Madison, Wisconsin).
2.2. Cloning and sequencing of the HSP90B cDNA The complete cDNA corresponding to the HSP90B gene from B. emersonii was isolated from a cDNA library obtained from cells submitted to heat shock (38 °C) during sporulation (Georg and Gomes, 2007a). The entire cDNA sequence was obtained using the Big Dye III kit (Applied Biosystem) and several synthetic oligonucleotides. The nucleotide sequences of the HSP90A gene and HSP90B cDNA, as well as their deduced amino acid sequences, were deposited in the GenBank/EMBL Data Bank and assigned accession numbers DQ787196 and EF559261, respectively. 2.3. Determination of transcription initiation sites Primer extension assays were performed using an 18nucleotide (nt) primer complementary to nt − 2 to + 16 of the coding region of the HSP90A gene which was 5′ end labeled with [γ-32P] ATP and T4 polynucleotide kinase, and then hybridized to 30 μg of total B. emersonii RNA isolated from cells at 0 h of sporulation incubated for 30 min either 27 °C or 38 °C. The annealing reaction was carried out in 25 μl of 100 mM piperazine-N, N′-bis (2-ethanesulfonic acid) buffer (pH 7.0) containing 1 M NaCl and 5 mM EDTA at 42 °C for 16 h. The nucleic acids were ethanol precipitated and resuspended in 100 μl of a solution containing 50 mM Tris–HCl buffer (pH 8.3); 3 mM MgCl2; 75 mM KCl; 20 mM dithiothreitol; 1 mM (each) dATP, dCTP, dTTP, and dGTP; and 40 U of RNase inhibitor (Boehringer Mannheim). The annealed primer was extended with 200 U of reverse transcriptase SuperScript RNase H− (GibcoBRL) and incubating at 42 °C for 90 min. The RNA was digested with 50 μg of RNase A (Pharmacia) and incubating at 37 °C for 30 min. The extension products were analyzed by denaturing polyacrylamide gel electrophoresis (7 M urea–7.5% polyacrylamide) followed by autoradiography. The extension fragments were sized by comparison to a sequencing ladder of M13mp18, using the universal primer forward − 40. 2.4. RNA isolation and Northern blot assays Total RNA, isolated from synchronized cells at different stages of B. emersonii development, was prepared by the Trizol method (Invitrogen). Possible DNA contamination of RNA samples used in qRT-PCR assays was eliminated by incubation with RNase–free DNase (Promega). For Northern blot assays RNA samples were resolved by electrophoresis on a 1% agarose–2.2 M formaldehyde gel and blotted onto Hybond N+ membranes (Amersham). The blots were pre-hybridized for 1 h at 42 °C in 120 mM sodium phosphate buffer (pH 7.2) containing 250 mM NaCl, 7% SDS, and 1 mM EDTA and hybridized for 16 h in the same solution with the 1.7 kb hsp90A cDNA fragment as probe (1 × 106 cpm/ml). The membranes were sequentially washed under high-stringency conditions, as previously described (de Oliveira et al., 1994). The membrane was air dried and exposed to X-ray film at − 80 °C in the presence of an intensifying screen.
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2.5. Quantitative real-time RT-PCR All quantitative real-time RT-PCR experiments (qRT-PCR) were performed using the GeneAmp 5700 Sequence Detection System (Applied Biosystems) equipment and the Platinum SYBR Green qPCR SuperMix UDG kit (Invitrogen). The thermocycling conditions comprised an initial step at 50 °C for 2 min, followed by 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. For each gene analyzed, two independent RNA samples were used. The gene encoding the mitochondrial RNA helicase-like protein was used as the calibrator gene in all experiments. The determination of the expression ratios was carried out using the method 2− ΔΔCT, described by Livak and Schmittgen (2001). 2.6. Western blot analysis Western blots were performed according to the method of Towbin et al. (1979) with modifications as follows. Cells from synchronized liquid cultures at different stages of B. emersonii development were collected by filtration through nytex cloth, suspended in cold 10% trichloroacetic acid, and incubated for 30 min at 4 °C. After centrifugation at 1500 ×g for 15 min, the precipitated proteins were resuspended by sonication, washed with cold chloroform and ethanol (1:1), dried, and dissolved in Laemmli buffer (Laemmli, 1970) for SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane, and the blot was incubated for 30 min in blocking buffer (10 mM Tris–HCl, pH 7.5 containing 150 mM NaCl, 5% non-fat dried milk, and 0.05% sodium azide). A monoclonal antibody against Hsp90 was diluted 1:400 in blocking buffer and the blot was incubated for 16 h at 4 °C. The membrane was then washed with TBS (10 mM Tris–HCl, pH 7.5; 150 mM NaCl) containing 0.05% Tween-20 followed by TBS alone and was incubated with anti-mouse immunoglobulin G antiserum conjugated with alkaline phosphatase (Sigma). Nitrobluetetrazolium and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside were used as substrates to visualize the reaction. 2.7. Immunofluorescence assay Immunofluorescence assays were carried out with a polyclonal antiserum anti-HSP90A from B. emersonii obtained from a rabbit immunized with a fusion protein expressed in Escherichia coli, corresponding to about 26 kDa of the central portion of B. emersonii HSP90A protein, and containing an Nterminal histidine tag encoded by the expression vector pPROEX-HTa (Invitrogen). For this experiment, 2 × 108 freshly collected zoospores were washed in sporulation solution (1 mM Tris–maleate, pH 6.8; 1 mM CaCl2) and fixed by incubation with one volume of 4% p-formaldehyde during 30 min at room temperature. After fixation, the cells were washed two times with PBS (137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.4 mM KH2PO4) and allowed to attach to glass slides at 37 °C, until dry. The slides were washed with PBS, then with double distilled H2O and dried at room temperature. The slides were incubated with PBS containing
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anti-HSP90A antiserum (1v/20v) for 30 min at room temperature. After washing 4 times for 5 min with PBS, the slides were incubated for 1 h with a secondary antibody against rabbit IgG conjugated with fluorescein isothiocyanate (1v/50v). The slides were then washed again with PBS, dried at room temperature and mounted with PBS/glycerol 1:1 pH 8.0. Immunostained cells were examined using a Nikon Microphot-FX microscope. 3. Results and discussion 3.1. Isolation and sequence analysis of cDNA and a genomic clone encoding the HSP90A from B. emersonii To isolate a cDNA encoding the HSP90A, a λgt11 cDNA library constructed with mRNA isolated from B. emersonii cells exposed to heat shock as previously described (Stefani and Gomes, 1995) was screened with using a monoclonal antibody against the human Hsp90. Sequence analysis indicated that the 1658 bp cDNA isolated was incomplete, corresponding to the C-terminal two thirds of the protein. Southern blot analysis using the HSP90A cDNA as a probe resulted in a single strong hybridization band for all different sets of endonuclease restriction digestions tested, indicating the presence of a single HSP90A gene in this fungus. To obtain the complete HSP90A gene, a partial genomic library was constructed based on the different restriction fragments observed during Southern blot analysis. A genomic clone containing a 4.0 kb BamHI/NotI fragment was isolated, and nucleotide sequence analysis demonstrated the presence of the entire coding region of the HSP90A gene including 5′ and 3′ flanking sequences. The coding region of the HSP90A gene is interrupted by a single intron of 184 bp in length, located near the aminoterminus of the protein (Fig. 1A). The position of the intron was confirmed by RACE-PCR, carried out using total RNA from sporulation cells exposed to 38 °C (data not shown). Although very few heat-inducible genes have been shown to contain intervening sequences, this is the second heat shock gene characterized in this fungus shown to present an intron. The other one was the HSP70 gene, and previous studies have indicated that the splicing machinery in B. emersonii seems to be quite heat stable (Stefani and Gomes, 1995). The putative translation start site for HSP90A deduced protein was inferred by translation in all three reading frames and by comparison with amino acid sequences of other eukaryotic HSP90 family members. The deduced protein sequence encompasses 710 amino acid residues with a calculated molecular mass of 80,792 Da and a pI of 4.85. We also searched for conserved domains in HSP90A putative amino acid sequence using Pfam program (http://pfam.sanger.ac.uk). We observed that B. emersonii HSP90A possesses an ATPase domain as well as an HSP90 domain, which is the putative region responsible by peptide binding (Fig. 1B). B. emersonii HSP90A protein contains all the amino acid signature sequences of cytosolic HSP90 proteins (Chen et al., 2006) as the motifs GLIINT (residues 18 to 23), NNLGTIA (92 to 98), SMIGQFGVGFYA (117 to 128), AGG (154 to 156), RGT (171 to 173), KHFSVEGQLEF (304 to 314),
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Fig. 1. A — Schematic representation of the HSP90A gene. The figure shows a partial restriction map of the 4.0 kb genomic fragment NotI/BamHI containing B. emersonii HSP90A gene. Some restriction sites are shown. Grey rectangles represent the coding region and the black rectangle represents the unique intron. The lines represent 5′ and 3′ non-coding regions of the gene. The position of the cDNA isolated from the λgt11 library is indicated. B — Scheme of the structural domains of B. emersonii HSP90A and HSP90B proteins. The domains shown were detected using Pfam program (http://pfam.sanger.ac.uk). The S box corresponds to the putative N-terminal signal sequence for the secretory pathway. C — Alignment between the deduced amino acid sequences of HSP90A and HSP90B from B. emersonii. The amino acid signature sequences characteristic of cytoplasmatic HSP90 proteins are underlined, and those from endoplasmic reticulum HSP90 proteins are in bold and italicized. The HDEL motif is shaded. The alignment was performed using ClustalW program (www.ebi.ac.uk/Tools/clustalw/).
VKK (394 to 396), HED (427 to 429), IDEY (502 to 505), QALRD (594 to 598) and MEEVD (706 to 710) (Fig. 1B and 1C). The MEEVD region is responsible for the cytosolic interaction of HSP90 with tetratricopeptide repeat (TPR) domains present in some of its co-chaperones (Pratt and Toft, 1997).
To confirm the subcellular localization of B. emersonii HSP90A, zoospores were observed by immunofluorescence using a confocal laser-scanning microscope. After incubation of the zoospores with anti-HSP90A antiserum followed by incubation with a secondary antibody against rabbit IgG labeled with
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fluorescein, a homogenous signal was found diffusely spread throughout the zoospore indicating that HSP90A is localized in the cytosol in B. emersonii (Fig. 2A). No fluorescence was observed when zoospores were incubated only with the secondary antibody, as a negative control (Fig. 2C). 3.2. Transcription start site of the HSP90A gene and its regulatory region The 5′ non-coding region of gene HSP90A was analyzed for potential basal promoter consensus elements, since HSP90 has important roles at physiological conditions, as well as for potential transcription factor binding sites involved in stress responses. For that we initially determined the transcription start site of the HSP90A gene by primer extension assay, using an 18-nucleotide (nt) primer complementary to the 5′ end of the coding region of the gene and RNA from sporulation cells at normal temperature or exposed to 38 °C for 30 min. As depicted in Fig. 3, a single start site
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was localized 65 nt upstream from the translation initiation codon both in heat shocked or non-heat shocked cells. As expected, the extension product obtained from heat shocked RNA was more abundant. The sequence upstream of the transcription initiation site was analyzed concerning the presence of putative transcription factor binding sites. A putative CAAT-box (CAAAT) was observed in the region between nucleotides -112 and -108, about 42 nt upstream from the transcription start site (TSS), but no consensus sequence for a TATA-box was identified (Fig. 3). The presence of DNA sequences known as binding sites for stress-related transcriptional activators was also found in the upstream region of the gene, including many repeats of the pentanucleotide 5′nGAAn-3′, known as the heat shock element (HSE), the binding site of the heat shock transcription factor (HSF), but none of them conforming to the canonical HSE (nnGAAnnTTCnnGAAnn). The 5′-CCCCT-3′ sequence motif, which is known as the stress response element (STRE) was also found about 29 nt from the TSS, as well as STRE-like motifs (5′-CCCT-3′ and 5′-AGGG-3′)
Fig. 2. Immunolocalization of HSP90A by indirect immunofluorescence using confocal laser-scanning microscopy. B. emersonii zoospores were fixed and incubated either in the presence (A and B) or in the absence (C and D) of anti-HSP90A antiserum followed by incubation with secondary antibody conjugated with fluorescein. Intense fluorescence is observed in the zoospore (A) and no fluorescence is observed in the control experiment without the primary antibody (C). (B) and (D) correspond to DIC images. Total zoom is 200×. The zoospore size is approximately 8 μm.
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around nucleotides 38 and 90 from the TSS, respectively (Fig. 3). STRE and STRE-like motifs serve as binding sites for transcriptional activators containing zinc fingers and are activated in response to a broad range of stresses (Ruis and Schüller, 1995; Grably et al., 2002). Putative GC boxes (5′-GGGCGG-3′) which are binding sites for transcription factor Sp1 were also found in the regulatory region of the HSP90A gene. Non-canonical HSEs and putative Sp1 binding sites were also found in the regulatory region of B. emersonii HSP70 gene, however no STRE-sequences were present (Stefani and Gomes, 1995). 3.3. Expression of the HSP90A gene during B. emersonii development and in response to heat shock Northern blot analysis was carried out to investigate changes in the amount of HSP90A mRNA during B. emersonii life cycle and
in response to high temperature. Significant changes in HSP90A transcript levels were observed during B. emersonii life cycle. As shown in Fig. 4, a large increase in the amount of HSP90A mRNA occurs during B. emersonii germination and early growth at 27 °C, the amount of transcript rising from very low levels in the zoospore to 20-fold higher levels 120 min after induction of germination. Changes in HSP90A mRNA levels were also observed during sporulation at normal temperature, with maximum levels observed after 90 min of starvation, the amount of transcript decreasing drastically after that (Fig. 4). Comparison of the maximum amounts of HSP90A mRNA in cells during early vegetative growth and during sporulation revealed 2-fold higher levels in sporulation cells (not shown). The pattern of variation of HSP90A mRNA levels during the life cycle of the fungus is very similar to the one previously determined for the HSP70-1 gene of B. emersonii, which is the most highly heat induced gene of the
Fig. 3. A — Primer extension mapping of the transcription start site of B. emersonii HSP90A gene. An 18 nt primer complementary to nt +16 to − 2 was 5′ end labeled with [γ-32P] ATP and hybridized to 30 μg of total RNA from 30 min sporulation cells maintained at 27 °C (lane 1) or heat shocked at 38 °C for 30 min (lane 2). The hybrids were then extended with reverse transcriptase, and the extension products were resolved by denaturing polyacrylamide gel electrophoresis and autoradiography. The sequencing ladder was generated with M13mp18 and the universal primer forward − 40. B — Nucleotide sequence of the 5′ region of the HSP90A gene. Nucleotide +1 is the A of ATG of the initiator methionine. The sequence complementary to the oligonucleotide used in the primer extension experiment is underlined. The transcription start site is depicted with an arrow over the sequence. Putative core sequences representing transcription factor binding sites are indicated: CAAT-box (double underline), GC box (underline), HSE (rectangles) and STRE and STRE-like (bold).
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Fig. 4. HSP90A mRNA levels during B. emersonii life cycle at normal temperature and after heat shock. Total RNA was isolated from cells at different stages of B. emersonii life cycle and samples (8 μg) were analyzed by Northern blot using the 1.7 kb HSP90A cDNA as a probe. A1 — Autoradiogram of the blot during germination and early growth; lane 1: zoospores; lanes 2 to 5: germinating cells 30, 60, 90 and 120 min after inoculation in DM4 medium; lanes 6–8: 30 min germinating cells submitted to heat shock (HS) at 38 °C during 30, 60 and 90 min. B1 — Relative amount of HSP90A mRNA, determined by densitometry scanning of the autoradiogram in A1. A2 — Autoradiogram of the blot during sporulation; lanes 1–6: sporulating cells 0, 30, 60, 90, 120 and 150 min after starvation; lane 7: zoospores; lanes 8–10: time zero sporulating cells submitted to heat shock (HS) at 38 °C during 30, 60 and 120 min. B2 — Relative amount of the HSP90A mRNA, determined by densitometry scanning of the autoradiogram in A2. The results shown are representative of three independent experiments.
HSP70 family (Stefani and Gomes, 1995; Georg and Gomes, 2007b), indicating that both genes should be important in the two differentiation stages of the cycle. B. emersonii cells exposed to heat shock both during germination and sporulation produced a large increase in the amount of HSP90A mRNA, with maximum levels observed after 60 min at 38 °C in both developmental stages. Transcript levels decreased significantly after 90 min at high temperature, indicating that heat shock induction of the HSP90A gene is transient (Fig. 4).
3.4. HSP90A protein levels during B. emersonii life cycle and heat shock To investigate if the changes observed in HSP90A mRNA levels during sporulation and germination resulted in different amounts of HSP90A protein during these stages, Western blot analysis was carried out. As depicted in Fig. 5, no significant changes were observed in the amount of HSP90A during germination at normal temperatures. Thus, the changes in transcript
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Fig. 5. HSP90A protein levels during life cycle at normal temperature and after heat shock. Total protein extracts from B. emersonii cells were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and the protein blots were probed with anti-HSP90A monoclonal antibody, as described in Materials and methods. A1 — Western blot during germination and early growth; lanes 1–5: 0, 30, 60, 90 and 120 min after inoculation in DM4 medium; lanes 6–8: 30 min germination cells exposed to 38 °C for 30, 60 and 90 min. Equal amounts of protein were applied to each lane. B1 — Relative amounts of HSP90A determined by densitometry scanning of the membrane shown in A1. A2 — Western blot during sporulation; lanes 1–6: sporulating cells collected 30, 60, 90, 120 and 150 min after starvation, lane 7: zoospores and lanes 8–10: time zero sporulating cells submitted to heat shock (HS) at 38 °C during 30, 60 and 90 min. B2 — Relative amounts of HSP90A determined by densitometry scanning of the membrane shown in A2. The results shown are representative of three independent experiments.
levels detected during germination are not accompanied by changes in HSP90A protein levels. However, during sporulation at normal temperatures, the amount of HSP90A was observed to present a variation, with its levels increasing about 2-fold by 90 min of sporulation and decreasing after that to reach very low levels in the zoospores (Fig. 5). This pattern of variation is similar to the changes in HSP90A mRNA observed during this stage. Exposure to 38 °C during germination and sporulation produced large increases in the amount of HSP90A protein, with levels 5-
fold higher after 60 min of heat shock during germination and 2.5fold higher after 90 min of heat stress during sporulation. 3.5. Isolation and sequence analysis HSP90B cDNA During the sequencing of a new B. emersonii stress cDNA library, constructed using mRNAs extracted of cells exposed to heat shock (Georg and Gomes, 2007b), an EST encoding another HSP90 protein from this fungus was observed. The complete
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cDNA sequence determined comprises 2440 bp and encodes a putative protein of 785 amino acids (Fig. 1B and C) with a calculated molecular mass of 87,547 Da and pI of 5.12. As performed for HSP90A protein, we also searched for conserved domains in HSP90B putative amino acid sequence using Pfam program (Fig. 1B). We observed that B. emersonii HSP90B also possesses an ATPase domain as well as an HSP90 domain. The deduced amino acid sequence of the second B. emersonii HSP90 displayed the amino acid signature sequences of endoplasmic reticulum (ER) HSP90 proteins (FLREL — residues 71 to 75; IGQFGVGFYS — 159 to 168 and FPLNVSRE — 429 to 436) as described by Chen et al. (2006) (Fig. 1C). HSP90B proteins do not possess a completely conserved motif that alone can distinguish them from the other proteins of this family. However, the simultaneous presence of these three conserved motifs, can separate HSP90B proteins from other subfamily members. A fourth motif, the C-terminal KDEL, that was thought to be the ER retention signal, is not completely conserved across all HSP90B subfamily members and therefore is not considered a signature sequence of ER HSP90 proteins (Chen et al., 2006). In fact, B. emersonii HSP90B protein possesses this HDEL motif in its Cterminal, which could be responsible by the retention of this protein in the ER (Fig. 1C). In addition, we used the targetP program (Emanuelsson et al., 2000 — www.cbs.dtu.dk/services/ TargetP), which predicts the subcellular location of eukaryotic proteins, and we identified a putative signal peptide for the secretory pathway in the N-terminus of the B. emersonii HSP90B protein (Fig. 1B and C). We also investigated if the gene encoding HSP90B was induced by heat shock in quantitative real-time RT-PCR assays. To compare the induction levels of HSP90B gene relative to the HSP90A gene, we performed qRT-PCR experiments for both genes (Fig. 6). We observed that HSP90B gene is also highly induced in response to heat shock both in sporulation and germination cells, its induction levels being as high as those of the HSP90A gene. Interestingly, the highest mRNA levels for both B. emersonii HSP90 genes were observed during heat stress in germination cells (Fig. 6). It is important to notice that until very recently, it was believed that ER HSP90 proteins were not present in fungi.
Fig. 6. Heat shock induction of HSP90A and HSP90B genes from B. emersonii evaluated by real-time RT-PCR. A — Induction ratios of HSP90 genes during sporulation. Cells were exposed to heat shock (38 °C) from 30 to 60 min after induction of sporulation. B — Induction ratios of HSP90 genes during germination. Cells were exposed to heat shock (38 °C) from 30 to 60 min after induction of germination.
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However, Chen et al. (2006) found in C. neoformans genome database a sequence encoding a representative of this type of HSP90 protein. Taking into account this finding the authors suggested that ER HSP90 has been lost in many fungal lineages. 4. Final conclusions HSP90 is a ubiquitous, abundant and evolutionarily conserved molecular chaperone (Young et al., 2001). Two homologues of HSP90A, a heat inducible and a constitutive isoform, are found in Saccharomyces cerevisiae and one in Neurospora crassa, Schizosaccharomyces pombe, Candida albicans, and Podospora anserina (Borkovich et al., 1989; Farrelly and Finkelstein, 1984; Girvitz et al., 2000; Aligue et al., 1994; Swoboda et al., 1995; Loubradou et al., 1997). No HSP90B has been found in these late diverging fungi, except for Cryptococcus neoformans that recently has been shown to possess a putative HSP90B gene (Chen et al., 2006). B. emersonii belongs to the Chytridiomycete class, which is at the base of the fungal phylogenetic tree. As we show here, this fungus has a single gene encoding a cytoplasmic HSP90A and sequences encoding a putative ER HSP90 protein were found during analysis of a stress cDNA library. The expression of an HSP90B gene in B. emersonii led us to investigate if this type of gene is found in other early diverging fungi. We searched for sequences encoding putative HSP90B proteins in the genomes of the recently sequenced zygomycetes Rhizopus oryzae and Phycomyces blakesleeanus using C. neoformans HSP90B protein sequence (accession number XP_571124.1) and the BlastP tool against local databases (www.broad.mit.edu/ annotation/fgi and http://genome.jgi-psf.org). Interestingly, we observed in both genomes genes encoding a putative HSP90B protein (accession numbers: RO3G_08307.1 for R. Oryzae and Phybl1|17326|e_gw1.3.60.1 for P. blakesleeanus). These data suggest that ER HSP90 proteins could have been lost in most late diverging fungi. Analysis of B. emersonii HSP90A expression during the fungus life cycle revealed a developmental regulation of the gene. Transcript levels were observed to increase during germination and early growth at 27 °C, although without significant changes in protein levels. In contrast, a transient increase in mRNA levels was observed during the sporulation phase, which is accompanied by an increase in HSP90A protein levels. Comparison of HSP90A mRNA levels between the two phases of B. emersonii life cycle demonstrated a two-fold higher amount of transcript during sporulation when compared to germination. The same difference was observed in the amount of HSP90A protein in both phases (data not shown). The fact that B. emersonii sporulation is induced by starvation, which could be considered a stress condition, would explain why HSP90 mRNA and protein levels are higher at this stage. In addition, transient heat shock induction was observed both during germination and sporulation, with a large increase in the amount of the protein during heat stress. This differential pattern of expression of the HSP90A gene during fungal differentiation and after heat shock was also observed for N. crassa, with
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HSP90 mRNA levels being much higher in mycelium cells at normal temperature and more highly induced by heat shock when compared to mature conidia cells (Girvitz et al., 2000). The HSP90B gene was also induced by heat shock in B. emersonii both during germination and sporulation, and as observed for the HSP90A gene, its highest induction ratios were detected in heat shocked germination cells. These results indicate that both HSP90 genes are important for the response to heat shock in the fungus. Higher levels of induction in germination than in sporulation cells were also observed after heat stress for other heat shock genes, as for instance, in the B. emersonii HSP70 gene family (Georg and Gomes, 2007b). This observation suggests that during temperature stress heat shock gene induction could be more important in germination than in sporulation cells. Acknowledgements The authors would like to thank Sergio R. Matioli for helpful discussions, and Luci D. Navarro and Sandra M.F. Fernandes for technical assistance. This work was supported by a grant from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP). L.P., R.C.G. and L.G.F. were FAPESP predoctoral fellows. S.L.G. was partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References Aligue, R., Akhavan-Niak, H., Russell, P., 1994. A role for Hsp90 in cell cycle control: Wee1 tyrosine kinase activity requires interaction with Hsp90. EMBO J. 13, 6099–6106. Borkovich, K.A., Farrelly, F.W., Finkelstein, D.B., Taulien, J., Lindquist, S., 1989. Hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol. Cell. Biol. 9, 3919–3930. Bose, S., Weikl, T., Bugl, H., Buchner, J., 1996. Chaperone function of Hsp90associated proteins. Science 274, 1715–1717. Chen, B., Zhong, B., Monteiro, A., 2006. Comparative genomics and evolution of the Hsp90 family of genes across all kingdom of organisms. BMC Genomics 7, 156. de Oliveira, J.C., Borges, A.C., Marques, M.V., Gomes, S.L., 1994. Cloning and characterization of the gene for the catalytic subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Eur. J. Biochem. 219, 555–562. Emanuelsson, O., Nielsen, H., Brunak, S., von Heijne, G., 2000. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol. 21, 1005–1016. Farrelly, F., Finkelstein, D., 1984. Complete sequence of the heat-inducible HSP90 gene of Saccharomyces cerevisiae. J. Biol. Chem. 259, 5745–5751.
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