Involvement of the TRAP-1 homologue, Dd-TRAP1, in spore differentiation during Dictyostelium development

Experimental Cell Research 303 (2005) 425 – 431 www.elsevier.com/locate/yexcr

Involvement of the TRAP-1 homologue, Dd-TRAP1, in spore differentiation during Dictyostelium development T. Morita1, H. Yamaguchi2, A. Amagai, Y. Maeda* Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan Received 30 July 2004, revised version received 6 October 2004 Available online 2 November 2004

Abstract Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. We have previously demonstrated that Dictyostelium discoideum TRAP1 (Dd-TRAP1) synthesized at the vegetative growth phase is retained during the whole course of D. discoideum development, and that at the multicellular slug stage, it is located in prespore-specific vacuoles (PSVs) of prespore cells as well as in the cell membrane and mitochondria. Thereupon, we examined the function of Dd-TRAP1 in prepore and spore differentiation, using Dd-TRAP1-knockdown cells (TRAP1-RNAi cells) produced by the RNA interference method. As was expected, Dd-TRAP1 contained in the PSV was found to be exocytosed during sporulation to constitute the outer-most layer of the spore cell wall. In the TRAP1-RNAi cells, PSV formation and therefore prespore differentiation were significantly impaired, particularly under a heat stress condition. Although the TRAP1-RNAi cells formed apparently normal-shaped spores with a cellulosic wall, the spores were less resistant to heat and detergent treatments, as compared with those of parental MB35 cells derived from Ax-2 cells. These findings strongly suggest that Dd-TRAP1 may be closely involved in late development including spore differentiation, as well as in early development as realized by its induction of prestarvation response. D 2004 Elsevier Inc. All rights reserved. Keywords: TRAP1; PSV; Prespore differentiation; Spore cell wall; Spore viability; Dictyostelium

Introduction TNF receptor associated protein 1 (TRAP-1) was initially identified using the yeast two-hybrid system as a novel protein that binds to the intracellular domain of the type 1 receptor for tumor necrosis factor (TNFR-1|C) [1]. TRAP-1 protein shows significant homology to 90-kDa molecular chaperone Heat Shock Protein 90 (HSP90) and is predominantly located in mitochondria in several cell lines, as expected from a mitochondrial localization sequence at its N-terminus [2]. However, TRAP-1 has also been

* Corresponding author. Fax: +81 22 217 6709. E-mail address: [email protected] (Y. Maeda). 1 Present address: Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita City, Osaka 5650871, Japan. 2 Present address: Department of Cell Genetics, National Institute of Genetics, Mishima, Shizuoka-ken 411-8540, Japan. 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.10.010

identified as an interacting partner for several extramitochondrial proteins, including the type 1 tumor necrosis factor receptor (TNFR-1), the retinoblastoma protein (Rb), EXT1, and EXT2 [1,3,4]. In addition, extramitochondrial localization of TRAP-1 has been observed in pancreatic zymogen granule, insulin secretory granule, cardiac sarcoma, the nucleus, and on the cell surface in mammalian cells [5]. These reports indicate that TRAP-1 functions outside as well as inside mitochondria, but its crucial roles in each case have not been determined. In Dictyostelium cells, a homologue of TRAP-1, Dd-TRAP1 has been reported to be localized in the cortical region of growing cells at a low cell density, and then translocated to mitochondria as the cell density increases, through induction of prestarvation response by which the expressions of several differentiation-specific genes are precociously augmented in growing cells [6]. Thus, the translocation of DdTRAP1 from the cell cortex to mitochondria seems to sense the cell density in growth medium and enhance the early

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developmental program through a novel prestarvation response [6,7]. The cellular slime mold Dictyostelium discoideum grows and proliferates as long as external nutrients are available. Upon deprivation of nutrients, however, starving cells aggregate by means of chemotaxis to cAMP [8] and EDTA-resistant cohesiveness [9]. The cells in the aggregate then form into two major types in a migrating pseudoplasmodium (slug): anterior prestalk and posterior prespore cells. The slug eventually culminates to form a fruiting body consisting of a mass of spores and a supporting cellular stalk. Prespore differentiation is characterized by the presence of PSVs (prespore-specific vacuoles) in which the lining membrane and fibrous structures are contained [10,11]. Moreover, the PSV is one of the most essential organelles to understand the structural basis of late differentiation of this organism because it is the sole organelle that exists only in one of the two types of cells [10]. The PSV is also a functionally crucial structure and it is exocytosed from the prespore cells to form the outer-most layer of spore cell wall during culmination [11–13]. The PSV has been shown to be constructed from a mitochondrion with the help of the Golgi complex [14]. Recently, we have demonstrated that Dd-TRAP1 is predominantly localized in PSVs as well as in mitochondria of differentiating prespore cells [15]. These findings suggested that Dd-TRAP1 might have critical functions beyond the regulation of prestarvation response, particularly in PSV formation and spore stability. Using Dd-TRAP1-knockdown cells, we report here that Dd-TRAP1 is actually involved in prespore differentiation and spore viability.

Materials and methods Cells and culture Vegetative cells of D. discoideum Ax-2 were grown axenically in PS-medium (1% Special Peptone (Oxoid: Lot. No. 333 56412), 0.7% Yeast extract (Oxoid), 1.5% d-glucose, 0.11% KH2PO4, 0.05% Na2HPO4 12H2O, 40 ng/ml vitamin B12, 80 ng/ml folic acid) at 228C. The conditional knockdown transformant (TRAP1-RNAi cells) of Dd-TRAP1 and its parental MB35 cells were grown axenically by shake culture in PS-medium containing 30 Ag/ml of G418, 10 Ag/ml of blasticidin S, and 10 Ag/ml of tetracycline. Before experiments using TRAP1-RNAi cells, they were pre-cultured for 2 days in the absence of tetracycline to suppress the expression of Dd-TRAP1. To allow cells to differentiate, cells were harvested at the exponential growth phase, washed twice in BSS (Bonner’s salt solution; 10 mM NaCl, 10 mM KCl, 2.3 mM CaCl2 [16]) as a starvation medium, and plated on 1.5% non-nutrient agar at a density of 2.5  105 cells/cm2. This was followed by incubation for various times at 228C or 288C.

d

Electron microscopy Migrating slugs and culminating cell masses were fixed in 1% osmium tetraoxide (OsO4) and dissolved in BSS for 20 min at room temperature, as described previously [17]. During subsequent dehydration in a graded series of ethanol (50%, 70%, 90%, 95%, and 100%), specimens were prestained with uranyl acetate saturated in 50% ethanol for 2 h at room temperature and were embedded in an epoxy resin, Epok-812 (TAAB, UK). After polymerization of the resin, ultrathin sections (60–80 nm thick) were cut with a Reichert-Nissei Ultracut S (VIM 535). The sections were stained with lead citrate according to Reynolds [18], and were observed with a transmission electronmicroscope (TEM) (Hitachi, H-8100). Immunoelectron microscopy Exponentially growing Ax-2 cells were harvested, starved, and developed on 1.5% non-nutrient agar. After 24 h of incubation, fruiting bodies were fixed with 2.0% formaldehyde and 0.1% glutaraldehyde in 10 mM Na/K phosphate buffer (pH 7.4) for 1 h at 228C. After dehydration in a graded series of ethanol (50%, 70%, 90%, 95%, and 100%), the specimens were embedded in LR-white (Polysciences Inc.). Polymerization of the LR-white resin was performed for 24 h at 508C. Ultrathin sections were cut and then mounted on nickel grids. The grids were immersed in a mixture of 50 times diluted anti-Dd-TRAP1 serum with PBS containing 1% BSA for 3 h at 228C. As a control, the treatment of the sections with the primary antibody was omitted. The grids were washed in PBS and incubated in EM grade 15-nm goldconjugated anti-rabbit IgG (H + L) Fab (goat) (100 times diluted with PBS containing 1% BSA, 0.1% sodium azide and 0.1% Tween 20; BBInternational) for 1 h at 228C. After thorough washings in PBS, the sections were stained with uranyl acetate saturated in 50% ethanol for 2 min and then Reynolds’ lead citrate for 5 min before observation under the TEM. Western blot analysis Cells were harvested and lysed in SDS sample buffer (2% SDS, 10% glycerol, 0.01% bromophenol blue, and 62.5 mM Tris–HCl (pH. 6.8)). Proteins were size fractionated on 10% acrylamide gel and blotted onto PVDF membranes (Millipore). After blotting, the membranes were gently shaken in TBS-T (20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5% Triton X-100) containing 1% skim milk, overnight at room temperature. Subsequently, the membranes were incubated in the primary antibody solution (1/ 5000 anti-Dd-TRAP1 antiserum in TBS-T containing 1% skim milk) for 1 h at room temperature. After washing with TBS-T, the membranes were incubated in the secondary antibody solution (1/30,000 HRP-conjugated anti-rabbit IgG, goat (Amersham Pharmacia Biotechnology) in TBS-

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T) for 1 h at room temperature. The chemical-enhanced chemiluminescence (ECL kit; Amersham Biosciences) was used for detection of the Dd-TRAP1 protein. Assay for spore viability The viability of spores was assayed, as previously described [19]. Starving MB35 cells and TRAP1-RNAi cells were separately developed on 1.5% non-nutrient agar for 24–96 h to form fruiting bodies. Spores were collected from the fruiting bodies and suspended in spore viability solution (10 mM EDTA, 0.1% Nonidet P-40, 10 mM phosphate buffer (pH 6.2)). After 30 min of incubation at 428C, the spores were diluted with Escherichia coli (B/r) suspension, and plated on 3 LP plate (0.3% Bacto peptone (Difco), 0.3% lactose, 2% agarose). Immunocytochemical detection of prespore-specific vacuoles (PSVs)

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sequence, the mature protein present in the whole course of development is derived from growth-phase cells by origin, thus giving a favorable situation to trace its developmental behavior. Even at the final developmental stage, fruiting bodies contained almost the same amount of 73-kDa Dd-TRAP1 as the original growth-phase cells (Fig. 1A). We have previously demonstrated that Dd-TRAP1 is localized in the cortex of cells growing at a low density, and then transferred to mitochondria as the density of growing cells increases [6]; and that in the prespore cells of a migrating slug, Dd-TRAP1 is mainly located in the lining membrane and fibrous structures of PSV [15]. In mature spores, the wall consists of three layers: a thick layer composed primarily of cellulose fibrils that are sandwiched between two electron-opaque layers (the inner layer and outer-most layer). Since the lining membrane of PSVs constitutes the outer-most layer of spore cell wall, coupling with exocytosis during sporulation, Dd-TRAP1 was

MB35 cells and TRAP1-RNAi cells were separately starved and developed on 1.5% non-nutrient agar at 288C. After 20–22 h of incubation, cell masses were chemically dissociated by shaking gently in pronase-BAL solution for 10 min [20]. The dissociated cells thus obtained were washed twice in BSS, pre-fixed in ice-cold 50% methanol, and then fixed in absolute methanol for 10 min on an icebath. The fixed cells were dried on cleaned coverslips. They were dipped in PBS (140 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM K2HPO4, pH 7.2) for 5 min and stained with the FITC-conjugated anti-D. mucoroides spore IgG in a moisture chamber for 45 min at room temperature, according to the method of Takeuchi [21]. The samples were washed three times in PBS (5 min for each) and mounted in PBS containing 20% glycerol. The percentages of PSV-containing cells (prespore cells) were determined by fluorescent microscopy.

Results and discussion Dd-TRAP1 is located in the outer-most layer of spore cell wall Northern and Western analyses have demonstrated that the mRNA for Dd-TRAP1 is exclusively expressed in the growth phase and rapidly lost after starvation, while the amount of Dd-TRAP1 protein is invariably retained during the whole course of development [7,15]. The precursor protein (80 kDa) of Dd-TRAP1 synthesized in the cytoplasm is rapidly transferred to mitochondria because it has the mitochondrial localization sequence at the N-terminus. Immediately after the transfer, the localization sequence is cleaved to form the mature form (73 kDa) of Dd-TRAP1 [6,7]. Since the anti-Dd-TRAP1 antibody recognizes the 73kDa protein devoid of the mitochondrial localization

Fig. 1. Dd-TRAP1 retained constantly during Dictyostelium development and its localization in a mature spore. (A) Exponentially growing Ax-2 cells were harvested at the exponential growth phase, starved by washings in BSS, and incubated on 1.5% non-nutrient agar for 24 h to obtain fruiting bodies. Total proteins from exponentially growing cells and fruiting bodies were separated by SDS-PAGE and analyzed by Western blots using the anti-Dd-TRAP1 antibody. As a loading control, the amount of actin in each lane (stained with CBB) is shown. (B) Ultrathin sections of spores embedded in LR-white were incubated with the anti-Dd-TRAP1 antibody and then 15 nm-gold-conjugated anti-rabbit IgG (H + L) Fab (goat). It is clear that the gold particles (arrows) are predominantly located in the outermost layer (arrowhead) of spore cell wall. Scale bar, 1 Am.

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expected to locate in the outer-most layer of spores. This was confirmed with an immunoelectron microscope, using the anti-Dd-TRAP1 antibody (Fig. 1B). In this connection, it is of interest to note that many spore coat proteins such as HSP70 and Dd-GRP94 contained in PSVs are also exocytosed during sporulation to locate in the outer-most layer of spore cell wall [13]. Dd-TRAP1 plays an important role in spore maturation To examine the function of Dd-TRAP1 in spore formation, morphogenesis of the conditional knockdown mutant (TRAP1-RNAi cells) of Dd-TRAP1 was observed and compared with that of parental MB35 cells. Since Dd-TRAP1 is necessary for cell growth, we have previously isolated TRAP1-RNAi cells by a combination of an RNA interference technique and a tetracycline-regulated gene expression system [6]. In TRAP1-RNAi cells, the expression of dsRNA against dd-trap1 is regulated by the tetracycline-regulated gene expression system, and therefore the expression of DdTRAP1 is severely suppressed in the absence of tetracycline (Fig. 2). Although starved TRAP1-RNAi cells exhibited slightly delayed differentiation from parental MB35 cells, they showed normal culmination after 22 h of incubation, eventually forming fruiting bodies (Fig. 3). Although TRAP1-RNAi cells and MB35 cells formed apparently normal fruiting bodies with a certain sorus/stalk ratio, the former tended to form the smaller and irregular-shaped spores compared to the latter (Fig. 4). This raised a possibility that Dd-TRAP1 might play a crucial role in spore maturation. Involvement of Dd-TRAP1 in assembly of the outer-most layer of spore coat

Fig. 3. Development of starved TRAP1-RNAi and MB35 cells. TRAP1RNAi cells, which had been cultured for 2 days in the absence of tetracycline, and parental MB35 cells were separately harvested, washed in BSS, and plated on 1.5% non-nutrient agar at a density of 2.5  105 cells/cm2. This was followed by incubation for the indicated times at 228C to allow the cells to develop. Scale bar, 500 Am.

Cellulose is one of the components of a spore coat and is also known to play an important role in spore maturation of Dictyostelium cells. A null mutant of catalytic subunit of cellulose synthase (dcsA) never synthesizes cellulose, and therefore forms small and round-shaped spores [22]. There-

Fig. 2. Expression of Dd-TRAP1 in TRAP1-RNAi cells and parental MB35 cells. TRAP1-RNAi cells, which had been cultured for 2 days in the absence of tetracycline to suppress the expression of Dd-TRAP1, and parental MB35 cells were incubated for 3 h at the indicated temperature. Total proteins extracted from them were separated by SDS-PAGE and analyzed by Western blots using the anti-Dd-TRAP1 antibody. As a loading control, the amount of actin in each lane (stained with CBB) is shown.

Fig. 4. Morphology of spores constituting fruiting bodies of TRAP1-RNAi cells and MB35 cells. TRAP1-RNAi cells and MB35 cells, both of which had been prepared as described in the legend of Fig. 3, were incubated for 48 h at 288C to obtain fruiting bodies. Spores were collected and incubated with 0.1% Calcofluor White for 5 min to stain cellulose. Samples were observed under a phase-contrast and fluorescence microscope. Scale bar, 10 Am.

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Table 1 Viability of spores derived from TRAP1-RNAi cells and parental MB35 cells Cells

Spore viability (average F SD)a 24 h

MB35 TRAP1-RNAi Pb

48 h 2

4.1 F 1.2  10 2.8 F 1.3  10 b0.04

96 h 4

3.6 F 0.6  10 7.1 F 2.3  103 b0.01

3.4 F 0.5  104 9.1 F 2.7  103 b0.01

a

Starved TRAP1-RNAi cells and MB35 cells were developed on 1.5% non-nutrient agar for the indicated times, and the formed spores were separately harvested from fruiting bodies. Spores, 5.0  104, were suspended in the test solution for spore viability and incubated at 428C for 30 min. Subsequently, the spores were diluted in a suspension of Escherichia coli (B/r) and plated on 3LP plates at a low density. After 3 days of incubation, the number of plaques formed was counted. Data from three independent experiments. b Statistical significance ( P) between the two at each time-period was evaluated with a Student’s t test.

To further analyze the effects of TRAP1-RNAi on spore coat formation, TRAP1-RNAi cells and parental MB35 cells were differentiated on 1.5% non-nutrient agar for 24 or 48 h and then compared, with special emphasis on the fine structures of PSVs in prespore cells and the cell wall of

Fig. 5. Fine structures of PSVs in prespore cells and the cell wall in mature spores. Starved TRAP1-RNAi and MB35 cells were separately incubated on 1.5% non-nutrient agar at 228C. After 20 or 48 h of incubation, resulting slugs or fruiting bodies were fixed in 1% OsO4, dehydrated and embedded in Epoxy resin for electron-microscopic observations, as described in Materials and methods. In prespore cells of slugs derived from TRAP1RNAi cells and MB35 cells, the lining membrane (arrowheads) of PSV is fairly fragmented as shown in A and B, contrasting with that observed previously in the PSV of parental Ax-2 cells [14]. The width of the fragmented membrane seems to be slightly thinner in TRAP1-RNAi spores than in MB35 spores (compare the arrowheads of B with those of A). In mature spores formed by 48 h of incubation, it is evident that both of the electron density and width of the outer-most layer (arrows) of spore coat are slightly less in TRAP1-RNAi spores (D) than in MB35 spores (C). Scale bar, 0.5 Am.

fore, to examine whether TRAP1-RNAi cells are able to synthesize normally cellulose in sporulation, the spores derived from TRAP1-RNAi cells were harvested and stained with a cellulose-specific dye, Calcofluor White, and compared with those derived from parental MB35 cells. Despite the aberrant shape of TRAP1-RNAi spores, they were well stained with Calcofluor White, as was the case for MB35 spores (Fig. 4). The majority of spore coat proteins are synthesized in PSVs of differentiating prespore cells and then secreted by exocytosis during sporulation to form the outer-most layer of spore coat [23], while cellulose is synthesized by a cellulose synthase enzyme complex on the cell membrane of differentiating spores and accumulates between the cell membrane and outer-most layer of spore coat [13,22]. Thus, it is unlikely that Dd-TRAP1, which is located in PSV and the outer layer of spore coat, is involved in cellulose synthesis.

Fig. 6. Developments of TRAP1-RNAi and MB35 cells starved under a heat-stress condition. TRAP1-RNAi cells (a, b, c, d), which had been cultured for 2 days in the absence of tetracycline, and parental MB35 cells (e, f, g, h) were separately harvested, washed in BSS, and plated on 1.5% non-nutrient agar at a density of 2.5  105 cells/cm2. This was followed by incubation at a relatively higher temperature (288C) for the indicated times to allow cells to develop. Magnified images of (c) and (g) are shown in (d) and (h), respectively. Scale bar, 500 Am.

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Table 2 Effect of heat stress on PSV formation in TRAP1-RNAi cells and parental MB35 cells Cells

Percentages of PSV-containing prespore cells (average F SD)a

MB35 TRAP1-RNAi

43.6 F 5.9%b 23.8 F 2.3%b

a Mounds and slugs derived from TRAP1-RNAi cells and MB35 cells were dissociated and stained with the FITC-conjugated anti-D. mucoroides spore IgG, as described in Materials and methods. This was followed by estimation of the number-ratio of cells stained with the FITC-antibody under a fluorescence microscope. b Data from three independent experiments. The difference between the two is statistically significant ( P b 0.03).

mature spores. The lining membrane of PSV in prespore cells derived from TRAP1-RNAi cells and MB35 cells was fairly fragmented as shown in Figs. 5A and B, contrasting with that observed previously in the PSV of parental Ax-2 cells [14]. Interestingly, however, the electron density and width of the outer-most layer of spore coat as well as of the PSV lining membrane was slightly less in TRAP1-RNAi spores than in MB35 spores (Figs. 5C, D). The width of PSV lining membrane in MB35 prespore cells was 24.8 F 3.5 nm (mean F SD), while that in TRAP1-RNAi prespore cells was 16.6 F 2.8 nm (mean F SD): The difference was statistically significant ( P b 0.03). Such morphologically incomplete spore coat formation may affect the resistance of spores to heat and detergent treatments, as previously reported by Zhang et al. [22]. To test this possibility, spores were collected from MB35 and TRAP1-RNAi fruiting bodies and incubated in 20 mM phosphate buffer (pH 6.4) containing 0.1% NP-40 for 30 min at 428C. MB35 spores showed high resistance to both detergent and heat treatments in young fruiting bodies (at least until 48 h of incubation), while TRAP1-RNAi spores showed much lower viability compared to MB35 (Table 1). In older fruiting bodies formed after 96 h of incubation, however, TRAP1-RNAi spores exhibited high resistance to detergent and heat (Table 1). These data suggest that Dd-TRAP1 may be involved in the stabilization of spores through an assembly of the outer-most layer of spore coat. Alternatively, it is also possible that Dd-TRAP1 may simply give a wider looking outer-most layer. Since the PSV contains several heat shock proteins such as HSP70 and Dd-GRP94 [23], it is most likely that these heat shock proteins including Dd-TRAP1 function as chaperones for structural integration of spore coat proteins. A possible function of Dd-TRAP1 in a late development under heat stress Although TRAP-1 belongs to the heat shock protein 90 family, its expression has been reported not to be induced by heat shock in monkey kidney cells (CV1) [3]. In Dictyostelium cells, however, the expression of Dd-TRAP1 was considerably induced by heat shock (at 288C for 3 h)

(Fig. 2), suggesting that Dd-TRAP1 functions as a molecular chaperone for proteins denatured under stress. To determine the roles of Dd-TRAP1 under stress, MB35 cells and TRAP1-RNAi cells were developed at 288C. In TRAP1-RNAi cells, the expression of Dd-TRAP1 was strikingly repressed and scarcely induced by heat stress (Fig. 2). When TRAP1-RNAi cells and MB35 cells were starved and developed on non-nutrient agar at 288C, they initiated an aggregation after almost the same time of starvation (Figs. 6a, e). Although MB35 cells formed migrating slugs and eventually developed to fruiting bodies after 48 h of incubation (Figs. 6g, h), most of the TRAP1-RNAi cells failed to culminate and either stopped developmental progress at the mound stage or formed aberrant final structures (Figs. 6c, d). To determine whether TRAP1RNAi cells formed prespore-specific vacuoles (PSVs) under this condition, TRAP1-RNAi cells and MB35 cells differentiated for 20–22 h at 288C were separately harvested, fixed with methanol and stained with FITC-conjugated antispore antibody. As a result, only 23.8% of TRAP1-RNAi slug cells were found to be prespore cells containing PSVs, compared to 43.6% of MB35 slug cells (Table 2). These marked phenotypes under stress may be caused by the emphasized effect of suppression of Dd-TRAP1 expression. Studies of yeast illustrate that reductions in Hsp90 have no apparent effects on cell growth or metabolism under normal conditions [24–27]. However, conditions that cause general protein damage (for example, high temperature) can divert Hsp90 from its normal targets to other denatured proteins, and therefore the effects of reductions in Hsp90 rise to the surface [28–31]. These raise a possibility that Dd-TRAP1 is tightly involved in prespore and spore differentiation not only under stress but also under normal conditions. Acknowledgments We thank Choe Juenn for her critical reading and insightful comments. We are grateful to the Dictyostelium cDNA project in Japan with support from JSPS (RFTF96L00105) and Ministry of Education, Science, Sports and Culture of Japan (No. 08283107) for their kind gift of the cDNA clone SLB414. This work was supported by a Grant-in-Aid (No. 16370030 and 16657020) from JSPS. This work was also funded by the Mitsubishi Foundation. References [1] H.Y. Song, J.D. Dunbar, Y.X. Zhang, D. Guo, D.B. Donner, Identification of a protein with homology to hsp90 that binds the type 1 tumor necrosis factor receptor, J. Biol. Chem. 270 (1995) 3574 – 3581. [2] S.J. Felts, B.A. Owen, P. Nguyen, J. Trepel, D.B. Donner, D.O. Toft, The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties, J. Biol. Chem. 275 (2000) 3305 – 3312. [3] C.F. Chen, Y. Chen, K. Dai, P.L. Chen, D.J. Riley, W.H. Lee, A new member of the hsp90 family of molecular chaperones interacts with

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