The Small Heat Shock Protein Hsp22 of Drosophila melanogaster Is a Mitochondrial Protein Displaying Oligomeric Organization*

Drosophila melanogaster has four main small heat shock proteins (Hsps), D. melanogaster Hsp22 (DmHsp22), Hsp23 (DmHsp23), Hsp26 (DmHsp26), and Hsp27 (DmHsp27). These proteins, although they have high sequence homology, show distinct developmental expression patterns. The function(s) of each small heat shock protein is unknown. DmHsp22 is shown to localize in mitochondria both in D. melanogaster S2 cells and after heterologous expression in mammalian cells. Fractionation of mitochondria indicates that DmHsp22 resides in the mitochondrial matrix, where it is found in oligomeric complexes, as shown by sedimentation and gel filtration analysis and by cross-linking experiments. Deletion analysis using a DmHsp22-EGFP construct reveals that residues 1–17 and an unknown number of residues between 17–28 are necessary for import. Site-directed mutagenesis within a putative mitochondrial motif (WRMAEE) at positions 8–13 shows that the first four residues are necessary for mitochondrial localization. Immunoprecipitation results indicate that there is no interaction between DmHsp22 and the other small heat shock proteins. The mitochondrial localization of this small Hsp22 ofDrosophila and its high level of expression in aging suggests a role for this small heat shock protein in protection against oxidative stress.

Exposure of organisms to various stresses activates a subset of genes encoding for phylogenically conserved proteins known as the heat shock proteins (Hsps). 1 These proteins are commonly divided into families on the basis of molecular weight and sequence homologies. Members of the Hsp70, Hsp90, and Hsp60 families have been shown to act as molecular chaperones in protein folding or refolding following aggregation, and some assist protein translocation through a number of cellular membrane systems. Their chaperone activities are ATP-dependent, and the process is often assisted by co-chaperones. The sHsps comprise a more diverse and less conserved group of proteins, the number and sequences of which vary between and within species. They share a common structural domain in their C termini that shows high homology to the ␣Aand ␣B-crystallins (1). There is evidence for a chaperone-like activity of some sHsps in vitro, where they have been shown to interact with nonnative proteins in an ATP-independent manner (2)(3)(4)(5). However, sHsps seem to be rather inefficient chaperones, and they have been suggested to act as reservoirs of nonnative refoldable proteins, which can be refolded in collaboration with other chaperones, such as Hsp70 (6,7).
The role(s) of sHsps in vivo remain unclear. Landry et al. (8) reported that overexpression of human Hsp27 could protect cells from the adverse effects of heat, a phenomenon referred to as thermotolerance. One of the sHsps of Drosophila melanogaster, DmHsp27, also conferred thermal resistance in the same assay (9). This finding is consistent with an early report (10) showing that induction of the Drosophila sHsps by the molting hormone ecdysone was sufficient to confer a thermostable phenotype to fruit flies. The cellular mechanisms by which these sHsps protect cells in vivo remain debated. Hamster Hsp27 has been proposed to protect the actin cytoskeletal network making it more resistant to disruption by stress (11,12). Mehlen et al. (13) suggested that sHsps protected cells through modulation of their glutathione levels. Although the chaperone-like activity of sHsps in vitro has been proposed to protect cells by helping the refolding of unfolded proteins in cooperation with other cellular chaperones (2), evidence for a chaperone activity in vivo is still lacking.
Although the role of sHsps in stress tolerance attracted attention for many years, there are other studies suggesting that these proteins may have other function(s) in vivo within unstressed cells. In addition to their action on microfilament organization in avian and mammalian cells (11,14), sHsps have been reported to modulate apoptosis mediated by the Fas/Apo1 death receptor (15) and to be involved in cell growth and differentiation (16). In Drosophila, sHsps have been shown to be individually expressed in a tissue-, cell-, and development-specific manner (see Refs. 17 and 18 for reviews). In this organism, there are four main sHsps (DmHsp22, DmHsp23, DmHsp26, and DmHsp27), the genes of which are localized in a cluster of seven heat-inducible genes at locus 67B (19). These fours sHsps show sequence homology in the ␣-crystallin-like domain of ϳ100 amino acids at the C terminus. At the N terminus, there is a hydrophobic region of 15 amino acids common to three sHsps but absent from DmHsp22 (20,21). Why there are four distinct albeit highly structurally similar sHsps in this organism is unknown. Studies of sHsp expression during embryogenesis in D. melanogaster have shown that each member of this family is expressed in a cell-specific and developmental stage-specific manner and that this pattern of expression is uncoordinated in contrast to the heat shock situation when all sHsps are coordinately induced (22)(23)(24)(25). Like that of the other three sHsps, DmHsp22 expression is develop-mentally regulated. Hsp22 mRNA reaches a peak in late third instar larvae and disappears in early pupae, whereas the other sHsps are expressed during pupation and in young flies (26 -28). DmHsp22 was recently reported to be the most abundantly expressed heat shock protein in aging D. melanogaster (29,30). In young adults, low amounts of DmHsp22 mRNA are found in the thorax. In aged flies, the level of DmHsp22 expressed in the head is higher than that found after a heat shock treatment (30).
We have previously shown that DmHsp27 localizes mainly to the nucleus in cultured cells (31) and in female and male gonads (24,25). DmHsp26 and DmHsp23 are both localized in the cytosol, where they can form large multimeric complexes. Little is known about the localization, the native state, and the function(s) of the smallest member of this family, DmHsp22. Here, we report that DmHsp22 is a mitochondrial protein that localizes to the mitochondrial matrix. Using deletion analysis and site-directed mutagenesis, we show that the first 28 amino acids at the N terminus end and, in particular, the first four residues WRMA of the motif WRMAEE at positions 8 -13 are important for intramitochondrial localization. Sedimentation and gel filtration data along with cross-linking experiments provide evidence that this small mitochondrial Hsp is present as an oligomer within this organelle.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection-The lung Chinese hamster cell line CCL39 was transfected with pRcCMV-Hsp22 (pRcCMV; Invitrogen) containing the full-length Hsp22 cDNA from D. melanogaster. A stable cell line (CCL39 -22C5), expressing DmHsp22, was grown in special Dulbecco's modified Eagle medium (Life Technologies, Inc.) supplemented with 5% (v/v) fetal bovine serum (Immunocorp Sciences), 100 units/ml penicillin G sodium, 100 g/ml streptomycin sulfate (Life Technologies, Inc.), and 4 g/ml Geneticin sulfate (Life Technologies, Inc.) for plasmid selection. CV1, HeLa, or COSm6 cells were grown in modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibiotics. Cells in exponential growth were transfected with FuGENE 6 (Roche Diagnostic), LipofectAMINE (Life Technologies, Inc.), or Lipofectin (Life Technologies, Inc.). The Schneider cell line 2 (S2 cells) was obtained from the American Type Culture Collection (CRL-1963) and was grown in suspension at 25°C as described previously (32). For heat shock, S2 cells were incubated at 35°C for 1 h and allowed to recover for 2 to 12 h at 25°C.
Immunofluorescence Microscopy-Indirect immunofluorescence was performed using standard methods. Briefly, cells grown on plastic or cover glasses were fixed for 20 min in ice-cold methanol (S2, CV1, and CCL39) or in 4% paraformaldehyde in phosphate-buffered saline (140 mM NaCl, 3 mM KCl, 8 mM Na 2 HPO 4 ⅐H 2 O, 1.5 mM KH 2 PO 4 ) for 20 min at room temperature (COSm6) and processed for immunofluorescence staining. Antibodies against DmHsp22 were produced in rabbits by immunization using a bacterial DmHsp22 fusion protein in the pET3 vector (Novagen). The primary antibodies used were the anti-DmHsp22 (diluted 1:1,000) or mouse monoclonal antibodies against cytochrome oxidase (diluted 1:250, Santa Cruz Biotechnology) or against cytochrome c (diluted 1:25, Pharmingen). Fluorescein isothiocyanate-, Alexa-, or Texas Red-conjugated goat secondary antibodies against mouse or rabbit IgG (Molecular Probes, Jackson Immunoresearch Laboratories) were used. Cells were examined on a fluorescence microscope (LEITZ DMR) or on a laser scanning confocal microscope (Fluoview, Olympus).
Isolation and Fractionation of Mitochondria-Mitochondria were isolated from cells essentially as described by Enríquez and Attardi (33) with two extra washes of mitochondria. Briefly, cell membranes were lysed by hypotonic shock (10 mM Tris-HCl, pH 6.7, KCl 10 mM, 0.15 mM MgCl 2 ), sucrose was added (final concentration, 0.25 M), and mitochondria were isolated by differential centrifugation. After removing nuclei, mitochondria were pelleted at 8100 ϫ g for 10 min at 4°C. Isolated mitochondria from CCL39 -22C5 cells were digested with trypsin as described by McMullin and Hallberg (34). Both the pellet and the released proteins were analyzed by immunoblotting.
Two different mitochondrial fractionation methods were used to localize DmHsp22. In the first, pelleted mitochondria from CCL39 -22C5 cells were lysed with 0.5% Nonidet P-40 to yield a mitochondrial soluble fraction (matrix and soluble mitochondrial intermembrane space (IMS) material) and an insoluble membrane fraction comprising both the mitochondrial outer membrane (MOM) and the mitochondrial inner membrane (MIM). Isolated mitochondria were resuspended in four pellet volumes of lysis buffer (300 mM NaCl, 10 mM CaCl 2 , 100 mM Tris-HCl, pH 8.5, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride). The solution was centrifuged for 30 min at maximum speed of a IEC/Micro Max centrifuge. All steps were done at 4°C. The second technique used digitonin for mitochondrial fractionation to separate the mitoplasts (matrix and MIM) from the combined MOM and IMS fractions (35,36).
Site-directed Mutagenesis and Deletion Mutants-DmHsp22 was fused with the enhanced green fluorescent protein (EGFP) in two constructs. pEGFP-Hsp22(1-174) contains the entire DmHsp22 coding sequence fused at the C terminus of EGFP. The DmHsp22 cDNA in pRcCMV-Hsp22 was digested with EcoRI and XhoI and inserted into pEGFP-C3 (CLONTECH) at the corresponding sites. In pHsp22(1-174)-EGFP, the DmHsp22 coding sequence is located at the N terminus of EGFP. The polymerase chain reaction product was digested with EcoRI and BamHI and inserted into pEGFP-N1 (CLONTECH) at the corresponding sites. Deletion mutants were prepared by polymerase chain reaction amplification using pHsp22(1-174)-EGFP or pRcCMV-Hsp22 as a template. Using combinations of primers, regions corresponding to the nucleotides 1-51, 1-86, and 16 -173 were prepared and ligated to the pEGFP-N1 plasmid as described above.
Site-directed mutagenesis of residues in the WRMAEE region was performed using the QuickChange site-directed mutagenesis kit (Stratagene) on the pHsp22(1-174)-EGFP plasmid. Each mutated insert was sequenced. The deleted or mutated constructs were transfected in COSm6 or CV1 cells respectively. 24 h posttransfection, the expressed mutated proteins were visualized by EGFP fluorescence or by indirect immunofluorescence with an anti-DmHsp22.
Sedimentation and Gel Filtration Analysis-The native state of Dm-Hsp22 was determined by density gradient centrifugation and gel filtration. Cell extracts were prepared from heat-shocked S2 (1 h at 35°C ϩ 2 h recovery at 25°C) or from CCL39 -22C5. S2 cells were washed twice in hybridoma phosphate-buffered saline (150 mM NaCl, 5 mM KCl, 10 mM Na 2 HPO 4 .7H 2 O, 5 mM NaH 2 PO 4 ⅐H 2 O, 0.2% dextrose, 0.001% phenol red) and twice in wash buffer (100 mM NaCl, 10 mM CaCl 2 , 10 mM Tris-HCl, pH 7.2). Cell pellets were resuspended in lysis buffer (see Isolation and Fractionation of Mitochondria) at a final concentration of 50 ϫ 10 6 cells/ml. The suspension was vortexed for 9 min and centrifuged at 1000 ϫ g for 10 min, and the supernatant applied to the top of the gradient. For CCL39 -22C5 cells, a mitochondrial fraction was first prepared as described above. Mitochondria were lysed in the presence of 0.5% Nonidet P-40, and the soluble extract applied to the gradient.
Sucrose was diluted in isolation buffer (200 mM NaCl, 1.5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.2) to final concentrations of 10 and 40%. Gradients (12 ml) were made in Sorval TH641 ultracentrifuge tubes with a Biocomp Gradient Master 105 settled at 81.5°for 108 s at speed 17 and allowed to cool down at 4°C. After a 39,600 rpm ultracentrifugation (Beckman, L8 -70M) of 22 h at 4°C, fractions of nine drops were collected from the bottom of the gradient, and refraction indexes measured on an Erma refractometer (Optical Works). Gel filtration was performed on Sepharose CL-6B columns as described (38). Proteins from 2.5-ml fractions were precipitated with trichloroacetic acid (final concentration, 10%), incubated for 15 min at 40°C, and washed in 5% trichloroacetic acid and in 99% ice-cold acetone. Dried pellets were resuspended in SDS sample buffer. Sedimentation constants calculations were done as described by McEwen (39).
Glutaraldehyde Cross-linking-Glutaraldehyde cross-linking was performed on Nonidet P-40 soluble fraction of CCL39 -22C5 mitochondria by varying the amount of freshly prepared glutaraldehyde added to the samples as described by Lambert et al. (40). Samples were run on a 10% SDS gel (41). Cross-linked DmHsp22 species were detected by immunoblotting with an anti-DmHsp22 antibody.
Immunoprecipitations-Immunoprecipitations were done as de-scribed by Faure et al. (42) on a soluble extract from S2 cells. Immunocomplexes were collected on protein A-Sepharose beads. Immunoprecipitates and the corresponding supernatants were analyzed on SDS gels. Antibodies used for the experiments were two polyclonal anti-DmHsp22 antibodies (antibodies 36 and 798) and an unrelated antibody.

RESULTS
Polyclonal antibodies raised against DmHsp22 were specific for this sHsp, as shown by immunoblot of heat-shocked S2 cells and of the CCL39 -22C5 clone expressing DmHsp22 (Fig. 1, CCL) and do not recognize the other sHsps of Drosophila (Fig.  5A). To determine the pattern of expression of DmHsp22 and its stability in cells following heat shock, S2 cells were shocked 1 h at 35°C and allowed to recover at 25°C for 2 to 12 h. Immunoblot analysis reveals that DmHsp22 expression began rapidly following heat shock, reaching a maximum after 6 h of recovery (Fig. 1). The level of DmHsp22 then decreased, but it was still present after 12 h.
DmHsp22 Colocalizes with Mitochondrial Markers-Staining of heat-shocked S2 cells with the anti-DmHsp22 shows a cytoplasmic labeling with a punctate appearance. This pattern is similar to that seen when cells are stained with Mito Tracker Red (CM-H 2 XRos) or with the mitochondrial vital stain rhodamine 123 (data not shown). Fixed cells were doubly stained for DmHsp22 and mitochondrial proteins, cytochrome c (S2), or cytochrome oxidase (CCL39 -22C5). In S2 cells, the cytoplasmic dot-like structures were labeled by both antibodies (Fig. 2A,  a-c), and merging of the two pictures reveals an exact colocalization of the two proteins. In the CCL39 -22C5 clone, Dm-Hsp22 shows a localization identical to that of cytochrome oxidase ( Fig. 2A, d-f). The mitochondrial localization of Dm-Hsp22 is observed in different transfected mammalian cell lines (HeLa and Chinese hamster ovary, data not shown). These results indicate that DmHsp22 is a mitochondrial protein and that the signals involved in its localization are recognized in mammalian cells.
Mitochondrial Membranes Protect DmHsp22 from Trypsin Digestion-To investigate whether DmHsp22 is located at the mitochondrion outer face (exposed to cytosol), mitochondria isolated from CCL39 -22C5 cells were incubated with trypsin in the absence or presence of SDS, which disrupts membranes. Immunoblotting was performed on proteins from the treated mitochondrial pellet and from the supernatant of each sample (Fig. 2B). Lanes 1 and 5 of Fig. 2B show that DmHsp22 is found in the pellet of intact mitochondria. When mitochondrial membranes are broken down by SDS treatment, DmHsp22 is released in the supernatant (Fig. 2B, lanes 2 and 6). In intact mitochondria, DmHsp22 is protected from trypsin digestion (Fig. 2B, lane 3), whereas it is degraded when the mitochondrial membranes are destroyed by SDS (lanes 4 and 8). Identical results were obtained using the S2 cells (data not shown).
From this experiment, we conclude that DmHsp22 is located within mitochondria as it is not accessible to external proteases.
DmHsp22 Is Located in the Mitochondrial Matrix-The submitochondrial localization of DmHsp22 was next examined by means of two distinct fractionation methods (Fig. 2C). First, mitochondria were treated with the nonionic detergent Nonidet P-40, which breaks down mitochondrial membranes to release a soluble fraction (matrix and IMS) and an insoluble fraction (MIM and MOM) of membranes. The identity and purity of each submitochondrial fraction was monitored using antibodies against mitochondrial markers, Hsp60 (a matrix protein (34)), cytochrome oxidase (MIM), cytochrome c (IMS), and TOM20 (MOM (Ref. 43; for review, see Ref. 44)). In the Nonidet P-40 fractionation method, Hsp60 fractionates with the soluble fraction as expected for a matrix protein, whereas TOM20 is associated with the membrane insoluble fraction. From this fractionation, we conclude that the majority of DmHsp22 is localized in the mitochondrial Nonidet P-40 soluble fraction. To distinguish between a matrix or an IMS localization, a digitonin fractionation method was used in which digitonin solubilizes the outer membrane, leaving the inner membrane with the matrix intact (mitoplasts). The localization of TOM20 with mitoplasts likely indicates that TOM20 remained attached to the mitochondrial pore system, as this protein is a component of the channel between the inner and outer membranes. In this fractionation, DmHsp22 is largely located in the mitoplasts fraction (Fig. 2C). This result and the data from the Nonidet P-40 treatment are consistent with a localization of DmHsp22 in the mitochondrial matrix.
A Matrix-targeting Sequence Is Located at the N Terminus of DmHsp22-Immunoblot or [ 35 S]methionine labeling experiments performed in S2 cells failed to provide any evidence for a precursor form of DmHsp22 (data not shown). Deletion and mutational experiments were designed to identify sequences involved in the mitochondrial localization of this sHsp. To easily follow DmHsp22 in mammalian cells, DmHsp22 was fused either at the C or the N terminus of EGFP. Only the DmHsp22-EGFP construct showed a mitochondrial localization (compare Fig. 3, A-C), suggesting that the target signal of DmHsp22 is at its N terminus. To identify the motif involved in targeting, deletion mutants were generated on the DmHsp22-EGFP construct (Fig. 3, D-F). Fusion constructs with residues 1-58 (not shown) and 1-28 (Fig. 3D) localized to mitochondria. Deleting amino acids 18 -174 (1-17, Fig. 3E) or the first five amino acids (6 -58, Fig. 3F) resulted in the lost of the mitochondrial localization. Mutants containing residues 10 -58, 14 -58, or 17-58 failed to localize to mitochondria (data not shown). These experiments indicate that the mitochondrial target signal of DmHsp22 is located at the N terminus. Residues 1-17 and others between 17 and 28 are necessary for import in the organelle.
Analysis of DmHsp22 using the PSORT program (63) revealed an N terminus motif that might be implicated in its mitochondrial localization. The motif (WRMAEE) is located at amino acid residues 8 -13 (Fig. 4C). Each residue within this motif was substituted by mutagenesis, and the localization of the mutated proteins was monitored by EGFP fluorescence after transfection. Expression of DmHsp22-EGFP in CV1 cells shows that four out of six amino acids are important for mitochondrial localization. Single mutations W8G, R9G, M10K, and A11D in DmHsp22 (Fig. 4, D-G) or a double mutation (R9G/ E13A; data not shown) led to a loss of mitochondrial targeting ability, whereas E12A and E13A mutants retained their mitochondrial localization (Fig. 4, H and I). Two other amino acids at the N terminus were mutated: a R2G mutation abolished the  mitochondrial localization, whereas a R16G mutation had no effect (data not shown). We conclude that DmHsp22 contains a mitochondrial targeting signal located within the first 28 amino acids of the polypeptide and that Arg-2, Trp-8, Arg-9, Met-10, and Ala-11 are essential for the import of this protein in mitochondria.
DmHsp22 Forms Oligomers under Its Native State-Many of the sHsps form large multimeric aggregates that are in a dynamic state (7). To see whether DmHsp22 interacts with the other sHsps, immunoprecipitation with antibodies to Dm-Hsp22 was carried out on S2 cells extracts. Both polyclonal antibodies immunoprecipitated DmHsp22, but not the other sHsps (Fig. 5A). From the corresponding stained gel, no other major cellular proteins are present in the immunoprecipitated fraction. Thus, DmHsp22 does not seem to interact with the other sHsps in these cells, a finding consistent with its mitochondrial localization.
Next, sucrose gradient centrifugation, gel filtration, and glutaraldehyde cross-linking were used to determine the native structure of DmHsp22 in vivo. Soluble extracts from heatshocked S2 cells and mitochondrial soluble fractions from the CCL39 -22C5 cell line were centrifuged on 10 -40% sucrose gradients. Immunoblotting of fractions showed two main peaks of DmHsp22 at 7 and 10 S in heat-shocked S2 cells (Fig. 5B) and some DmHsp22 on top of the gradient. In CCL39 -22C5 cells (Fig. 5B), DmHsp22 was found in two peaks of 8 and 12 S. There did not seem to be free DmHsp22 in CCL39 -22C5 cells. These sedimentation coefficients argue for an oligomeric native state of DmHsp22, and these values are similar, although not identical, to the ones obtained for DmHsp26 and DmHsp27 oligomers (data not shown). The reason for the small difference in sedimentation of the DmHsp22 complex in both cell types is unknown at this time.
To further characterize the supramolecular organization of DmHsp22, protein cross-linking with glutaraldehyde was done on the Nonidet P-40 mitochondrial extract of CCL39 -22C5. As the glutaraldehyde concentration increased, DmHsp22 was cross-linked in a ladder type distribution with bands of molecular masses multiples of 22 kDa (Fig. 5C). The first crosslinked species consists of a doublet of 42 and 43 kDa. Other cross-linked species were seen at 68, 95, and 115 kDa and at higher molecular mass at the top of the gel with increased cross-linking. These may correspond to DmHsp22 oligomers or to subunits of larger oligomeric forms that are close enough to be linked by glutaraldehyde.
To get further information on the native organization of DmHsp22, the Hsp was expressed in HeLa cells and analyzed by gel filtration. On a Sepharose CL-6B column, DmHsp22 showed an heterodisperse distribution between 200 and over 1000 kDa, with a peak at 850 kDa (Fig. 5D). Native gel electrophoresis gave a distribution between 200 and 900 kDa (data not shown). Together with the sedimentation analysis, these results suggest that DmHsp22 can form large oligomeric structures in vivo. DISCUSSION We have determined the intracellular localization of Dm-Hsp22. This sHsp is localized in the mitochondrial matrix. The first 28 amino acid residues at the N terminus are capable of targeting a fusion DmHsp22-EGFP protein into mitochondria. Five amino acid residues, Arg-2, Trp-8, Arg-9, Met-10, and Ala-11, four of which are within a putative targeting motif (WRMAEE), are shown to be important for mitochondrial import of DmHsp22. A number of other sHsps have been reported to be localized in mitochondria; these include a 22-kDa temperature-induced protein in Leishmania (45), an Hsp22 in pea leaves (46), an Hsp22 with a putative mitochondrial transit signal in soybean (47), a light-stress regulated Hsp of 23 kDa in Chenopodium rubrum (48), and an Hsp30 in Neurospora crassa (49). A cytosolic Hsp22 induced by oxidative stress in Lycopersicon esculentum and presumed to be mitochondrial on the basis of its partial homology with C. rubrum Hsp23 (50) may be identical with Hsp23.8, the gene for which shows a mitochondrial target signal using PSORT software analysis (51). A preliminary sequence analysis of these sHsps does not show much sequence conservation with the exception of small regions within the so-called ␣-crystallin domain. In addition, only Hsp23 from C. rubrum and Hsp23.8 from L. esculentum have a putative mitochondrial target signal, as predicted by PSORT. However the mitochondrial localization of these other sHsps has not been proven, and the present work is, to our knowledge, the first demonstration that a peptide signal within a sHsp can actually target a reporter protein (here EGFP) in mitochondria.
The targeting sequence identified in DmHsp22 comprises six positively charged, three hydroxylated, and many hydrophobic residues within the first 28 amino acids. Site-directed mutagenesis indicates that changing the two negatively charged residues Glu-12 and Glu-13 in the WRMAEE motif has no effects on localization, which is consistent with the fact that negatively charged residues do not seem to be important in presequences (52).
Small Hsps in diverse systems have been shown to form large multimeric structures thought to be important for chaperone activity (3). Changes in the oligomeric state of these proteins have been reported to modulate their biochemical activities both in vitro (7,40) and in vivo (38,53). Some members of the sHsp family in Drosophila have been reported to form large aggregated structures (54,55), and DmHsp23 is found in complexes sedimenting between 10 and 20 S (56). Sucrose density centrifugation, gel filtration, and glutaraldehyde cross-linking show that DmHsp22 also organizes in an oligomeric form in vivo. In the cross-linking experiments, four of the five Hsp22 cross-linked species had molecular masses that were multiples of 22 kDa. The sedimentation measurements consistently gave two peaks at 7 and 10 S. The reason for this double peak is unknown. These sedimentation values correspond to masses around 300 and 600 kDa. Gel filtration indicates a polydisperse distribution with molecular masses between 200 and over 1000 kDa, with a peak at 850 kDa. Thus, the size estimate for the native form of DmHsp22 is in the range of those determined for human Hsp27 (200 -800 kDa (40,53)) and ␣B-crystallin (280 -2000 kDa, with a peak at 800 kDa (3)).
The association of DmHsp22 with other chaperones like Hsp60 seems unlikely, as antibodies to Hsp22 fail to immunoprecipitate Hsp60 (data not shown). However, the possibility cannot be excluded that DmHsp22 interacts with other mitochondrial chaperones, such as the mitochondrial member of the FIG . 5. Hsp22 is present as an oligomer in vivo. A, left, Western blot of immunoprecipitates with anti-DmHsp22 antibodies. S2 cell extracts were immunoprecipitated using two different DmHsp22 antiseras (antibodies 36 and 798) and an unrelated serum. Immunoblotting was performed with anti-DmHsp22 (antibody 36) (bottom) and a monoclonal antibody recognizing DmHsp23, DmHsp26, and DmHsp27 (top). Lane 1, total extract of S2 cells; lanes 2 and 3, immunoprecipitate with anti-DmHsp22 antibodies 36 and 798, respectively; lane 4, immunoprecipitate with unrelated antibody; lanes 5-7, corresponding supernatants. Right, corresponding stained gel. B, Western blot of sucrose density gradient fractions. A Nonidet P-40 soluble extract of heat-shocked S2 cells (top) was layered on a 10 -40% sucrose and fractions immunoblotted with an anti-DmHsp22. The Nonidet P-40 soluble fraction of isolated CCL39 -22C5 mitochondria (bottom) was analyzed as above. S, soluble cell extract; P, Nonidet P-40 insoluble mitochondrial fraction. C, cross-linking with glutaraldehyde. Mitochondria from CCL39 -22C5 cells were isolated and lysed in Nonidet P-40. Aliquots were cross-linked with increasing glutaraldehyde concentrations. Samples were analyzed on 10% SDS gels, and cross-linked species were revealed by Western blot with an anti-DmHsp22 antibody. D, Western blot analysis of gel filtration fractions. HeLa cells were transfected with a plasmid encoding the full-length DmHsp22 (pRcCMV-Hsp22). The lysed cell extract was loaded on a Sepharose CL-6B column, and fractions were analyzed on SDS gels by immunoblotting using an anti-DmHsp22.
Hsp70 family (57), or with the recently discovered Hsp90-like mitochondrial protein TRAP1 (58). Interestingly, it has recently been reported that disruption of a small ␣-crystallinrelated sHsp, Hsp30 in N. crassa, caused defects in import of proteins in mitochondria (59).
There are four main sHsps in D. melanogaster. We have previously shown that DmHsp27 is a nuclear protein both in cultured S2 cells (31) and in gonads of adult flies (24,25). Two of the other main sHsps in this organism, DmHsp23 and Dm-Hsp26, are localized in the cytosolic fraction (54,55,60), where they can form large aggregates. However, DmHsp23 and Dm-Hsp26 do not form complexes of identical sedimentation values, suggesting that they may be associated with different components within this compartment. 2 Moreover antibodies to Dm-Hsp23 do not stain the same structures in flies as antibodies to DmHsp26 (23). The localization of DmHsp22 in mitochondria is of particular interest, as it shows that the four sHsps of D. melanogaster have distinct locales within the cell. It is tempting, then, to suggest that these four structurally similar proteins have functions in different intracellular compartments.
mRNA for DmHsp22 has been reported to be expressed at different developmental stages and sequences required for expression of its gene during heat shock and normal development are partly defined (26,61). King and Tower (30) showed that DmHsp22 was one of the most abundant heat shock protein in aged flies. One of the most consistent changes associated with aging is the accumulation of abnormal proteins or enzymes damaged by oxidative stress (see Ref. 62 for review). Damaged proteins seem to be one of the main inducer of heat shock genes. The induction of DmHsp22 might result from such damages, and in fact, King and Tower (30) showed that functional heat shock elements in the promoter sequence of the DmHsp22 gene were required for induction of this Hsp during aging. These authors suggested that the stress causing aging-specific induction of DmHsp22 might be damage at specific organelles such as mitochondria. This is a particularly interesting possibility given that the present data shows a mitochondrial localization of this Hsp. It will be interesting to test whether DmHsp22 can protect against damages induced by reactive oxygen species and whether it has a chaperone activity in vivo. Experiments are presently under way to test these possibilities.