Cloning and expression of murine high molecular mass heat shock proteins, HSP105.

We have shown that the 105-kDa heat shock protein (HSP105) and the 42 degrees C-specific heat shock protein (42 degrees C-HSP) constitute high molecular mass heat shock proteins. To elucidate the structure of these heat shock proteins, we have screened a cDNA library constructed with poly(A)+ RNA derived from mouse FM3A cells preheated at 42 degrees C for 2 h using an antibody against murine HSP105. Two full-length cDNA clones were obtained: the pB105-1 insert encoded an 858-amino acid protein, and the pB105-2 insert encoded an 814-amino acid protein and lacked 44 amino acids found in pB105-1. The two clones contained the amino acid sequence found in the 17-kDa polypeptide fragments from HSP105 and 42 degrees C-HSP by lysylendopeptidase digestion. In vitro translation products of the RNA transcripts from pB105-1 and pB105-2 migrated to the same positions of HSP105 and 42 degrees C-HSP, respectively, on SDS-polyacrylamide gel electrophoresis. Northern blot analysis showed that the transcript was approximately 4 kilobases in murine FM3A cells and was strongly induced by heat shock and by treatment with arsenite or an amino acid analog. By reverse transcription-polymerase chain reaction analysis using primers by which deletion of 132 nucleotides in pB105-2 could be detected, the polymerase chain reaction product corresponding to pB105-2 was increased only after heat shock at 42 degrees C, whereas the product corresponding to pB105-1 was induced by heat shock at either 42 or 45 degrees C and also by other stresses. Thus, the cDNA clones pB105-1 and pB105-2 encode HSP105 and 42 degrees C-HSP, respectively, and HSP105 and 42 degrees C-HSP (a short form of HSP105) are suggested to be produced by alternative splicing. Here, HSP105 and 42 degrees C-HSP are renamed HSP105 alpha and HSP105 beta, respectively. A protein sequence homology search revealed that HSP105 shares 54, 34, and 25% amino acid identity with human HSP70RY, the sea urchin egg receptor for sperm, and murine inducible HSP70, respectively. Furthermore, by Northern blot analysis, HSP105 mRNA was revealed to be present in most murine tissues and to be highly expressed in the brain.

We have shown that the 105-kDa heat shock protein (HSP105) and the 42°C-specific heat shock protein (42°C-HSP) constitute high molecular mass heat shock proteins. To elucidate the structure of these heat shock proteins, we have screened a cDNA library constructed with poly(A) ؉ RNA derived from mouse FM3A cells preheated at 42°C for 2 h using an antibody against murine HSP105. Two full-length cDNA clones were obtained: the pB105-1 insert encoded an 858-amino acid protein, and the pB105-2 insert encoded an 814-amino acid protein and lacked 44 amino acids found in pB105-1. The two clones contained the amino acid sequence found in the 17-kDa polypeptide fragments from HSP105 and 42°C-HSP by lysylendopeptidase digestion. In vitro translation products of the RNA transcripts from pB105-1 and pB105-2 migrated to the same positions of HSP105 and 42°C-HSP, respectively, on SDS-polyacrylamide gel electrophoresis. Northern blot analysis showed that the transcript was ϳ4 kilobases in murine FM3A cells and was strongly induced by heat shock and by treatment with arsenite or an amino acid analog. By reverse transcription-polymerase chain reaction analysis using primers by which deletion of 132 nucleotides in pB105-2 could be detected, the polymerase chain reaction product corresponding to pB105-2 was increased only after heat shock at 42°C, whereas the product corresponding to pB105-1 was induced by heat shock at either 42 or 45°C and also by other stresses. Thus, the cDNA clones pB105-1 and pB105-2 encode HSP105 and 42°C-HSP, respectively, and HSP105 and 42°C-HSP (a short form of HSP105) are suggested to be produced by alternative splicing. Here, HSP105 and 42°C-HSP are renamed HSP105␣ and HSP105␤, respectively. A protein sequence homology search revealed that HSP105 shares 54, 34, and 25% amino acid identity with human HSP70RY, the sea urchin egg receptor for sperm, and murine inducible HSP70, respectively. Furthermore, by Northern blot analysis, HSP105 mRNA was revealed to be present in most murine tissues and to be highly expressed in the brain.
Upon exposure to heat shock, living cells from bacteria to humans synthesize a set of proteins called heat shock proteins (HSPs). 1 Since HSPs are also induced by a variety of stresses, these proteins are also called stress proteins. However, HSPs are expressed in considerable amounts in nonstressed cells. HSPs are involved not only in cell protection and repair of cell damage caused by a variety of stresses, but also in normal cellular functions (for recent reviews, see Refs. 1-3). These proteins have been classified into several families according to their apparent molecular mass: high molecular mass HSP, HSP90, HSP70, HSP60, HSP47, and low molecular mass HSP. The families such as HSP70, HSP60, and HSP90 have been studied extensively. These proteins are found to interact with other proteins to mediate protein folding, unfolding, assembly, and disassembly of proteins as molecular chaperones.
The members of the mammalian high molecular mass HSP family are not well characterized, although yeast HSP104 has been cloned and sequenced (4,5). Deletion of the HSP104 gene has little effect on growth at a normal temperature, and the cells die at the same rate as wild-type cells when exposed to a high temperature. However, the mutant cells do not acquire tolerance to heat and other forms of stress (4 -6). Interestingly, HSP104 exhibits homology to bacterial ClpA/ClpB proteins, which appear to play a role in an ATP-dependent protein degradation process (7). Recently, ClpA was found to function as a molecular chaperone like DnaK (8).
The 110-kDa HSP (HSP110) from Chinese hamster ovary cells (9, 10) and 105-kDa HSP from murine cells (11,12) have been reported to be the mammalian high molecular mass HSP. Chinese hamster HSP110 was found to be localized in the nucleoli of nonstressed and heat-stressed murine cells by indirect immunofluorescence (9). Since HSP110 is associated with actively transcribed rRNA genes by immunoelectron microscopy, HSP110 was inferred to participate in ribosome assembly (10). On the other hand, by indirect immunofluorescence, we have shown that HSP105 is localized to the cytoplasm and nuclei under both nonstressed and stressed conditions in murine cells and has never been found in the nucleoli, unlike HSP110 (11). Furthermore, we have found a specific 90-kDa HSP that is synthesized only when mammalian cells are heatshocked continuously at 42°C (42°C-HSP) (13). An anti-HSP105 serum reacts not only with HSP105, but also with 42°C-HSP (11,14). By amino acid sequence analysis, HSP105 and 42°C-HSP were found to be similar proteins containing a particular sequence similar to an adenosine-binding domain of HSP70 family proteins and actin (12).
Herein, we report the cloning of HSP105 and 42°C-HSP from murine cells and reveal that HSP105 and 42°C-HSP are highly homologous to human HSP70RY and moderately homologous to the sea urchin egg receptor for sperm and less so to * This work was supported by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) D67016 and D67017.
¶ To whom correspondence should be addressed. Tel.: 81-75-751-3848; Fax: 81-75-751-4645. murine inducible HSP70. The N-terminal half of these proteins contained an ATP-binding domain similar to that of HSP70 family members and was more conserved among these proteins. Northern blot analysis of various murine tissues revealed that HSP105 mRNA was present in most murine tissues and was highly expressed in the brain.

EXPERIMENTAL PROCEDURES
Construction and Screening of a cDNA Library-Mouse mammary carcinoma FM3A cells (supplied by the Japan Cancer Research Resource Bank) were maintained in Eagle's minimal essential medium supplemented with 10% calf serum (15). Total RNA was isolated from FM3A cells pretreated at 42°C for 2 h by a guanidine thiocyanate method (16), and poly(A) ϩ RNA was purified from total RNA by using an oligo(dT)-cellulose column (type 3, Collaborative Research). The cDNA library was constructed from 5 g of poly(A) ϩ RNA by using a ZAP cDNA synthesis kit (Stratagene) according to the manufacturer's instructions. Approximately 1 ϫ 10 6 plaques were screened using anti-HSP105 serum (14), and signals were detected by using the ECL system (Amersham Corp.). To obtain full-length cDNA, a cDNA library was screened using a partial cDNA as a probe essentially as described previously (17).
Nucleotide Sequence Analysis-The cDNA clones were converted into pBluescript vector by in vivo excision. Fragments cut with various restriction enzymes were subcloned into the pGEM7 vector (Promega). Sequences were determined for both strands by using an ALFred™ DNA sequencer II (Pharmacia Biotech Inc.) after performing a dideoxy chain termination reaction by using an AutoRead sequencing kit (Pharmacia Biotech Inc.). Sequences were analyzed and assembled with GeneWorks (IntelliGenetics, Inc.).
Analysis of in Vitro Translated Products-The cDNA clones pB105-1 and pB105-2 were linearized with KpnI. RNAs were produced by transcribing linearized cDNA constructs in vitro by standard procedures. The RNA transcripts were translated in rabbit reticulocyte lysates (Promega) in the presence of [ 35 S]methionine (specific activity Ͼ 800 Ci/mmol; DuPont NEN). The reaction mixtures were subjected to 8% SDS-PAGE and fixed with 50% trichloroacetic acid. After soaking in 1 M sodium salicylate, the gels were dried, and fluorography was performed.
Western Blot Analysis-FM3A cells were lysed in lysis buffer containing 1% Nonidet P-40, 0.15 M NaCl, 50 mM Tris-HCl, 5 mM EDTA, 2 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 1 mg/ml pepstatin. Twenty micrograms of the cell lysate was subjected to 8% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% dry milk/phosphate-buffered saline for 1 h, the membrane was incubated with a 1:1000 dilution of anti-HSP105 serum in 2% dry milk/phosphate-buffered saline. This was then washed and incubated with a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Cappel) in 2% dry milk/phosphate-buffered saline. After washing with 0.1% Tween 20/phosphatebuffered saline, signals were detected by using the ECL system.
Northern Blot Analysis-Total cellular RNA was isolated by the acid guanidium thiocyanate/phenol/chloroform method (18). Ten micrograms of RNA was electrophoresed and transferred to GeneScreen Plus membrane (DuPont NEN). Filters were washed with 2 ϫ SSC; baked; and hybridized in solution containing 50% formamide, 5 ϫ SSC, 0.6 ϫ Denhardt's solution, 1% SDS, and 100 g/ml salmon sperm DNA for 24 h at 42°C. Denatured 32 P-labeled probes were added to the solution for an additional 16 h at 42°C. Mouse HSP105 cDNA (3.2-kb EcoRI/XhoI fragment of pB105-2), human HSP70 DNA (2.3-kb HindIII/BamHI fragment of pH2.3; a gift of Dr. R. I. Morimoto) (19), and human ␤-actin cDNA (2.0-kb BamHI fragment of pHF␤A-1) (20) were used as probes. Filters were washed in 2 ϫ SSC and then in 0.5% SDS, 2 ϫ SSC at 65°C. For analyzing expression in various tissues, a membrane filter containing 20 g of total RNA from each tissue (a kind gift from Drs. Y. Kaneko and J. Fujita, Clinical Molecular Biology, Kyoto University) was hybridized using cDNAs as probes.
PCR Analysis-Total RNA (1 g) extracted from FM3A cells by the acid guanidium thiocyanate/phenol/chloroform method was treated with RNase-free DNase (Promega) at 37°C for 15 min and was used for first strand synthesis primed with random 9-mers according to the manual for the RNA PCR kit (Takara). To generate the PCR product, PCR was performed using a sense oligonucleotide (5Ј-AGAGTGAAG-GTCAAAGTG-3Ј) corresponding to the region between positions 1483 and 1500 of pB105-1 and an antisense oligonucleotide (5Ј-TTAAGAAG-GTCTCTCCCT-3Ј) complementary to the coding region between positions 1875 and 1892 of pB105-1 as primers. PCR was performed using Taq DNA polymerase for 18 cycles (each cycle consisted of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1.5 min) in the presence of [␣-32 P]dCTP (3000 Ci/mmol; Amersham Corp.). The products were electrophoresed on a 3.5% polyacrylamide gel, and the gel was dried and autoradiographed at Ϫ80°C. Molecular size markers (100-base pair DNA ladder, Life Technologies, Inc.) were used for calibration of the sizes of the PCR products.

RESULTS
Cloning and Nucleotide Sequence of HSP105-The cDNA library was constructed with poly(A) ϩ RNA derived from mouse FM3A cells that were treated at 42°C for 2 h. The cDNA library was screened using an anti-mouse HSP105 antibody (11,14). Among several reactive clones, one clone (ES105) with an ϳ1.5-kb insert was isolated. The partial cDNA insert of ES105 was used to further screen the library, which resulted in two full-length cDNA clones. Nucleotide sequence analysis revealed that pB105-1 was 3345 nucleotides long and had a single open reading frame of 858 amino acids beginning with the ATG codon at nucleotide 55 and terminating with the in-frame TAG codon at nucleotide 2628 (Fig. 1). The other clone, pB105-2, was 3268 nucleotides long and had a single open reading frame of 814 amino acids beginning with the ATG codon at nucleotide 142 and terminating with the in-frame TAG codon at nucleotide 2584. pB105-2 lacked the region between nucleotides 1642 and 1773 of pB105-1 (boxed in Fig. 1). Both clones contained the predicted amino acid sequence (shaded in Fig. 1) found in the 17-kDa polypeptide fragments of HSP105 and 42°C-HSP generated by lysylendopeptidase digestion (12).
Structural Features of HSP105-A homology search for protein sequence similarities revealed that murine HSP105 was highly homologous to human HSP70RY and the sea urchin egg receptor for sperm (Fig. 2). Among these proteins, two regions were highly identical: the N-terminal region between amino acids 1 and 500 and the region between amino acids 612 and 713. The N-terminal region of murine HSP105 contains a single ATP-binding site consisting of the conserved VVGLDVGS (amino acids 3-10), EKLK (amino acids 271-274), IEIVG-GATRIPAVKE (amino acids 338 -352), and D (amino acid 369) sequences (underlined in Fig. 1) (21). Since HSP105 and 42°C-HSP clearly contained an ATP-binding domain, we examined the ATP binding ability of HSP105 and 42°C-HSP by ATPagarose column chromatography, but these proteins were found not to bind to the column under the conditions used for ATP binding of HSP70 (data not shown) (22).
Murine HSP105 was 54% identical to human HSP70RY (23,24) and 34% to the sea urchin sperm receptor (25) and only 25% to murine inducible HSP70 (26). In general, HSP70 family members are conserved in the N-terminal ATP-binding domain and less so in the C-terminal putative peptide-binding domain (27)(28)(29). The N-terminal 500-amino acid sequence of HSP105 that contained an ATP-binding domain was more homologous to those of human HSP70RY (72% identity) and the sea urchin egg receptor (50%) and less to that of murine HSP70 (32%). Another region between positions 612 and 713 of HSP105 was also highly homologous to HSP70RY (amino acids 594 -695; 66% identity) and the sea urchin sperm receptor (amino acids 649 -750; 49% identity), but the homologous region was not found in murine inducible HSP70.
In Vitro Transcription and Translation of pB105-To examine protein products of pB105-1 and pB105-2, these cDNAs were transcribed in vitro with bacteriophage RNA polymerase and translated in rabbit reticulocyte lysate. The resulting [ 35 S]methionine-labeled proteins were analyzed by SDS-PAGE (Fig. 3). The translation products of pB105-1 and pB105-2 were both separated to three protein bands, and the largest proteins of the translation products migrated to the same positions as HSP105 and 42°C-specific HSP, respectively, indicating that pB105-1 and pB105-2 encode HSP105 and 42°C-HSP, respectively.
Stress Inducibility of HSP105 mRNA-We next examined whether the level of HSP105 mRNA is affected by various stresses. First, mouse FM3A cells were incubated at 42°C for 15-300 min or were incubated at 37°C after exposure to heat shock at 42°C for 60 min or at 45°C for 15 min, and total cellular RNA was analyzed by Northern blotting. Using pB105-2 as a probe, 4-kb RNA species were detected in both RNAs from nontreated and heat-treated FM3A cells (Fig. 4A). HSP105 mRNA began to increase after heating the cells for 60 min at 42°C and markedly increased after 5 h. When FM3A cells that were heated at 42°C for 60 min were incubated at 37°C, the mRNA increased significantly even after 15 min. By contrast, there is a significant increase in the amount of HSP70 mRNA after 15 min during continuous heating at 42°C. As reported previously (30), the maximum induction of HSP70 mRNA was observed after 120 min of heat shock. When FM3A cells were incubated at 37°C after heat shock at 45°C for 15 min, HSP105 mRNA began to increase at 3 h, while HSP70 mRNA increased at 1 h (Fig. 4B). Furthermore, when FM3A cells were treated with 100 M arsenite, HSP105 mRNA significantly increased at 1 h, and HSP70 mRNA also increased at 1 h after exposure to arsenite (Fig. 4C). Both HSP105 and HSP70 mRNAs increased at 3 h after exposure to 20 mM azetidine-2-carboxylic acid (Fig. 4D). Actin mRNA did not change significantly under these conditions.
RT-PCR Analysis-Since we had cloned two species of cDNAs (pB105-1 and pB105-2), we next performed RT-PCR analysis using primers by which the deletion of 132 nucleotides in pB105-2 could be detected. These primers were supposed to give PCR products of 410 and 298 nucleotides for the transcripts corresponding to pB105-1 and pB105-2, respectively. As shown in Fig. 5, two PCR products of approximately 410 (band a) and 280 (band b) nucleotides were detected in total RNAs prepared from cells treated at 42°C. Bands a and b increased approximately 2-and 3-fold, respectively, after incubation at 42°C for 5 h (Fig. 5, lanes 1-5), and the increase in the sum of both bands was similar to that of HSP105 mRNA analyzed by Northern blot analysis (Fig. 4). On the other hand, only one product (band a) was observed in the RNA prepared from cells treated at 45°C (Fig. 5, lanes 6 -10). Under these conditions, band a increased drastically as observed by Northern blot analysis. Treatment of the cells with arsenite or azetidine-2carboxylic acid did not induce band b, while it induced a dramatic increase in band a (Fig. 5, lanes 11 and 12, respectively). Since mRNA from pB105-2 was only induced by heat shock at 42°C, pB105-2 was supposed to encode 42°C-HSP.
Tissue Specificity of HSP105 Expression-To determine whether HSP105 mRNA is expressed in murine tissues, total cellular RNAs from various murine tissues were analyzed by Northern blotting using pB105-2 as a probe (Fig. 6). Fourkilobase species, as observed in FM3A cells, hybridized to the probe in RNA from almost all tissues. HSP105 mRNA was highly expressed in the brain and moderately expressed in the lungs, heart, thymus, spleen, liver, and small intestine. These findings were also confirmed by Western blot analysis. 2

DISCUSSION
We have previously reported that HSP105 and 42°C-HSP, which are serologically related to each other, have an ATPbinding domain similar to that of the HSP70 family and actin (11)(12)(13)(14). Here, we describe the cloning of the cDNAs encoding HSP105 and 42°C-HSP from mice.
We have screened a cDNA library constructed with mRNA from heat-shocked mouse FM3A cells using an anti-HSP105 antibody and have obtained two full-length cDNA clones. pB105-1 consists of 858 amino acids, and pB105-2 encodes 814 amino acids. Both clones were exactly the same except that pB105-2 lacked 44 amino acids in the region between positions 530 and 573 of pB105-1, and both contained the amino acid sequence found in the 17-kDa polypeptide fragments from HSP105 and 42°C-HSP digested by lysylendopeptidase (12). From in vitro translation experiments and RT-PCR analysis, the cDNA clones pB105-1 and pB105-2 were suggested to encode HSP105 and 42°C-HSP, respectively. HSP105 mRNA was strongly induced by heat shock at 42 or 45°C and by treatment FIG. 2. Amino acid sequence homology between mouse HSP105 and human HSP70RY, sea urchin egg receptor, and mouse HSP70. Region I of mouse HSP105, which contained an ATP-binding domain, is highly similar in these four proteins, whereas region III is similar in HSP105, HSP70RY, and the sperm receptor, and the identity of these regions is shown in the respective boxes. Region II represents the spliced-out region in pH105-2. The identity between HSP105 and other proteins is shown at the right. Numbers on the top indicate the positions of amino acid residues for each protein. with arsenite or an amino acid analog, whereas 42°C-HSP mRNA was only induced by heat shock at 42°C.
The 42°C-specific HSP consists of at least two polypeptides (basic and acidic) with molecular masses of ϳ90,000 (31). Pulse-chase experiments suggested that the acidic protein originated from the basic protein by post-translational modification. HSP105 is induced by heat shock either at 42 or 45°C, whereas 42°C-HSP is synthesized only when heated continuously at 42°C (13,14). Since the amino acid sequences of HSP105 and 42°C-HSP deduced from the cDNA sequences were the same except that 42°C-HSP lacked 44 amino acids in the region between positions 530 and 573 of HSP105, 42°C-HSP and HSP105 may be produced by alternative splicing from the same transcript. This possibility was also confirmed by the RT-PCR experiment (Fig. 5). From these findings, we renamed HSP105 and 42°C-HSP as HSP105␣ and HSP105␤, respectively. Previously, we reported that heat shock induces an alternative splicing in the 5Ј-noncoding region of the collagen-specific stress protein, HSP47 (32). Similarly to the observation for HSP105 (Fig. 5), the alternative splicing of HSP47 was not observed by treatment with other stress inducers like arsenite or azetidine. The finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression. To elucidate the mechanism for this heat shock-induced alternative splicing, we are now cloning the genomic DNA encoding HSP105, which will reported in the near future.
Murine HSP105␣ and HSP105␤ contained a single ATPbinding site similar to that of HSP70 family members, as has been suggested from our protein sequencing data (12). By comparison with GenBank TM protein sequences, HSP105␣ was found to be 54% identical to human HSP70RY, 34% to the sea urchin sperm receptor, and 25% identical to murine inducible HSP70. The N-terminal 500-amino acid sequence of HSP105␣ that contained an ATP-binding domain similar to that of HSP70 family members shared more identity with those of human HSP70RY (72% identity) and the sea urchin sperm receptor (50%). Human HSP70RY is a novel HSP70 of 701 amino acids cloned from a human B lymphocyte cell line (23). The HSP70RY gene has been mapped to human chromosome 5, whereas genes encoding major HSP70 are localized at chromo-  7-9). B, cells were treated without (lane 1) or with heat shock at 45°C for 15 min and then incubated at 37°C for 0, 1, 3, and 6 h (lanes 2-5). C, cells were treated with 100 M sodium arsenite for 0, 1, 3, and 6 h (lanes 1-4). D, cells were treated with 20 mM azetidine-2-carboxylic acids for 0, 3, 6, and 12 h (lanes 1-4). Total RNAs were extracted from these stress-treated FM3A cells, and 10 g of RNA was electrophoresed on 1% agarose containing 6% formaldehyde. After blotting onto nylon membranes, the RNAs were hybridized with the cDNA insert from pB105-2 (top panels), human HSP70 DNA (middle panels), and ␤-actin cDNA (bottom panels). The positions of 28 S and 18 S rRNAs are indicated at the right. HSC70 represents heat shock cognate 70.  6. Northern blot analysis of HSP105 mRNA from various murine tissues. Total cellular RNAs (20 g each) from mouse brain, lung, heart, thymus, spleen, liver, stomach, small intestine, large intestine, kidney, uterus, testis, and bone were electrophoresed on a 1% agarose gel; stained with ethidium bromide (bottom panel); and analyzed by Northern blotting using pB105-2 as a probe. The positions of 28 S and 18 S rRNAs are indicated at the right (top panel). somes 6, 14, and 21 (24). The function of HSP70RY is not clear, but it may have distinctive functions in antigen processing and presenting of B lymphocytes. On the other hand, the sea urchin egg receptor for sperm is a transmembrane glycoprotein of 1184 amino acids with a short cytoplasmic domain (25). The extracellular sperm-binding domain of the receptor shows sequence similarity to HSP70 family members. The extracellular portion of the receptor binds to the sperm protein, bidin, and also inhibits fertilization in a species-specific manner. In addition, the region between amino acids 612 and 713 of HSP105␣ was also highly similar to HSP70RY and the sea urchin sperm receptor between amino acids 594 and 695 and between amino acids 649 and 750, respectively, but not to murine inducible HSP70. In the sea urchin sperm receptor, the second homologous region was also localized to the extracellular sperm-binding domain of the receptor. Thus, since HSP105␣, HSP70RY, and the sperm receptor all contain not only an ATP-binding domain but also the second homologous region, these proteins may constitute a distinct protein family.
Recently, the cDNA of the murine APG protein was isolated and sequenced. 3 The APG protein is 838 amino acids and is found only in germ cells during spermatogenesis in mice. The amino acid sequence of the APG protein was 57% identical to murine HSP105␣ and also shared high identity at the N-terminal 500 amino acids and the second homologous region. The APG protein was testis-specific and was not induced by heat shock, although HSP105␣ was expressed in most murine tissues and was significantly induced by heat shock. Thus, the APG protein may be a testis-specific member of the HSP105 family in mice, as is testis-specific HSP70 (HSP70T) in the HSP70 family (33).
HSP104 is a high molecular mass heat shock protein in yeast, and the antibody against yeast HSP104 cross-reacts with the high molecular mass heat shock protein from human cells and Chinese hamster cells (5). Yeast HSP104 is a protein of 908 amino acids, and the deduced amino acid sequence revealed the presence of two nucleotide-binding sites in HSP104. Deletion of the HSP104 gene in yeast cells has little effect on growth at a normal temperature, but has an effect on the acquirement of resistance to heat and other various stresses (4 -6). The two nucleotide-binding domains of HSP104 are essential for thermotolerance (5). Recently, HSP104 was found to function to resolubilize heat-aggregated proteins (34). Furthermore, yeast HSP104 exhibits homology to bacterial ClpA/ClpB proteins, which appear to play a role in an ATPdependent protein degradation process (7). In addition, ClpA functions as a molecular chaperone like DnaK (8). However, murine HSP105␣ did not show any identity to yeast HSP104 or bacterial ClpA/ClpB proteins.
When preparing this report, the cDNA sequence of Chinese hamster HSP110 was found in the GenBank TM protein sequence data base and has recently been reported (35). The cDNA of Chinese hamster HSP110 encodes 858 amino acids, and the predicted amino acid sequence shared 96% identity with murine HSP105␣. Chinese hamster HSP110 is a high molecular mass heat shock protein that was first reported by Subjeck et al. (9). This heat shock protein is constitutively expressed at low levels and appears to increase with heat shock. When visualized by indirect immunofluorescence, HSP110 is present in nuclei and essentially in nucleoli. Since HSP110 is released from the nucleoli by RNase treatment, it is probably complexed to the RNA component of the nucleoli and possibly participates in ribosome assembly (9,10). However, when we carefully examined the cellular distribution of HSP105 by immunofluorescence using the anti-HSP105 antibody, we found that HSP105 is mainly localized in the cytoplasm and nuclei and is never found in the nucleoli in either nonstressed or stressed cells (11). It would also be interesting to clarify the differences between our murine HSP105 and Chinese hamster HSP110.