©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Subcellular Localization of Human Mitochondrial hsp70 (*)

(Received for publication, September 14, 1994; and in revised form, November 7, 1994)

Timothy Bhattacharyya (§) Anthony N. Karnezis Shawn P. Murphy (¶) Thuc Hoang Brian C. Freeman (**) Benette Phillips (§§) Richard I. Morimoto (¶¶)

From the Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We report the cloning, nucleotide sequence, and localization of mitochondrial hsp70, a member of the human hsp70 multi-gene family. The human mthsp75 gene was cloned by screening an expression library with monoclonal antibody 3A3 that recognizes three members of the human hsp70 family (hsp70, hsc70, and a 75-kDa protein with characteristics identical to that previously established for mitochondrial hsp70). The identity of the 75-kDa protein was confirmed by subcellular fractionation of HeLa cells and the demonstration that the 3A3-reactive 75-kDa protein co-fractionates with mitochondrial localized proteins. The nucleotide sequence of the respective cDNA clone revealed an open reading frame of 679 amino acids with extensive sequence identity with members of the human hsp70 family. The derived amino-terminal pre-sequence shares features common to other mitochondrial targeting sequences. The identity of the cDNA was unequivocally established by introduction of an epitope-tag at the carboxyl terminus of the cloned gene, transfection and analysis by immunofluorescence. The tagged 75-kDa protein localizes to mitochondria, thus providing conclusive evidence that it corresponds to the human mitochondrial hsp70, referred to here as mthsp75.


INTRODUCTION

The hsp70 class of molecular chaperones have multiple activities in escorting of unfolded nascent polypeptide chains, the assembly of multi-protein complexes, and in membrane translocation(1, 2) . In humans, the hsp70 multi-gene family consists of at least four members: hsp70, hsc70, grp78 (BiP), and mthsp75. hsp70 and hsc70 are found in the cytosol and nucleus and are involved in the chaperoning of nascent polypeptides and protection against the accumulation of malfolded proteins(1, 2) . These proteins are key components of the cytosolic endoplasmic reticulum and mitochondrial import machinery where they maintain pre-proteins in a translocation-competent form(3) . Grp78/BiP is confined to the endoplasmic reticulum where it receives the imported endoplasmic reticulum and secretory proteins and is involved in the translocation and folding of the nascent chain(4) . mthsp75 (also identified as Grp75) is located in the matrix of the mitochondria and is involved as a chaperone in the import and folding of newly synthesized, nuclear and mitochondrial encoded proteins(5, 6) .

Three members of the human hsp70 family (hsp70, hsc70, and grp78) have been previously cloned and are well characterized. Three recent independent studies have described the isolation of a cDNA corresponding to a fourth member of the hsp70 family; however, the data regarding the localization of the respective protein has yielded inconsistent results. The same cDNA has been referred to as p66-mortalin, CSA, and PBP74 and has been reported to encode a cytoslic, mitochondrial, and vesicular protein, respectively(7, 8, 9, 10, 11) .

In the present study we report the characteristics of a monoclonal antibody that recognizes three members of the hsp70 family, including mthsp75. We confirm that mthsp75 co-fractionates with mitochondria and use antibody 3A3 to clone the corresponding cDNA. The nucleotide sequence of mthsp75 is identical to that reported for p66-mortalin, PBP74, and CSA; however, we demonstrate by transfection of an epitope-tagged mthsp75 cDNA and visualization by immunofluorescence that mthsp75 is localized to the mitochondria.


MATERIALS AND METHODS

Preparation of Monoclonal Antibody 3A3

Recombinant human hsp70 was overexpressed in Escherichia coli and native hsp70 was purified by ATP-affinity chromatography as described previously (12) . Female BALB/c mice were injected twice with 70 µg of purified hsp70 and bled 28 days after the primary injection to check for an efficient immune response. Spleen cells from mice with the highest titers of hsp70 antibodies were fused to SP/0 myeloma cells using polyethylene glycol(13) . Fusions between the myeloma cells and spleen cells were selected for by growth in hypoxanthine/aminopterin/thymidine medium (Sigma). Colonies were screened for production of hsp70 antibodies using a combination of enzyme-linked immunosorbent assay, Western blot analysis, and immunofluorescence. Positive colonies were cloned by three successive rounds of dilution cloning. Antibody 3A3 represents one of the positive colonies that exhibited reactivity to hsp70.

Two-dimensional Gel Electrophoresis and Western Blot Analysis

Whole cell extracts (50 µg of protein) prepared from HeLa cells grown at 37 °C or heat shocked for 2 h at 42 °C were subjected to two-dimensional electrophoresis as described elsewhere (14) using ampholines in the pH range 4-7. Proteins were electroblotted to nitrocellulose and the filters were blocked for 1.5 h with PBS, (^1)1% BSA. Filters were then incubated for 1 h at room temperature with monoclonal antibody 3A3 (supernatants were diluted 1:100 in PBS, 1% BSA), washed three times for 10 min each with PBS, 0.5% Nonidet P-40, and incubated for 45 min with alkaline phosphatase conjugated goat anti-mouse IgG antibodies (Boehringer-Mannheim) diluted 1:1000 in PBS, 1% BSA. The filters were washed three times for 10 min each with PBS, 0.5% Nonidet P-40, incubated in alkaline phosphatase substrate nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Promega), and exposed to film. All other Western blot analysis was performed using a protocol adapted from the Amersham ECL kit. After blocking for 1 h with PBS, 2.5% nonfat dry milk, filters were rinsed with PBS, 0.1% Tween 20 and incubated with 3A3 diluted 1:10,000 in PBS/ascites for 1 h. Filters were washed twice for 5 min each with PBS, 0.1% Tween 20, and incubated in goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Promega) diluted 1:20,000 in PBS, 2.5% nonfat dry milk. Filters were washed three times for 10 min each in PBS, 0.3% Tween 20 and subjected to ECL according to the manufacturer's protocol.

Subcellular Organelle Fractionation

HeLa S(3) cells (1 times 10^7) were fractionated using a modification of the protocol in(15) . A post-nuclear supernatant was prepared as follows: Cells were incubated for 10 min at 4 °C in hypotonic lysis buffer (10 mM NaCl, 3 mM MgCl(2), 10 mM Tris, pH 7.4) to swell the cells, pelleted by centrifugation, and resuspended in 1 ml of 0.25 M sucrose, Tris, pH 7.4. Cells were then homogenized by 20 strokes in a Dounce homogenizer and centrifuged to remove nuclei. The post-nuclear supernatant was loaded onto a discontinuous density gradient containing 1.7 ml of post-nuclear supernatant, 1.68 ml of 6% Percoll, 0.70 ml of 17% metrizamide, and 0.70 ml of 37% metrizamide (all solutions in 0.25 M sucrose, Tris, pH 7.4) and centrifuged 30 min at 23,000 rpm in a Beckman SW25i rotor. The gradients were fractionated from top to bottom in 200-µl aliquots. Cytochrome c oxidase assay was performed (15) using 10 µl (2.7 mg/ml) of reduced cytochrome c and 100 µl of gradient fractions. Hexosaminidase assays were performed (16) using 20 µl of gradient fractions.

Expression Library Screening

A HeLa cDNA library in ZAPII (Stratagene) was a gift from G. Dreyfuss. E. coli BB4 cells were infected with the phage at 10^6 plaques/25-cm plate. The plaques were transferred to nitrocellulose filters incubated briefly in 10 mM IPTG, blocked with 2.5% BSA for 1 h, and probed as the two-dimensional Western blots described previously. The positive colonies were enriched and cloned through two successive rounds of screening. The 5` fragment of a partial clone for mthsp75 was used to rescreen the library for the full length cDNA. The 5` fragment was labeled with P using the Amersham Random-Primed kit and hybridization reactions were performed in 6 times SSC, 5 times Denhardt's reagent, 0.5% SDS, and 1 mg/ml salmon sperm DNA at 65 °C overnight. Filters were washed four times in 2 times SSC, 0.1% SDS followed by two washes of 0.2 times SSC, 0.1% SDS at 65 °C(17) . Positive plaques were excised and purified by two more rounds of screening. The full length clone was rescued by in vivo excision to the pBluescript phagemid using the Stratagene protocol. Southern blots with hsp70 (pH 2.3) and grp78 probes confirmed that none of the clones hybridized at high stringency (data not shown).

DNA Sequencing and Analysis

Dideoxy sequencing was performed on CsCl-purified, double-stranded plasmid DNA using the U. S. Biochemical Sequenase 2.0 kit. Alignment and analysis of sequences was performed using GeneWorks 2.0 (IntelliGenetics). Published sequences were downloaded from GenBank.

Expression of Recombinant Proteins in E. coli

pET HSP70 has been described previously(12) . pET-hsc70 was a kind gift from N. Imamoto (Osaka University Medical School). For pET-mthsp75, oligonucleotides containing either NdeI or BamHI sites were synthesized to PCR amplify DNA corresponding to amino acids 51-679. The resulting fragment was blunt-end cloned into pBluescript, and then subcloned using NdeI and XbaI into pET21b (Novagen). After transformation into BL21/DE3 E. coli, proteins were induced by adding 0.5 mM IPTG to log growth cells (OD = 0.95) for 3 h. Cells were lysed in SDS sample buffer and subjected to Western blotting with 3A3.

Construction of mthsp75tag

The eukaryotic expression construct pbeta-actin-neo-wthsp70tag has been described(18) . To construct pbetaactin-neo-wtmthsp75tag, oligonucleotide primers were synthesized to PCR amplify DNA corresponding to amino acids 1-679 of mthsp75. The stop codon was modified to a SalI site and a HindIII site was introduced at the 5` end. The resultant PCR fragment was cloned into pGEM1 and the 5` end was sequenced up to the NsiI site and the 3` end up to the BstBI site to confirm that no errors were introduced by Taq polymerase. The internal NsiI-BstBI fragment was replaced by non-PCR cloned DNA to eliminate all possible PCR mutations. The resulting construct was called pGEM1-1-679*. The EcoRI-BamHI fragment of pbeta-actin-neo-wthsp70tag (18) , which contains the epitope for the polyclonal anti-lactate dehydrogenase (LDH) and sequences of the hsp70 3`untranslated region, was subcloned into pBluescript to create pBKS-tag, and confirmed by nucleotide sequencing. pGEM1-1-679* was digested with SalI, and the recessed end was filled with the Klenow fragment of DNA polymerase I and then digested with HindIII. pBKS-tag was digested with EcoRI, filled in with Klenow, and digested with HindIII. Thus the 1-679* HindIII-SalI fragment was cloned into the pBKS-tag, placing the LDH-tag sequences in frame with the open reading frame of mthsp75.

In vitro transcription and translation demonstrated that a protein of approximately 79-kDa protein was produced. The junction region was confirmed by nucleotide sequence analysis and the mthsp75tag gene was subcloned into pbeta-actin-neo and transfected into HeLa cells. Whole cell extracts of transfected cells were subjected to Western blot analysis with anti-LDH and showed expression of the tagged cDNA (data not shown).

Transfection of Tagged cDNA

Transfection of HeLa cells was performed using 20 µg of wthsp70tag or mthsp75tag plasmid DNA per 10-cm dish containing 10^6 cells using the CaPO(4) method as described previously(18) . After incubating with the DNA precipitate for 18 h, cells were washed two times with PBS and incubated with fresh medium for 18 h before any assay was performed. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% calf serum (Life Technologies, Inc.).

Immunocytochemistry

HeLa cells were plated on glass coverslips, transfected with either pbeta-actin-neo-wthsp70tag or pbeta-actin-neo-wtmthsp75tag. Mitochondria were specifically stained by incubation for 30 min at 37 °C with 25 nM CMXR (Molecular Probes), a derivative of the mitochondrial stain rhodamine 123 (19) which is negatively charged and sequestered by actively respiring mitochondria. Cells were washed three times with media at 37 °C for 5 min each, fixed with 3% paraformaldehyde, PBS for 10 min at room temperature, and permeated for 15 min with PBS, 0.2% Triton X-100. After blocking with PBS/BSA (5 mg/ml) for 1 h, coverslips were incubated with anti-LDHtag antibody diluted 1:400 in PBS/BSA for 30 min at 37 °C. Coverslips were rinsed with PBS and incubated with preabsorbed goat anti-rabbit-FITC conjugated secondary antibody (Promega) for 30 min at 37 °C. Coverslips were mounted on FITC-Guard and visualized under a Zeiss Axiophot microscope with an RITC or FITC filter and photographed using Kodak T-MAX 400 ASA film.


RESULTS AND DISCUSSION

Monoclonal Antibody 3A3 Recognizes the Human Mitochondrial Member of the hsp70 Family

Two-dimensional gel electrophoresis of S-labeled extracts from unstressed, heat shocked, and glucose-deprived HeLa cells has previously identified a protein of approximately 75 kDa, referred to as grp75(5, 14) . Monoclonal antibody, 3A3, was obtained following injection of a recipient mouse with human hsp70 and characterized by Western blot analysis of a two dimensional gel of extracts from HeLa cells. As shown in Fig. 1, 3A3 cross-reacts with three proteins corresponding to hsp70, hsc70, and a 75-kDa protein that co-migrates with grp75. The identity of hsp70 and hsc70 were independently established using other well characterized anti-hsp70 antibodies (C92 and 7.10). The immunological cross-reactivity of the 75-kDa protein with known members of the hsp70 family together with the calculated pI and M(r) implied that the 75-kDa cross-reactive protein was grp75.


Figure 1: Monoclonal antibody 3A3 recognizes hsp70, hsc70, and a 75-kDa protein. Western blot analysis of whole cell extracts from control (top panel) and heat shocked (bottom panel, 42 °C for 2 h) HeLa cells resolved by two-dimensional electrophoresis and detected by incubation with antibody 3A3. hsp70 and hsc70 were identified by their M(r) and pI.



Grp75 was previously suggested from studies using biochemical fractionation to be human mitochondrial hsp70, therefore analogous to the yeast SSC1 and trypanosome mitochondrial hsp70(5, 6, 20) . We initially established whether the 75-kDa, 3A3-cross-reactive protein was associated with mitochondria by subcellular fractionation using differential gradient centrifugation and analysis of gradient fractions by Western blot analysis with antibody 3A3. Additionally, aliquots of each fraction were assayed for enzymatic activity to establish the gradient position for lysosomes (hexosaminidase) and mitochondria (cytochrome oxidase). As shown in Fig. 2, the 75-kDa protein detected by antibody 3A3 is in fractions (11, 12, 13) containing the mitochondrial marker cytochrome oxidase and not in fractions containing the lysosome marker hexosaminidase(8, 9, 10) . In contrast, hsp70 and hsc70 are dispersed throughout the gradient, presumably due to their interactions with polyribosomes and organelles. The co-fractionation of the 3A3 reactive 75-kDa protein with mitochondrial markers offers independent corroborative evidence for the mitochondrial member of the hsp70 family. For clarity, we will use the name mthsp75 rather than grp75 in subsequent sections.


Figure 2: The antibody 3A3-reactive 75 kDa protein co-fractionates with mitochondrial marker proteins. A post-nuclear supernatant was prepared from HeLa S(3) cells and separated on a discontinuous density gradient (see ``Materials and Methods''). 200 µl fractions were collected from top to bottom. A, hexosaminidase activity (normalized to percent maximum levels) identifies the lysosomes in fractions 8, 9, and 10. B, Cytochrome c oxidase activity (normalized to percent maximum levels) identifies the mitochondria in fractions 11, 12, and 13. C, Fractions from the gradient were resolved by SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose, and probed with 3A3. The 75-kDa protein (indicated with arrow) is detected only in fractions 11, 12, and 13. D, same as C, but corresponding to a lighter exposure.



Molecular Cloning and Sequence Analysis of the mthsp75 cDNA

The cDNA clone for mthsp75 was selected by immunoscreening a ZAPII HeLa cDNA library with antibody 3A3. Based on the ability of 3A3 to recognize hsp70, hsc70, and mthsp75, we enriched for clones corresponding to mthsp75 by counterselection for hsp70- and hsc70-positive cDNA clones by hybridization with the cloned human hsp70 and hsc70 genes. The resulting recombinant bacterial clones containing cDNAs exhibiting nucleotide sequence relatedness but not identical to hsp70 or hsc70, as determined by partial sequence analysis, were analyzed further. A 2.8-kilobase cDNA was selected and analyzed for complete nucleotide sequence (Fig. 3). The contiguous open reading frame contains 679 amino acids and upon analysis of the sequence corresponds to a full-length cDNA clone for mthsp75 (GenBank accession no. L17189). In addition, the cDNA contains a 51-residue amino-terminal leader sequence that is rich in in basic, hydroxylated, and hydrophobic amino acids, which is characteristic of a mitochondrial targeting signal sequence (Fig. 4).


Figure 3: Physical organization of the mthsp75 cDNA. mthsp75 cDNA (clone 13), isolated as described under ``Materials and Methods'' was physically characterized and gene was sequenced. The segments of DNA corresponding to subclones are indicated by thick lines and the arrows indicate regions and directions sequenced. Arrows that begin with open circles are from oligonucleotide primers. The gray box indicates the 679-amino acid open reading frame.




Figure 4: The amino terminus of mthsp75 has characteristics of a mitochondrial targeting sequence. The amino-terminal 63 residues of mthsp75 were compared to known mitochondrial targeting sequences. Hydrophobic (bullet), basic (+), and hydroxylated (circle) amino acids are marked. Adapted from Hartl et al.(24) .



A comparative analysis of amino acid identities among the cloned members of the human hsp70 family, the bacterial homolog dnaK, and the yeast and Drosophila mitochondrial hsp70 proteins is presented in Table 1. Human mthsp75 is most closely related (72% identity) to the Drosophila mthsp70 (hsc5a), followed by the yeast mthsp70 (62%). Furthermore, that mthsp75 is more closely related to the bacterial dnaK (54%) than to human hsp70 (43%) provides additional support for the endosymbiotic origin of mitochondria(21) . An alignment of the amino acid sequences of the known members of the human hsp70 family according to the functional domains, as defined by biochemical analysis of hsp70 (22, 23) (^2)is shown in Fig. 5. The amino-terminal ATP binding domain is highly conserved among all members of the human hsp70 family with the exception of three regions corresponding to hsp70 residues 96-103, 188-195, 281-297. These regions are in domains IB, IIA, and IIB, respectively, of the structure of bovine hsc70 and would correspond to regions away from the ATP binding cleft(25) . Comparison of the protein substrate binding domain reveals distribution of variable residues in an alternating pattern with regions that are highly conserved separated by sequences of complete divergence. The carboxyl-terminal oligomerization domain contains very little homology among family members, in contrast to the high conservation of the other two domains.




Figure 5: Comparison of the amino acid sequences of the cloned human hsp70 family members (mthsp75, hsp70, hsc70, and grp78). Amino acid sequences were aligned using GeneWorks 2.0. Identical residues are boxed and conservative substitutions are shaded. The functional domains of hsp70 as defined by Wang et al.(22) , Tsai and Wang(23) , and B. C. Freeman, M. P. Myers, and R. I. Morimoto (manuscript in preparation) are indicated.



To directly confirm that the 3A3 cross-reactive protein corresponds to the protein encoded by the mthsp75 cDNA, we introduced the cDNA into a bacterial expression vector and analyzed the expressed protein by Western blot analysis with antibody 3A3. As shown in Fig. 6, the Western blot analysis of extracts from bacterial cells overexpressing the mthsp75 cDNA reveals that the expressed protein was recognized by antibody 3A3.


Figure 6: Recombinant mthsp75 cross-reacts with antibody 3A3. The mthsp75 cDNA corresponding to residues 51-679 (residues 1-51 correspond to the mitochondrial leader sequence) was cloned into the pET bacterial expression vector and described under ``Materials and Methods.'' Crude extracts were resolved by 8% SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose, and probed with antibody 3A3. As positive controls, pET-hsp70 and pET-hsc70 expressing recombinant human hsp70 and human hsc70, respectively, were included. As indicated in Fig. 1, both hsp70 and hsc70 are detected by antibody 3A3.



Subcellular Localization of mthsp75

The cDNA sequence established for mthsp75 is identical to that reported for human PBP74 and nearly identical to those for rat p66-mortalin and CSA (greater than 98% identity without consideration of the substitutions due to species differences). Despite these similarities, there is conflicting data on the subcellular localization of the protein encoded by this cDNA(5, 6, 7, 8, 9, 10, 11) .

Our approach to determine the localization of the translation product of the mthsp75 cDNA was to construct an epitope-tagged, eukaryotic expression vector for mthsp75. In this case, the epitope tag is a peptide from the sperm specific LDH-C for which cross reactive antisera are available. We have previously used the LDH-C peptide tag to examine the structure function of wild type and mutant hsp70 by transfection analysis(18) . The tagged mthsp75 gene was transfected into HeLa cells and the localization of the protein established by indirect immunofluorescence using an anti-LDH-C antibody followed by visualization with fluorescence conjugated antibody. To provide an unequivocal identification of subcellular localization, the cells were simultaneously stained with CMXR, a fixable derivative of rhodamine 123 that permeates the plasma membrane and specifically associates with actively respiring mitochondria(19) .

As shown in Fig. 7, cells transfected with hsp70tag show a diffuse cytoplasmic and nuclear staining while CMXR staining reveals the tubular network corresponding to mitochondria. In this example, hsp70tag acts as an independent control for the mthsp75tag localization. In mthsp75 transfected cells, the antibody to the LDH-tag (Panels B and C, right) recognizes a tubular network which is identical to that described for mitochondria visualized independently by CMXR staining (Panels B and C, left). Also seen in Panel B is that only those cells expressing the transfected mthsp75tag gene exhibit antibody cross-reactivity to the anti-tag antibody. Based on these results, we conclude that the translation product of the mthsp75 cDNA is, indeed, localized to the mitochondria.


Figure 7: mthsp75 is a mitochondrial protein. HeLa cells were transfected with pbeta-actin-neo-mthsp75tag and the cells were stained with the mitochondrial fluorescent dye CMXR and visualized using an RITC filter. mthsp75 tag was visualized by immunofluorescence microscopy with anti-LDHtag primary antibody and goat anti-rabbit-FITC secondary antibody. A, left and right, shows a HeLa cell transfected with hsp70tag and observed under the RITC (to detect CMXR) and FITC (to detect the LDH-tag) filters; non-transfected cells are visible in the background. B and C, left and right, show HeLa cells transfected with mthsp75tag and observed as described.



Given the conflicting data on the subcellular localization of mthsp75, we sought to identify its locale using a direct approach employing the minimum number of assumption regarding reagent specificity. It was necessary that the localization studies detect only the protein encoded by the cloned mthsp75 cDNA and to avoid potential difficulties introduced by the use of anti-hsp70 antibodies which could exhibit cross-reactivity with other hsp70 proteins and thus obscure identification. Although numerous monoclonal antibodies have been described for which the specificity to various members of the hsp70 family has been established by Western blot analysis or immunoprecipitation assays, immunolocalization protocols employ distinct protein fixatives that could potentially obscure the apparent specificities of the respective antibodies. Therefore, the choice of the mitochondrial dye CMXR avoided another level of unintentional cross-reaction and simultaneously allowed us to visualize the entire mitochondrial apparatus, while the use of the epitope-tag provided unequivocal identification of human mthsp75.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
This work was submitted by this author as fulfillment of an honor's thesis in Biological Sciences at Northwestern University.

Supported by National Institutes of Health postdoctoral fellowship. Present address: Dept. of Molecular Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263.

**
Supported by a National Institutes of Health predoctoral fellowship.

§§
Supported by a National Institutes of Health postdoctoral fellowship. Present address: Dept. of Obstetrics and Gynecology, Northwestern University School of Medicine, 4200 Prentice CH HNMH, Chicago, IL 60611.

¶¶
Supported by National Institutes of Health grants GM47150 and GM38109. To whom correspondence should be addressed: Dept. of Biochemistry, Molecular and Cell Biology, Northwestern University, 2153 Sheridan Rd., Evanston, IL 60208. Tel.: 708-491-3340; Fax: 708-491-4461.

(^1)
The abbreviations used are: PBS, phosphate-buffered saline; BSA, bovine serum albumin; IPTG, isopropyl-1-thio-beta-D-galactosidase; PCR, polymerase chain reaction; LDH, lactate dehydrogenase; RITC, rhodamine B isothiocyanate; FITC, fluorescein isothiocyanate; CMXR, chloromethyl X-rosamine.

(^2)
B. C. Freeman, M. P. Myers, and R. I. Morimoto, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank E. Goldberg for the gift of the anti-LDH antibody, Sue Fox for excellent technical support, and the members of our laboratory (especially the undergraduates) for advice and support.


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