(Received for publication, September 14, 1994; and in revised form, November 7, 1994)
From the
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.
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.
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 p-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).
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 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 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.
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
(), basic (+), and hydroxylated (
) 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) ()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.
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
p-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.