(Received for publication, May 24, 1995; and in revised form, June 23, 1995)
From the
Utilizing the ability of bacterial chaperonin 60 (GroEL) to functionally interact with chaperonin 10 (Cpn10) homologues in an ATP-dependent fashion, we have purified substantial amounts of mammalian, chloroplast, and thermophilic Cpn10 homologues from their natural host. In addition, large amounts of recombinant rat Cpn10 were produced in Escherichia coli and found to be identical to its authentic counterpart except for the lack of N-terminal acetylation. By comparing these two forms of Cpn10, it was found that acetylation does not influence the oligomeric structure of Cpn10 and is not essential for chaperone activity or mitochondrial import in vitro. In contrast, N-terminal acetylation proved crucial in the protection of Cpn10 against degradation by N-ethylmaleimide-sensitive proteases derived from organellar preparations of rat liver. The availability of large amounts of both affinity-purified and recombinant Cpn10 will facilitate not only further characterization of the eukaryotic folding machinery but also further scrutiny of the reported function of Cpn10 as early pregnancy factor.
The partnership between Escherichia coli Cpn10 ()(GroES) and Cpn60 (GroEL) in the folding of polypeptides
has been well documented. GroEL associates with GroES in an
ATP-dependent manner and appears to quantize the ATPase activity of
GroEL during repeated binding and release of the folding polypeptide
substrates (Todd et al.,1994).
The presence of GroES and GroEL homologues has also been established in eukaryotic systems such as fungi, plants, and animals (reviewed by Martinus et al., 1995), but these systems are much less characterized despite some striking differences to their E. coli counterparts. For example, as opposed to the tetradecameric GroEL, mammalian Cpn60 forms a heptamer and has a strict requirement for mammalian Cpn10 (Viitanen et al., 1992). Furthermore, unlike GroEL, the ATPase activity of yeast Cpn60 is not inhibited by Cpn10 (Rospert et al., 1993). Cpn10 homologues also differ in their oligomeric structure. For example, it was reported that the chloroplast homologue, Cpn21 may exist as a dimer or trimer of identical 21-kDa subunits (Bertsch et al., 1992) as opposed to the likely heptameric structure of the bacterial (Chandrasekhar et al., 1986) and mammalian (Hartman et al., 1992) counterparts.
Perhaps most surprisingly and excitingly, it has recently been concluded that EPF, a secreted substance that exhibits growth factor and immunosuppressive properties, is identical to Cpn10. This surprising link between mammalian Cpn10 and EPF was defined by bioassays in which bacterial Cpn10 (GroES) does not exhibit activity (Cavanagh and Morton, 1994; Quinn et al., 1994). Thus, in order to further elucidate the relationship between Cpn10's role as a chaperone and its suspected role as EPF, there is an urgent need to establish a ready source of mammalian Cpn10. Past purification procedures have, however, proven very cumbersome and will only yield microgram (Cavanagh et al., 1994) to milligram (Hartman et al., 1992) quantities despite the use of large amounts of starting material. Since cDNAs encoding Cpn10 have been cloned (Pilkington and Walker, 1993; Ryan et al., 1994; Monzini et al., 1994), it therefore seems attractive to attempt synthesis of mammalian Cpn10 in a bacterial expression system. Indeed, two recent reports have detailed the synthesis of a soluble His-tagged mouse Cpn10 (Dickson et al., 1994) and a denatured but reconstitutable human Cpn10 in E. coli (Legname et al., 1995). A potential drawback is that bacterially synthesized Cpn10 lacks the N-terminal acetylation unequivocally demonstrated to be present on the rat homologue (Hartman et al., 1993a) and indirectly on the human homologue (Cavanagh and Morton, 1994).
We have now developed a single step affinity procedure for the purification of mg quantities of authentic Cpn10 from a variety of sources and further describe a recombinant expression system which allows the production and purification of several hundred milligrams of active mammalian Cpn10 in 1 day. The availability of these procedures has not only allowed us to further characterize and compare Cpn10 homologues in protein folding reactions but also allowed us to assess the importance of N-terminal acetylation for folding activity, mitochondrial import, and resistance to proteases. While the acetylation is shown not to be crucial for chaperone-mediated folding reactions and import into mitochondria in vitro, it is demonstrated that N-terminal acetylation drastically decreases the susceptibility of Cpn10 to degradation by NEM-sensitive proteases from rat liver organelles. By defining a functional significance for the acetylation event, this questions whether mammalian Cpn10 synthesized in bacteria without this post-translational modification can be used to reliably assess the link between its well established role in protein folding and its suspected role as a secreted growth factor with immunosuppressive characteristics (Cavanagh and Morton, 1994; Quinn et al., 1994).
The GroEL column can be used repeatedly and when stored in Buffer A at 4 °C it is stable for at least 1 month.
Anti-Cpn10 antibodies were prepared by precipitation of yolk-lipid using 4% (w/v) PEG 6000 in phosphate-buffered saline essentially as described previously (Gasman et al., 1990). The final precipitate containing the purified antibody mixture was resuspended in phosphate-buffered saline containing 0.05% sodium azide and stored at 4 °C. A dilution of 1:2500 was used for immunoblotting experiments.
Figure 1: Affinity purification of Cpn10 homologues. A, Coomassie Brilliant Blue-stained Tris-Tricine gel illustrating the purification of porcine Cpn10. Mitochondrial extract was passed through an anion-exchange column, and the unbound fraction (lane 3) was loaded onto the GroEL affinity column in the presence of ATP. The GroEL column was washed with buffer A (lane 4) and with buffer A containing 1 M NaCl (lane 5). Cpn10 was eluted with buffer A depleted of ATP, and fractions were collected (lanes 6-10). Molecular weight markers (lane 2) and purified GroEL (lane 1) were also analyzed. B, SDS-PAGE analysis of affinity purified T. brokii (lane 2), chloroplast (lane 4), porcine (lane 7), and bovine (lane 10) Cpn10 homologues. Starting materials (lanes 1, 3, 5, and 8) and fractions obtained following anion-exchange of the appropriate extracts were also analyzed (lanes 6 and 9).
Figure 2: Determination of the molecular mass of Cpn10 homologues by ESI-MS. The left-hand side panel shows the raw m/z spectra, and the right-hand side panels show the corresponding transformations onto a real mass scale of the data for the porcine 7-kDa polypeptide (A and B), the porcine 10-kDa polypeptide (C and D), the bovine 7-kDa/10-kDa polypeptide mix (E and F), and a mixture of affinity purified rat Cpn10 and rec-Cpn10 (G and H).
Purification of the GroES homologue from spinach chloroplasts yielded a 21-kDa protein (2 mg from 1.5 kg of spinach leaves, Fig. 1B, lane 4). A mass for chloroplast Cpn21 was not determined; however, automated Edman degradation (Fig. 3) revealed a single unambiguous N-terminal sequence which is similar to but clearly not identical with that deduced from a cDNA clone encoding spinach Cpn21 (Bertsch et al., 1992). Surprisingly, the N-terminal sequence for the Cpn21 isolated in this study exhibits a greater similarity to a partial pea sequence determined directly by Edman degradation (Bertsch et al., 1992). Given the extensive amino acid conservation of Cpn10 between mammalian species, the amino acid difference between these chloroplast Cpn21 species is unexpected and worthy of further investigation. A much less prominent 10-kDa protein which migrated with mammalian Cpn10 on SDS-PAGE co-purified with the 21-kDa chloroplast species. This may represent a genuine chloroplast Cpn10 homologue or alternatively arise from a minor mitochondrial contamination (Burt and Leaver, 1994). To investigate this proposition further, we attempted to sequence the 10-kDa homologue but without success, most likely due to N-terminal acetylation.
Figure 3: Amino acid sequence similarity of chloroplast Cpn21 homologues. The N-terminal amino acid sequence obtained from pea (Pisum vinosum; Bertsch et al.,1992) and the translation of the first 72 nucleotides encoding the mature portion of spinach (Spinacea oleracea (1) Cpn21 (Bertsch et al., 1992) were compared to the N-terminal amino acid sequence obtained from affinity-purified chloroplast Cpn21 (Spinacea oleracea(2) ; this study). Residues exhibiting positional identity are shaded gray.
The fifth Cpn10 homologue purified using
GroEL affinity chromatography was that from T. brockii. The yield (2 mg from 15 g of cells) was comparable to that
previously obtained by laborious standard chromatography (Truscott et al., 1994), and ESI-MS established its identity (M 10,251.74 ± 2.34).
Figure 4: In vitro refolding of porcine mitochondrial malate dehydrogenase using GroEL and affinity-purified Cpn10 homologues. Porcine mitochondrial malate dehydrogenase was chemically denatured and added to GroEL and ATP plus GroES, bovine (bov-) Cpn10, refolded porcine (por-) Cpn10, chloroplast Cpn21, thermophilic bacterial (T.b.-) Cpn10, rat Cpn10 or porcine 10-kDa/7-kDa polypeptide mix (por-Cpn7/10) as indicated and incubated at 36 °C. Porcine mitochondrial malate dehydrogenase activity was assayed at various time points and compared against the activity of non-denatured porcine mitochondrial malate dehydrogenase also incubated at that temperature for the same time.
Figure 5: Western blot analysis reveals a mitochondrial localization of Cpn10. Pure Cpn10 (1 µg; lane 1) and equal amounts of nuclear (lane 2), mitochondrial (lane 3), microsomal (lane 4), and cytosolic (lane 5) protein (100 µg) were electrophorized on a Tris-Tricine gel. Proteins were transferred to nitrocellulose and probed with chicken anti-Cpn10 antibodies and a horseradish peroxidase-conjugated secondary antibody.
Figure 6: Glutaraldehyde cross-linking of bovine Cpn10. Cpn10 (25 µg) was cross-linked with 0.000% (lane 3), 0.005% (lane 4), 0.010% (lane 5), 0.020% (lane 6), and 0.050% (lane 7) glutaraldehyde for 30 min at 25 °C. Reactions were terminated by the addition of SDS-PAGE loading dye followed by Tris-Tricine SDS-PAGE and Coomassie Brilliant Blue staining. Molecular weight markers (lanes 1 and 2) are shown and their sizes indicated.
Figure 7: Overexpression and purification of rec-Cpn10. After induction, the E. coli cells were lysed and the supernatant (lane 2) was passed through an anion-exchange column. Initial pass-through fractions containing purified rec-Cpn10 were pooled and concentrated (lane 3). Molecular weight markers (lane 4) are indicated.
Both amino acid sequencing and mass spectrometry revealed that rec-Cpn10 lacked N-terminal acetylation, the only respect in which it differed from rat Cpn10 purified by GroEL-affinity chromatography (Fig. 2, G and H).
Figure 8: Non-acetylated Cpn10 is active with GroEL in the in vitro refolding of porcine mitochondrial malate dehydrogenase. Porcine mitochondrial malate dehydrogenase was chemically denatured and added to GroEL and ATP plus affinity purified Cpn10, rec-Cpn10, or GroES as indicated, and incubated at 36 °C. Porcine mitochondrial malate dehydrogenase activity was assayed at various time points and compared against the activity of non-denatured porcine mitochondrial malate dehydrogenase also incubated at that temperature for the same time.
We recently established that acetylation of
Ala stabilizes the N-terminal amphiphilic helix in a
synthetic peptide representing residues 1-25 of rat Cpn10 and
that the mitochondrial targeting signal resides in these residues
(Jarvis et al., 1995). Since mitochondrial targeting
efficiency correlates well with helical amphiphilicity (von Heijne,
1986), the ability of acetylated and non-acetylated Cpn10 to traverse
the mitochondria was investigated (Fig. 9). The qualitative
results clearly show that acetylation is not a prerequisite for
mitochondrial import in vitro.
Figure 9:
Both I-labeled rat Cpn10 and
rec-Cpn10 are imported into mitochondria in vitro.
I-rat Cpn10 and
I-rec-Cpn10 were added
to mitochondria and incubated at 30 °C for 30 min with or without
the presence of proteinase K and/or 2,4-dinitrophenol as indicated.
Import reactions were stopped by the addition of SDS-PAGE sample buffer
and analyzed by Tris-Tricine SDS-PAGE followed by PhosphorImage
analysis (Molecular Dynamics). prot.k, proteinase
K.
Most, if not all cellular
compartments including mitochondria contain proteases required for
protein turnover (Goldberg and John, 1976). We therefore investigated
whether the naturally acetylated rat Cpn10 and non-acetylated rec-Cpn10
differed in their susceptibility to proteolytic degradation when added
to lysates of crude mitochondria (Fig. 10A). It was
repeatedly found that rec-Cpn10 underwent proteolysis with a half-life
of about 1 h whereas rat Cpn10 remained intact over an 8-h period under
the same conditions. The presence of endogenous proteases present in
the rec-Cpn10 preparation only was ruled out, since both rat Cpn10 and
rec-Cpn10 were previously denatured and purified by reverse-phase HPLC.
Additionally, it was found that rec-Cpn10 was stable when incubated at
37 °C for a number of hours in the absence of mitochondrial
lysates. Rec-Cpn10 was also observed to have a shorter half-life than
rat Cpn10 when employing non-denatured Cpn10 species (data not shown).
The nature of the proteolytic activity is unknown, but a preliminary
characterization shows that it is NEM-sensitive and not dependent on
EDTA-chelatable metal ions or ATP (Fig. 10B). The
precise cellular location of the proteolytic activity was not defined,
but it was established that loss of proteolytic activity correlated
well with the loss of -glucuronidase, a lysosomal marker (Stahl
and Touster, 1972; data not shown).
Figure 10: Recombinant Cpn10 is a substrate for a NEM-sensitive proteolytic activity. A, purified rat Cpn10 and rec-Cpn10 were added to separate crude mitochondrial lysates (previously depleted of endogenous Cpn10) and incubated at 37 °C for various times as indicated. Incubations were terminated by the addition of SDS-PAGE sample buffer. Following electrophoresis and Western transfer, anti-Cpn10 antibodies were used to visualize residual Cpn10. B, rec-Cpn10 was incubated at 37 °C for 4 h as above but with or without the presence of apyrase (5 units), 1 mM NEM, or 5 mM EDTA as indicated. Remaining rec-Cpn10 was visualized by immunoblotting as described in panel A and in Fig. 5.
It has frequently been stated that purification of eukaryotic Cpn10 is difficult (Martin et al., 1992; Cavanagh and Morton, 1994; Dickson et al., 1994). Accordingly, only Hartman et al.(1992) and Viitanen et al.(1992) have described the purification to homogeneity of organellar Cpn10 and demonstrated its activity in chaperone-mediated protein folding. We report here on the fast purification of native Cpn10 homologues in high yields from three mammalian species, spinach chloroplasts, and thermophilic bacteria. The procedure has previously been applied to extracts from bacteria (Torres-Ruiz and McFadden, 1992) and could most likely be extended to detect, purify, and characterize Cpn10 from species in which chaperonins have yet to be identified.
While all these homologues are functionally compatible with E. coli GroEL, other Cpn60 homologues are less promiscuous with respect to the choice of Cpn10 partners. Thus, only yeast, chloroplast, and mammalian Cpn10 species have so far been found to satisfy the requirements of mt-Cpn60 albeit to different degrees (Rospert et al., 1993), whereas little is known about the specific requirements of the chloroplast Cpn60 homologues. In this context the general purification procedure presented here may greatly facilitate studies on structure-function relationships of eukaryotic chaperones.
The availability of large amounts of authentic chaperones allowed us to characterize its oligomeric state through glutaraldehyde cross-linking and gel filtration chromatography. The cross-linking of bovine Cpn10 with glutaraldehyde clearly established its heptameric nature, a fact that often had been assumed but not demonstrated by a direct means for any Cpn10 homologue. By contrast, the chloroplast homologue appeared not to constitute a heptamer. Previous work had established that the spinach chloroplast Cpn10 homologue Cpn21 is composed of 21-kDa monomers. Each monomer contains a tandem repeat of ``traditional'' Cpn10 units, and the native molecular mass for the active chaperone was estimated to be 55 kDa as judged by gel filtration (Bertsch et al., 1992). In this study, gel filtration revealed that Cpn21 co-elutes exactly with the 76-kDa bovine Cpn10 heptamer. This suggests that Cpn21 is trimeric or tetrameric in vitro and therefore does not exhibit the 7-fold symmetry observed for Cpn60 homologues. However, like Bertsch et al.(1992), we also observed a 10-kDa protein which copurified with Cpn21. Due to its low abundance we have not been able to characterize this component in detail. It may either constitute a mitochondrial contaminant (Burt and Leaver, 1994) or alternatively represent a genuine chloroplast Cpn10 homologue. Such a homologue may either form a traditional heptamer or alternatively complex with three Cpn21 monomers to form a hetero-oligomer with 7-fold symmetry. Admittedly, such an arrangement would be unprecedented for a Cpn10 homologue, and it is possible that gel filtration, as performed in this study and by Bertsch et al.(1992), is not a reliable indicator of the oligomeric state of Cpn21 in vivo. This could be due either to nonspecific interactions with the column or a dissociation event. Baneyx et al.(1995) performed electron microscopy of purified spinach Cpn21 negatively stained with uranyl acetate and observed circular molecules with an apparent hole in the center as seen previously for E. coli GroES (Chandrasekhar et al., 1986). Unfortunately individual Cpn21 monomers were not distinguishable, and the question of the oligomeric state of native chloroplast Cpn21 therefore remains unresolved.
Despite the relative ease with which Cpn10 homologues could be purified from natural sources as described above, it was also decided to express rat Cpn10 in E. coli. Due to the very high expression levels and the high pI of Cpn10 (9.65), several hundred milligrams of pure non-acetylated Cpn10 could be obtained within 1 day. The simultaneous availability of acetylated rat Cpn10 and non-acetylated recombinant Cpn10 allowed us to rigorously test the effect of this modification in three different assay conformations.
First, chaperone-mediated refolding of porcine mitochondrial malate dehydrogenase was tested using the two Cpn10 species and GroEL as a co-chaperone. As established earlier, acetylation was not required for chaperone function (Dickson et al., 1994; Legname et al., 1995) and had no effect on the rate of folding under the in vitro conditions employed here.
Second, since we have previously shown that acetylation stabilizes the amphiphilic N-terminal helix which constitutes the mitochondrial targeting signal of rat Cpn10 (Jarvis et al., 1995), the import of rat Cpn10 and rec-Cpn10 into mitochondria was compared. Although a difference in rate of translocation cannot be ruled out, the results clearly show that both molecules are imported into isolated mitochondria in vitro.
Third, the effect of acetylation on protease susceptibility was also assessed. In this case acetylation had a dramatic effect. While rat Cpn10 showed no sign of proteolytic breakdown, rec-Cpn10 was degraded with a half-life of 1 h. The nature of the protease was not established, but preliminary experiments indicated it may be of lysosomal origin and NEM-sensitive. Given these characteristics and the effect of N-terminal modification, amino-peptidases such as Cathepsin J (Nikawa et al., 1992) are likely to be responsible for the rapid turnover of rec-Cpn10. The importance of acetylation for proteolytic resistance has also been established for a number of neuropeptides (Veber and Freidinger, 1985) and more recently during the development of potent inhibitors of herpes simplex virus ribonucleotide reductases (Paradis et al., 1991). If the proteolytic activity detected in this study resides in an extramitochondrial compartment, what is the significance of this finding? A number of recent reports (Cavanagh and Morton, 1994; Quinn et al., 1994) suggest that Cpn10 is identical to EPF. EPF performs an extramitochondrial function as a growth factor and a peptide hormone which is believed to initiate a cascade of events which ultimately results in an immunosuppressive response of benefit to the developing fetus (Morton et al., 1987; Noonan et al., 1979; Athanasas-Platsis et al., 1989, 1991). This unexpected and exiting dual role of Cpn10 is far from understood but the findings in this study clearly indicate that the naturally acetylated Cpn10 would have a much longer half-life and therefore greater biological potential than non-acetylated Cpn10 produced by means of recombinant-DNA technology. With the availability of simple procedures to simultaneously obtain large quantities of both acetylated and non-acetylated Cpn10, these and other pertinent questions concerning the mt-Cpn10/EPF system can now be pursued.