Temperature interactions of the molecular chaperone Hsc70 from the eurythermal marine goby Gillichthys mirabilis
Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA
*e-mail: ghofmann{at}asu.edu
Accepted May 25, 2001
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Summary |
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Key words: molecular chaperone, Hsc70, ATPase, heat shock, temperature sensitivity, thermal stability, marine goby, Gillichthys mirabilis.
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Introduction |
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Although very little is known about the temperature relationships of molecular chaperones in ectotherms in natural populations, a great deal is known about the cellular role and biochemistry of the molecular chaperones in general (for reviews, see Hartl, 1996; Gething, 1997; Morimoto, 1998; Fink, 1999; Feder and Hofmann, 1999; Bukau, 1999). Molecular chaperones are ubiquitous and have been found in virtually every organism examined (Lindquist, 1986). The major classes of molecular chaperones include the six families of heat-shock proteins (Hsps); the Hsps most involved in protein folding are members of the Hsp40, Hsp60 and Hsp70 families (Gething, 1997; Fink, 1999). Within each Hsp gene family, there are different forms of the protein; for example, most organisms have around 12 isoforms of the 70kDa Hsp family, with members found in various cellular compartments (Gething, 1997). Molecular chaperones are also characterized as having different expression patterns; some isoforms are constitutively expressed (e.g. Hsc70) and others are stress-induced (e.g. Hsp70). Despite expression patterns that differ with respect to the physiological status of the cell, all chaperones function via a similar mechanism: molecular chaperones recognize exposed, hydrophobic regions of non-native proteins and bind specifically to partially folded proteins, preventing aggregation and misfolding (Hightower, 1991; Parsell and Lindquist, 1993; Hesterkamp and Bukau, 1998; Mogk et al., 1999). As a group, chaperones operate in a functional network in which some chaperones are holders and others are folders (Freeman and Morimoto, 1996: Buchberger et al., 1996; Johnson and Craig, 1997; Lüders et al., 1998; Veinger et al., 1998; Netzer and Hartl, 1998).
Despite major advances in the field of chaperone biology, our understanding of how Hsp function varies with environmental temperature in organisms in nature is tenuous. Nevertheless, a few points are clear. First, since all living organisms synthesize proteins, molecular chaperones across the phyla participate in protein folding over a wide range of temperatures literally, the temperatures over which life exists, from approximately -2°C to +113°C (Somero, 1995). Second, from studies on model systems, it is clear that temperature affects not only the rate of chaperone activity (e.g. McCarty and Walker, 1991) but also the degree to which proteins will denature and aggregate (e.g. Mogk et al., 1999). Thus, organisms living in environments characterized by variable temperatures will have chaperoning needs that are almost certainly different from those of mammalian cells at 37°C. Whether molecular chaperones from ectotherms, particularly eurythermic ectotherms, are structurally different so as to compensate for temperature effects is unknown.
This latter point begs the question of how the biochemical function of Hsps as molecular chaperones relates to the environmental temperature range over which an organism must perform physiologically. To this end, we have examined the temperature interactions of a molecular chaperone from a marine goby, Gillichthys mirabilis. G. mirabilis occurs from the Northern Gulf of California to Tomales Bay, California (Miller and Lea, 1972). In its estuarine habitat in Mexico, G. mirabilis encounters annual environmental temperatures ranging from 5 to 30°C and appears to avoid higher temperatures by burrowing into the estuarine sediment (Barlow, 1961). Because of its extreme eurythermality, G. mirabilis was an ideal study organism for our investigation of molecular chaperone function in ectotherms. For biochemical characterization, we purified Hsc70, a cytoplasmic molecular chaperone, from G. mirabilis white muscle and tested the thermal sensitivity and stability of the ATPase activity, a weak activity exhibited by members of the 70kDa Hsp gene family (McKay et al., 1994). Using in vitro 32P-based ATPase assays, our results confirmed that purified native Hsc70 from G. mirabilis displayed biochemical activity across the range of temperatures encountered by G. mirabilis in nature and that the Hsc70 protein itself was thermally stable, displaying ATPase activity at temperatures that greatly exceeded physiologically relevant temperatures.
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Materials and methods |
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Protein purification
Hsc70 was purified from G. mirabilis white skeletal muscle according to a modified two-step purification protocol described previously (Welch and Feramisco, 1985). For each purification procedure, 5060g of white muscle was homogenized in 5 volumes of homogenization buffer (10mmoll-1 Tris-HCl, pH7.5, 10mmoll-1 NaCl, 0.1mmoll-1 EDTA, 1.0mmoll-1 phenylmethylsulfonyl fluoride, PMSF) in a Waring industrial blender. The homogenate was centrifuged at 10700g for 20min at 4°C. The supernatant was then applied to a DEAE anion-exchange column (2.5cmx20cm; DEAE Sephacel, Pharmacia Biotech) in DEAE column buffer (DEAE-CB; 10mmoll-1 Tris-HCl, pH7.5, 10mmoll-1 NaCl, 0.1mmoll-1 EDTA). The DEAE column was then washed with 200ml of DEAE-CB containing 50mmoll-1 NaCl and 15mmoll-1 2-mercaptoethanol. Elution of Hsc70 from the DEAE column was performed by washing the column with 150ml of DEAE-CB containing 200mmoll-1 NaCl and 15mmoll-1 2-mercaptoethanol. Fractions were pooled and then applied to an ATPagarose affinity column (approximately 5ml; Sigma). The ATP affinity column was washed with 45ml of affinity column buffer (A-CB; 10mmoll-1 Tris-HCl, pH7.5, 10mmoll-1 NaCl, 0.1mmoll-1 EDTA, 15mmoll-1 2-mercaptoethanol, 3mmoll-1 MgCl2) followed by 50ml of A-CB containing 0.5moll-1 NaCl and then with 45ml of A-CB to remove the high concentration of salts. Low-molecular-mass chaperones were removed by washing the affinity column with 1mmoll-1 GTP in A-CB. Hsc70 was eluted with 3mmoll-1 ATP in A-CB, and the resulting Hsc70-containing fractions were pooled. The pooled fractions were concentrated using Centricon-30 centrifugal concentrators (Millipore) at 5000g for 30min. Protein content was determined using a modified Bradford assay (Coomassie Plus; Pierce), and the purity of each preparation was determined by two-dimensional SDSPAGE separation followed by silver staining (BioRad Silver Stain Plus kit). Western blotting (as described in Hofmann and Somero, 1995), using a monoclonal anti-Hsp70/Hsc70 primary antibody (MA3001; Affinity Bioreagents), was performed to identify the purified protein.
Luciferase protection assays
Luciferase protection assays were performed using a modified protocol from Lu and Cyr (Lu and Cyr, 1998). Immediately prior to the assay, luciferase was diluted to 0.2nmoll-1 in 100µl of refolding buffer (10mmoll1 Mops, pH7.2, 50mmoll1 KCl, 5mmoll-1 MgCl2, 1mmoll1 ATP) and then 1µmoll-1 Hsc70, 0.2µmoll-1 mammalian Hsp40 and 50µmoll-1 ATP were added. These mixtures were incubated at 38°C for 40min. Activity was measured at 5min intervals by combining 10µl samples with 50µl of luciferase assay reagent (Promega). Relative light units were then immediately measured using a luminometer (Turner Designs).
ATPase assays
ATP hydrolysis was assayed by measuring the formation of [-32P]ADP from [
-32P]ATP. In vitro activity assays were assembled on ice and consisted of 2µg of Hsc70, 50µl of assay buffer (AB; 20mmoll-1 Hepes-KOH, pH7.0, 25mmoll-1 potassium chloride, 1.0mmoll-1 (NH4)2SO4, 2mmoll-1 magnesium acetate, 0.1mmoll-1 EDTA, 1.0mmoll-1 dithiothreitol and 50µmoll-1 ATP) and 3.7x105Bq of [
-32P]ATP. In some assays, reduced carboxymethylated
-lactalbumin (RCMLA; Sigma), an unfolded stable protein, was used as a target to test the stimulation of the intrinsic ATPase activity of Hsc70. Duplicate or triplicate samples were incubated with 80µmoll-1 RCMLA for selected times and temperatures, and 1µl samples were removed for nucleotide separation. Nucleotides were separated by thin-layer chromatography (TLC) on Baker·flex PEI-cellulose plates (J. T. Baker) in 0.5moll-1 LiCl and 1moll-1 formic acid (OBrien and McKay, 1993). Levels of product, [
-32P]ADP, were quantified using a Storm PhosPhor Imager system (Molecular Dynamics), and densitometric analysis was performed using ImageQuant software. For all figures in the Results section, the resulting densitometric data are expressed as absorbance volume, the mean pixel intensity of a standard area defined and analyzed by the ImageQuant software.
The effect of temperature on Hsc70 ATPase activity was expressed as a Q10 value; Q10 was calculated using the vant Hoff equation as follows:
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where k1 and k2 are rate processes and t1 and t2 are temperatures. A Q10 value is the ratio of the rate of a reaction at a given temperature to its rate at a temperature 10°C lower (see Randall et al., 1997).
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Results |
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Discussion |
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Overall, Hsc70 from G. mirabilis displayed biochemical characteristics that are consistent with those reported for other Hsc70 homologs. Similar to Hsp70-class chaperones from Escherichia coli, Thermus thermophilus and homeothermic vertebrates (McCarty et al., 1995; Motohashi et al., 1990; Motohashi et al., 1996; Russell et al., 1998; Zylicz et al., 1983), Hsc70 from G. mirabilis had a low intrinsic ATPase activity in the absence of its substrate, unfolded proteins (Fig.4). However, the ATPase activity of the G. mirabilis Hsc70 was stimulated by 28% by the addition of reduced carboxymethylated -lactalbumin (RCMLA), a stable unfolded form of
-lactalbumin that acts as a substrate for Hsc70 in vitro (Palleros et al., 1991; Fig.4). Other studies of Hsp70-class chaperones have demonstrated unfolded-protein-dependent activation of the ATPase activity in vitro. Human Hsp70 was activated by 40% by the addition of RCMLA (Freeman et al., 1995), and bovine brain Hsc70 exhibited a twofold increase in ATPase activity in response to apocytochrome c (Sadis and Hightower, 1992). A general explanation for the stimulatory effect of unfolded proteins on Hsc70 ATPase activity can be found in the kinetics of the chaperoning cycle. Although the precise nature of Hsc70 activity is still a subject of debate, the ATPase cycle is closely coupled to the binding and release of unfolded peptides during which the hydrolysis of ATP to ADP fosters a more stable complex of Hsc70 and substrate peptides (for a review, see Hightower et al., 1994). This interaction creates the driving force for increased hydrolysis in the presence of unfolded peptides (Flynn et al., 1989; Jordan and McMacken, 1995).
A second significant finding of this study was that G. mirabilis Hsc70 displayed an ATPase activity that was moderately temperature sensitive and that Hsc70 was functional over the ecological temperature range of this species of goby (Fig.5). Hsc70 ATPase activity increased approximately fourfold between 10 and 35°C, with the maximal rate occurring at 45°C (Fig.5). Interestingly, the preferred temperature for G. mirabilis is reported to be 23°C (Barlow, 1961), and the ATPase activity of Hsc70 displayed very low thermal sensitivity between 15 and 25°C (Fig.5). The activity of Hsc70 ATPase increased between 25 and 45°C (Fig.5), although it should be noted that 40°C and above is a thermal extreme that we believe G. mirabilis is unlikely to experience in the field (see Fig.1A). Finally, the ATPase activity of Hsc70 was measured under two conditions, an intrinsic rate that was measured in the absence of substrate and a substrate-stimulated rate in the presence of RCMLA (Fig.5). The relative similarities of the intrinsic and the RCMLA-stimulated ATPase activity of Hsc70 indicate that the ATPase domain and the peptide-binding domain of G. mirabilis Hsc70 are not different. Thus, there was no measurable differential thermal sensitivity of Hsc70 when both the ATP- and substrate-binding domains were taken into consideration.
Although there are few data sets with which to compare, members of the 70kDa Hsp gene family have been shown to display thermal sensitivity. In E. coli, the ATPase activity of DnaK, the bacterial Hsp70 homolog, varied over the range of physiologically relevant temperatures for growth of the bacterium. However, the ATPase activity of DnaK exhibited a significantly stronger temperature dependence than Hsc70 from G. mirabilis and increased 80-fold across the temperature range 2053°C (McCarty and Walker, 1991). Other studies of DnaK from another species, the thermophilic bacterium Thermus thermophilus, have not found such extreme thermal sensitivity and reported a modest threefold increase in ATPase activity between 25 and 75°C (Klostermeier et al., 1998).
In addition to displaying a degree of thermal sensitivity, Hsc70 from G. mirabilis was a relatively thermally stable protein (Fig.6). During incubations across the temperature range 5080°C, the ATPase activity of Hsc70 was functional up to 62.5°C (Fig.6). However, activity declined abruptly at 65°C, and the protein was completely non-functional at higher temperatures (Fig.6). Similar results have been recorded for DnaK from E. coli, in which ATPase activity declined abruptly between 47.5 and 53°C (McCarty and Walker, 1991). These data indicate that the molecular chaperone under study here was not extraordinarily thermally stable compared with other cellular proteins. For comparison, Fields and Somero (Fields and Somero, 1997) have shown that G. mirabilis A4-lactate dehydrogenase rapidly lost activity between 50 and 55°C.
Ultimately, the biological consequences of folding proteins in a variable-temperature environment will depend on the interactions between the principal components in the chaperone cycle: the non-native protein and the chaperone. From the perspective of the non-native protein, higher relative temperatures will tend to increase the breathing of proteins and result in the destabilization of pre-existing proteins in the cell. In addition, since hydrophobic interactions are strengthened as temperature increases, nascent polypeptides will increasingly aggregate at elevated temperatures. Consequently, thermal stress can be viewed as a double-jeopardy situation for the cell in which the levels of non-native proteins and the likelihood of protein aggregation both increase as temperature rises. From the perspective of the molecular chaperone, temperature will interact with both the rate of the chaperone cycle and the nature of substrate binding. The efficacy of a molecular chaperone could be influenced directly by temperature via the thermal sensitivity of the ATPase activity. Alternatively, temperature could alter the way in which molecular chaperones bind target molecules. Since molecular chaperones interact with unfolded proteins via hydrophobic interactions (e.g. Rüdiger et al., 1997), elevated temperature will strengthen target binding and lower temperatures will weaken hydrophobic interactions.
Taken together, the above observations suggest that there are numerous functional properties of a molecular chaperone that could be modulated by environmental temperature. The results presented here provide data regarding the interaction between temperature and the biochemical function of Hsc70 from a non-model organism, the eurythermal goby Gillichthys mirabilis. Specifically, the ATPase activity of Hsc70 was temperature sensitive, with increasing activity over the environmentally relevant temperature range for this species, indicating that the molecular chaperone cycle would accelerate as temperature-driven protein denaturation increased. Whether this increase is sufficient to accommodate the increasing number of non-native, and potentially aggregating, proteins is currently unknown.
Our data, however, do not address another major functional aspect of a molecular chaperone cycle. Namely, how does Hsc70 interact and bind non-native proteins as a function of temperature? Freeman and Morimoto (Freeman and Morimoto, 1996) have shown that the refolding activity of human Hsp70 was inhibited at temperatures above 41°C, a threshold only 4°C above human body temperature. Whether target binding and protein refolding by molecular chaperones from ectothermic animals are similarly affected by temperature is not known. If there are temperature-dependent differences in the protein-refolding ability of ectothermic chaperones, an important question is whether function varies in a manner that correlates with species evolutionary thermal history. In future experiments, we hope to address this and other unanswered questions regarding the holding and folding of proteins in nature.
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Acknowledgments |
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