From the Department of Molecular Genetics and Cell
Biology and the
Howard Hughes Medical Institute, The University
of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Hsp104 is crucial for stress tolerance in
Saccharomyces cerevisiae, and both of its
nucleotide-binding domains (NBD1 and NBD2) are required. Here, we
characterize the ATPase activity and oligomerization properties of
wild-type (WT) Hsp104 and of NBD mutants. In physiological ionic
strength buffers (pH 7.5, 37 °C) WT Hsp104 exhibits Michaelis-Menten kinetics between 0.5 and 25 mM ATP (Km
~5 mM, Vmax ~2 nmol
min1 µg
1). ATPase activity is strongly
influenced by factors that vary with cell stress (e.g.
temperature, pH, and ADP). Mutations in the P-loop of NBD1 (G217V or
K218T) severely reduce ATP hydrolysis but have little effect on
oligomerization. Analogous mutations in NBD2 (G619V or K620T) have
smaller effects on ATPase activity but impair oligomerization. The
opposite relationship was reported for another member of the HSP100
protein family, the Escherichia coli ClpA protein, in
studies employing lower ionic strength buffers. In such buffers, the
Km of WT Hsp104 for ATP hydrolysis decreased
10-fold and its stability under stress conditions increased, but the
effects of the NBD mutations on ATPase activity and oligomerization remained opposite to those of ClpA. Either the functions of the two
NBDs in ClpA and Hsp104 have been reversed or both contribute to ATP
hydrolysis and oligomerization in a complex manner that can be
idiosyncratically affected by such mutations.
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INTRODUCTION |
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HSP100/Clp proteins are found in eubacteria, fungi, plants, and animals including man. They function in a wide variety of biological activities such as stress tolerance, proteolysis, DNA transposition, and gene regulation. These diverse activities involve a common biochemical mechanism, disassembling quaternary protein structures and aggregates (1). For example, the ClpA protein of Escherichia coli regulates the activity of the ClpP protease, promoting the hydrolysis of specific substrates by ClpP in response to ATP (2, 3). In vitro, in the presence of ClpP, ClpA promotes the degradation of RepA (the origin-binding protein of plasmid P1); but in the absence of ClpP, ClpA activates RepA for DNA binding by disassembling inactive dimers and releasing them in an active, monomeric form (4). Thus, it appears that the function of ClpA in proteolysis is to alter the conformational state of its substrates so that they become accessible to the protease. The Hsp104 protein of Saccharomyces cerevisiae is critical for survival after exposure to extreme temperatures (50 °C; Ref. 5) or high concentrations of ethanol (20%; Ref. 6). These stresses cause protein denaturation, and recent work indicates that Hsp104 promotes survival by facilitating the resolubilization of heat-damaged, aggregated proteins (7, 8).1 Hsp104 also regulates the aggregation state of Sup35, the protein responsible for the prion-like inheritance of an extrachromosomal genetic element in yeast. Thus both ClpA and Hsp104 appear to utilize a similar mechanism for their widely divergent biological functions.
There are two classes of HSP100 proteins, and these are divided into eight subfamilies comprising more than 70 members identified to date (1). Hsp104 is a member of the B subfamily within class 1. The closest E. coli relative of Hsp104, ClpB, is a member of the same subfamily. It and many other members of this subfamily also function in thermotolerance (for review, see Ref. 1). The E. coli ClpA protein, too, is a class 1 protein but from a different subfamily. In biochemical terms, ClpA is currently the best characterized protein in this class.
All class 1 HSP100 proteins contain two predicted nucleotide-binding domains (NBD1 and NBD2)2 separated by a middle region of variable size and flanked by amino- and carboxyl-terminal regions (1, 9). Both NBDs contain classical Walker-type consensus sequences for the P-loop (10) that resemble those of N-ethylmaleimide-sensitive fusion protein and the P-type transporter families. Both NBD1 and NBD2 are highly conserved in all class 1 HSP100 proteins but, except for the few residues that constitute the nucleotide-binding consensus, NBD1 and NBD2 exhibit little homology to each other. For example, although ClpA and Hsp104 are from different subfamilies, they share 51% identity in NBD1 and 42% identity in NBD2, but NBD1 and NBD2 of Hsp104 itself share only 22% identity. This high degree of sequence conservation in each NBD, throughout all four subclasses of class 1 HSP100 proteins, has led to the assumption that the functional relationships between the domains would also be conserved.
All class 1 HSP100 proteins tested assemble into homo-oligomers in the presence of adenine nucleotides (12-15) and hydrolyze ATP in vitro at similar rates (11, 12, and data herein). Both ClpA and Hsp104 assemble into ring-shaped hexamers, as determined by cross-linking, sizing chromatography, and electron microscopy (13-16). In every case tested, ATP is required for efficient functioning of the HSP100/Clp proteins in vitro. Moreover, point mutations in the NBDs, constructed to interfere with ATP binding or hydrolysis, eliminate biological function in vivo (17-19). Elucidating the contributions of the two NBDs to oligomerization and ATP hydrolysis is therefore an important first step for deciphering the molecular mechanism by which class 1 HSP100/Clp proteins alter the conformational state of their substrates.
For ClpA, two independent studies have addressed the contributions of the two NBDs to oligomerization and ATP hydrolysis. Both are in general agreement. One study concludes that NBD2 is not required for hexamer formation and that more than 50% of the basal ATPase activity is derived from NBD2 (18). The other study suggests that NBD1 is responsible for hexamer formation, whereas NBD2 is essential for ATP hydrolysis (19). For Hsp104, we previously reported that a point mutation in NBD1 had little effect on oligomerization in vitro, whereas an analogous mutation in NBD2 strongly inhibited oligomerization (15), a surprising contrast with results for ClpA. No characterization of the ATPase activity of wild-type Hsp104 and the effects of these NBD mutations on ATP hydrolysis has been presented. Here we present this characterization and examine the effects of environmental conditions relevant to the role of Hsp104 in stress tolerance on its ATPase activity. We also test the effects of additional NBD mutations on oligomerization and ATP hydrolysis. Mutations in the highly conserved residues of NBD2 exert stronger effects on oligomerization, whereas analogous mutations in NBD1 exert stronger effects on ATP hydrolysis, results that contrast with similar experiments in ClpA. The same relationship between NBD1 and NBD2 mutations was observed when ATP hydrolysis and oligomerization were examined in physiological ionic strength buffers and in the lower ionic strength buffer employed in ClpA studies. Thus, in agreement with work on ClpA, the two domains of Hsp104 have distinct biochemical characteristics. However, the ways in which similar mutations affect those characteristics are different in ClpA and Hsp104. The assumption that the sequence similarities in the NBD domains of ClpA and Hsp104 predicates an identical distribution of functions is reevaluated in the "Discussion."
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction--
Point mutations that changed amino
acids in the ATP-binding domains were produced by site-directed
mutagenesis, using single-stranded plasmid DNA encoding the
HSP104 gene and the Muta-Gene M13 in vitro
Mutagenesis Kit (Bio-Rad). The G217V and G619V coding sequences were
inserted into the pJC45 vector (gift of J. Clos, Bernhard Nocht
Institute for Tropical Medicine, Hamburg, Germany), derived from the
pJC20 vector (20) that contained a 10-histidine extension under control
of the T7/lac promoter. To eliminate the possibility of extraneous
mutations, K218T and K620T were produced in a modified version of the
HSP104 gene, HSP104R, containing
nucleotide substitutions that introduced restriction sites
(BamHI at 3, SacII at 1125, SalI at
1626, SpeI at 2128; the natural sites EagI at 568 and SacI at 2842 were also used so that unique sites
occurred approximately every 500 base pairs) without changing the
encoded amino acids or significantly altering codon usage. After
mutagenesis, the segment containing the mutation was sequenced,
excised, and inserted into an unmutagenized version of
HSP104R. The K218TR and
K620TR coding sequences were inserted behind the
6-histidine extension of the pET28a vector (Novagen, Madison, WI) from
which an encoded T7 epitope tag had been removed.
Protein Purification--
Hsp104 was purified from yeast as
described (15). For purification from E. coli, proteins were
produced using the T7 expression system (21) pLysS strain (22) for
K218T and K620T and using BL21[DE3](pAPlacIQ) (20) for the other
mutants. Cells were grown to an A595 of <0.4
and then induced with 1 mM
isopropyl-1-thio--D-galactopyranoside for 1 h.
Induced cells were collected by centrifugation, sonicated in buffer A
(20 mM Tris, pH 8.0, 400 mM NaCl, 10 mM imidazole), and bound to nickel-nitrilotriacetic
acid-agarose (Qiagen, Chatsworth, CA). After washing in buffer A,
proteins were eluted with the same buffer containing 220 mM
imidazole, dialyzed against buffer B (20 mM Tris, pH 8.0, 10 mM MgCl2, 2 mM EDTA, 1.4 mM
-mercaptoethanol, 5% glycerol, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride), and applied onto 5-ml DEAE
columns (Amersham Pharmacia Biotech). Protein was eluted with a 50-300
mM KCl gradient in buffer B, dialyzed against buffer B
containing 10% glycerol (storage buffer), and either frozen for long
term storage or used immediately. WT, K218T, and K620T proteins were
diluted to equal concentrations in the storage buffer to equalize
potential inhibition by glycerol and in most assays were diluted into
buffer at least 10-fold. In comparing the wider range of mutants, all
proteins (including WT, K218T, and K620T) were first dialyzed against
and concentrated in 20 mM HEPES, pH 7.5, 140 mM
KCl, 15 mM NaCl, 10 mM MgCl2, 2 mM dithiothreitol using Ultrafree-15 Centrifugal Filter
Devices with a nominal molecular mass limit of 30,000 Da (Millipore,
Bedford, MA). Concentrated proteins were diluted to equivalent
concentrations with the same buffer.
Determination of Protein Concentration--
Protein
concentration was determined using the method of Bradford (23) with
bovine serum albumin as a standard. Subsequently, an extinction
coefficient was calculated for Hsp104 (A276, = 31,900 M
1 cm
1) that did not
vary with native or denatured protein (24) and matched data from amino
acid analysis but yielded lower values than the Bradford assay which
overestimates Hsp104 protein concentration by a factor of ~2.
Remaining frozen stocks of some proteins were analyzed using this
extinction coefficient and the others extrapolated for the values given
herein. Previously published work on Hsp104 utilized the Bradford assay
standardized against bovine serum albumin, so values therein are
overestimated.
ATPase Assays-- General characterization of Hsp104 ATPase activity (Tables I-III and Fig. 1) was performed in reaction buffer 1 (40 mM Tris adjusted to pH 7.5 at 37 °C, 175 mM NaCl, 5 mM MgCl2, 0.02% Triton X-100, and 5 mM ATP). For measurement of the ATPase activity at different pH values, 40 mM sodium acetate buffer was used for pH 4.5-6.0, 40 mM MOPS for pH 6.5-7.0, 40 mM Tris for pH 7.0-9.0, and 40 mM glycine-NaOH buffer for pH 9.5-10.5. Hsp104 was at a concentration of 0.01 mg/ml with a 7-min end point for all general characterization assays.
To relate oligomerization to ATP hydrolysis, assays comparing different mutant proteins and all Km experiments were performed in the same buffer employed in oligomerization experiments, reaction buffer 2 (20 mM HEPES, pH 7.5, 140 mM KCl, 15 mM NaCl, 10 mM MgCl2). Km and Vmax were identical whether assayed in reaction buffer 1 or 2, but HEPES buffer was employed because Tris buffer inhibits glutaraldehyde cross-linking. All reactions contained 5 mM ATP, pH 7.5, except for Km experiments where ATP concentration was varied. The reaction buffer for Km experiments included 5 mM MgCl2, and ATP was resuspended with equimolar MgCl2. All nucleotides were adjusted to pH 7.5 prior to ATPase assays. Experiments using low ionic strength buffers were performed in reaction buffer 3 (10 mM Tris, pH 7.5, 5 mM MgCl2, 0.02% Triton X-100) or reaction buffer 2 minus salt. All assays were performed at 37 °C in a 25-µl reaction volume in siliconized Eppendorf tubes so that Triton X-100 (used in reaction buffer 1 to reduce loss of proteins on the walls of reaction tubes) could be eliminated; this detergent slightly stimulates the ATPase activity of Hsp104. Reactions were terminated and released Pi quantified by the addition of 800 µl of Malachite Green Reagent (0.034% Malachite Green, catalog number M9636, Sigma; 1.05% ammonium molybdate; 1 M HCl, filtered to remove insoluble material; Ref. 25). After 1 min at room temperature, color development was stopped by addition of 100 µl of 34% citric acid. 200 µl of the sample was removed to 96-well assay plates and A650 determined with a Molecular Devices (Palo Alto, CA) vmax kinetic microplate reader with SoftMaxTM software. Values were calibrated against KH2PO4 standards and corrected for phosphate released in the absence of Hsp104. For each point at least 3 and typically 6 independent assays were used to calculate the mean ± S.D. Curves were generated using least squares fitting to the Michaelis-Menten equation with the KaleidagraphTM graphics program (Synergy Software, Reading, PA). Km and Vmax were calculated using the Kaleidagraph program, and similar values were also obtained using Lineweaver-Burk and Eadie-Hofstee plots.Cross-linking Assays-- WT and mutant proteins dialyzed against reaction buffer 2 with 2 mM dithiothreitol were cleared of aggregated protein by centrifugation and adjusted to 0.01 mg/ml in reaction buffer 2 containing dithiothreitol (2 mM) with or without ATP (5 mM). 200-µl reactions were incubated at 37 °C for 10 min, glutaraldehyde added (final concentration 0.01%), and cross-linking terminated at 0 or 12 min with 1 M glycine as described (15). Reaction buffer 2 lacking KCl and NaCl was employed for cross-linking reactions at low ionic strengths.
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RESULTS |
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Characterization of Wild-type Hsp104 ATPase Activity-- First, we characterized the ATPase activity of WT Hsp104 using a colorimetric assay to measure phosphate released. ATP hydrolysis required divalent cations. Hydrolysis was supported by Mg2+, Mn2+, Ni2+, and Co2+ but not by Ca2+, which supports hydrolysis by ClpA and ClpB (3, 12) (Table I). Surprisingly, hydrolysis in the presence of 5 mM CoCl2 was 4-fold higher than with 5 mM MgCl2. Although the closest E. coli homolog of Hsp104, ClpB, is reported to hydrolyze other nucleotides (12) (Table II), Hsp104 did not hydrolyze other nucleotides at a significant rate (Table II). GTP, CTP, and UTP moderately inhibited the ATPase activity of Hsp104, whereas AMP inhibited it only slightly. ADP, however, inhibited the ATPase activity of Hsp104 strongly (Table III).
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ATP Hydrolysis by hsp104 Point Mutants--
To investigate the
relative contributions of each nucleotide-binding site to ATP
hydrolysis, we examined the effects of point mutations in
nucleotide-binding consensus residues (Fig.
3A). One matching set of
mutants, K218T and K620T, carried threonine substitutions in the
conserved lysine that interacts directly with the - and
-
phosphates of bound nucleotide and is important for the structure of
the P-loop in other nucleotide-binding proteins with Walker consensus
motifs (27). Separately, matching glycine to valine substitutions
(G217V,G619V) were generated in the conserved glycines immediately
preceding these lysines. All of these residues are absolutely conserved
in both the Walker A P-loop consensus ((G/A)X4GK(T/S)) (10, 27) and in the
more specific HSP100 family consensus
(GX2GXGKT) of both NBDs.
When proteins carrying the lysine to threonine substitutions were
expressed in yeast cells, they were no more susceptible to proteolysis
than WT Hsp104, suggesting the mutations caused no global perturbation
in structure (15). Furthermore, the circular dichroism spectra of both
the K218T and the K620T mutants were very similar to WT protein, also indicating no gross loss of structure in the mutants (data not shown).
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Oligomerization of hsp104 Point Mutants--
We examined the
oligomerization properties of one of these mutant pairs previously,
finding that a lysine to threonine substitution in NBD2 (K620T)
impaired the protein assembly into hexamers in response to ATP, whereas
the analogous substitution in NBD1 (K218T) had little effect (15).
Surprisingly, an analogous lysine to threonine substitution in the NBD1
of ClpA impaired the assembly of the proteins into hexamers in response
to ATP, whereas the analogous substitution in NBD2 had little effect
(19). Here, to directly relate oligomerization and ATP hydrolysis, we
compared the effects of the Lys Thr substitutions, as well as the
new Gly
Val substitutions, on oligomerization using the same buffer employed in ATPase assays. This buffer mimics physiological ionic strength (140 mM KCl and 15 mM NaCl). The G619V
substitution, like the K620T substitution, impaired oligomerization of
Hsp104; the G217V substitution, like the K218T substitution, did not
(Fig. 5). Thus, substitutions in highly
conserved nucleotide-binding consensus residues of the NBD2 of Hsp104
affect oligomer assembly more than the same substitutions in NBD1.
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Effects of Salt Concentration on the ATPase Activity of Hsp104-- We characterized the ATPase activity of Hsp104 using buffers that mimic physiological conditions; however, Maurizi and colleagues (14) reported that the ATPase activity of ClpA was inhibited by high salt concentrations, and most published work on other HSP100 proteins has used low ionic strength buffers. To compare the ATPase activity of Hsp104 with that of other HSP100 proteins and to determine if the different effects of the ClpA and the Hsp104 mutations are due to the use of different buffer conditions, we also examined ATPase activity and oligomerization properties of Hsp104 in low ionic strength buffers.
The salt content of buffers strongly influenced ATPase activity. As the ionic strength of the buffer increased, ATPase activity decreased (Fig. 6A). In separate experiments the Km of Hsp104 was tested in buffer lacking added NaCl and KCl. The Km measured by ATP hydrolysis dropped to 0.6 mM in this low salt buffer, compared with a Km of ~5 mM in the physiological salt buffer (Fig. 7). The Vmax was unaffected.
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DISCUSSION |
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We have provided the first characterization of the ATPase activity of WT Hsp104 from S. cerevisiae and the effects of environmental conditions related to its biological function on that activity. We have also shown that mutations in conserved residues of the first nucleotide-binding domain of Hsp104 (NBD1) severely reduce the ATPase activity of Hsp104 but do not inhibit oligomerization, whereas analogous mutations in the second nucleotide-binding domain (NBD2) have a modest effect on ATP hydrolysis but inhibit Hsp104 oligomerization. Although the oligomerization properties and ATPase activity of WT Hsp104 are dramatically affected by differences in the ionic strength of buffers, the distinct effects of NBD1 and NBD2 mutations are retained.
Because the nucleotide-binding domains of Hsp104 are essential for its function in stress tolerance (17), we also examined the effects of parameters that vary with stress on ATPase activity of Hsp104. In physiological buffers, the ATPase activity of Hsp104 peaks at about 45 °C and is greatly reduced at 55 °C. It is inhibited by ethanol, very severely at 20%. Strikingly, it is in cells exposed to such conditions that Hsp104 is most critical for survival. The expression of Hsp104 increases survival in cells exposed to 44 °C by 10-fold but increases survival in cells exposed to 55 °C by 1000-fold (28). A similar relationship holds for ethanol (6). This result provides biochemical support for an earlier suggestion, based on very different types of evidence, that the key function of Hsp104 is to repair damage after stress, rather than to prevent damage during stress (7, 8). However, we also find that in low salt buffers, Hsp104 retains ATPase activity at higher temperatures and higher concentrations of ethanol. Thus, it remains possible that as yet undiscovered in vivo factors mimic this effect and allow Hsp104 to function even during severe stress.
The effects of pH, ATP concentrations, and ATP/ADP ratios may also be relevant to the biological function of Hsp104. ATP hydrolyzing activity is high at pH 6.5 and low at pH 7.5. 31P NMR measurements of chemical shifts in inorganic and sugar phosphates indicate that the cytoplasmic pH of yeast cells decreases from 7.5 at 23 °C to 7.0 at 37 °C and subsequently to 6.2 at 45 °C (26). Such changes in pH may increase the activity of Hsp104 during or immediately after stress and inhibit it again when normal physiology is restored. The Km for ATP hydrolysis by Hsp104 is ~5 mM, close to the normal physiological concentration of ATP (~4 mM; Refs. 29 and 30). The nucleotide hydrolyzing activity of Hsp104 is highly specific for ATP and is strongly inhibited by ADP. Thus, stress-induced fluctuations in ATP concentrations and ATP/ADP ratios may also regulate the activity of Hsp104 in vivo.
The Km of Hsp104 at physiological ionic strength is 20-fold higher than the Km reported for ClpA (Table IV). However, the buffers employed for Km measurements of ClpA did not contain NaCl or KCl (3). When the Km of Hsp104 was measured in a low ionic strength buffer, it was reduced 10-fold. Thus, under similar conditions, these different HSP100 proteins have very similar Km values. The Vmax of Hsp104 was not much affected by ionic strength and is similar to values reported for ClpA. The similarities in the ATPase activities of these two proteins underscore other, previously noted similarities as follows: (a) both form hexameric complexes in the presence of ATP (15, 16, 18, 19); (b) both promote the disassembly of protein aggregates or oligomers (4, 7); (c) both contain two nucleotide-binding domains that have little sequence homology with each other but are highly related to the corresponding domains of the other protein (1, 9).
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Given the similarities between ClpA and Hsp104, it is surprising that analogous mutations in their nucleotide-binding domains have such different effects on their most basic biochemical characteristics, oligomerization and ATP hydrolysis. In Hsp104, a lysine to threonine mutation in the P-loop of NBD1 (K218T) inhibits ATP hydrolysis more severely than the equivalent mutation in NBD2 (K620T), whereas the mutation in NBD2 inhibits oligomerization more severely than the equivalent mutation in NBD1. In ClpA the opposite relationship was reported; a lysine to threonine P-loop substitution in NBD1 most strongly affects oligomerization, whereas the equivalent mutation in NBD2 more strongly affects ATP hydrolysis (19).
We investigated this apparent contradiction in three ways. 1) Because site-directed mutagenesis might have introduced extraneous mutations, we recreated the K218T and K620T mutations in a manner that ensured that no extraneous mutations were present (see "Experimental Procedures," HSP104R). It seems unlikely that extraneous mutations in the ClpA proteins caused this discrepancy, as multiple mutants were tested in two different laboratories (18, 19). 2) We tested additional mutations in the NBDs, specifically, equivalent glycine to valine substitutions in the P-loop consensus sequences of each NBD. These mutants behaved similarly to our original lysine to threonine substitutions. 3) We used both low salt buffers and physiological ionic strength buffers for ATPase and oligomerization assays. (ClpA oligomerization assays had been performed in buffers containing 100-300 mM NaCl or KCl, whereas ATPase assays were performed in the absence of added salt (18, 19).) Although the ionic strength of the buffer affected both the oligomerization properties and ATPase activity of Hsp104, the NBD2 mutations still exerted stronger effects on the oligomerization of Hsp104, and the NBD1 mutations still exerted stronger effects on ATP hydrolysis.
One explanation for the different effects of equivalent mutations in ClpA and Hsp104 is that the two domains have independent functions, one responsible for ATP hydrolysis and the other for oligomerization, and that these functions have switched in the two proteins during the course of evolution. This switch might be due to the influence of specific amino acid substitutions in the nucleotide-binding domains or to the influence of flanking domains, which are highly divergent in different HSP100 subtypes. A precedent exists for similar nucleotide-binding domains evolving such diverse functions in N-ethylmaleimide-sensitive fusion protein, a protein that regulates vesicle fusion. N-ethylmaleimide-sensitive fusion protein has two NBDs that are similar to each other in sequence, yet nucleotide binding at one site is required for oligomerization, whereas ATPase activity at the other site is required for function. Furthermore, the order of the domains can be switched without altering their functions (34).
An alternative explanation is that both domains contribute to ATP hydrolysis and oligomerization in a complex, interdependent manner and that other differences between the two proteins idiosyncratically cause analogous mutations to perturb one of these functions more than the other. At least three lines of evidence support this notion. First, in both ClpA (18) and Hsp104,5 oligomerization increases ATPase activity. Second, class 2 HSP100 proteins have only one NBD, which more closely resembles the NBD2 of class 1 proteins, yet mutational analysis indicates that it functions both in oligomerization and ATP hydrolysis (35). Third, oligomerization occurs at very low nucleotide concentrations (µM ATP or ADP)6 such that in vivo nucleotide concentrations (mM ATP or ADP) should always be sufficient to maintain WT protein in an oligomerization-competent state. Thus, it seems likely that nucleotide binding (and perhaps hydrolysis) at the second site serves some other purpose beyond simply tethering the subunits together.
Understanding the reaction cycle utilized by Hsp104 will require a detailed understanding of the interplay between these sites. The multimeric nature of the protein, the possibility that both NBDs may contribute at different levels to ATP hydrolysis and oligomerization, the effects of environmental conditions on its behavior, and the as yet poorly explored effects of partner proteins and substrates make this a challenging problem. Results presented here provide the first step.
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ACKNOWLEDGEMENTS |
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We thank Edward Taylor, John Westley, Anil Cashikar, and John Glover for helpful discussions. We thank Joachim Clos for the kind gift of pJC45 vector and Olivier Fayet for strain BL21[DE3](pAPlacIQ). We thank Lisa Rosman, Laura Loftus, and Danielle Ware for technical assistance.
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FOOTNOTES |
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* This work was supported in part by Department of Energy Grant FG02-95ER20207.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by USPHS Grant 6 T32 GM07183-19 from the National Institutes of Health.
¶ Supported by Boehringer Ingelheim Fonds. This work has been presented as part of the requirements for a doctoral thesis.
** To whom correspondence should be addressed: 5841 South Maryland Ave., MC1028, Rm. AMB N339, Chicago, IL 60637. Tel.: 773-702-8049; Fax: 773-702-7254; E-mail: s-lindquist{at}uchicago.edu.
1 J. R. Glover and S. Lindquist, manuscript in preparation.
2 The abbreviations used are: NBD, nucleotide-binding domain; WT, wild type; MOPS, 4-morpholinepropanesulfonic acid.
3 E. C. Schirmer and S. Lindquist, unpublished results.
4 D. A. Parsell and S. Lindquist, unpublished observations.
5 E. C. Schirmer, C. Queitsch, A. S. Kowal, and S. Lindquist, manuscript in preparation.
6 A. S. Kowal, E. C. Schirmer, and S. Lindquist, unpublished observations.
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REFERENCES |
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