OF CHEMISTRY Vol. No. of May for USA. hsp70-Protein Complexes · THE JOURNAL OF BIOL~~ICAL...

THE JOURNAL OF BIOL~~ICAL CHEMISTRY 0 1994 by The American Society for Biochemistry ’ and Molecular ’ Biology, Inc.

Vol. 269, No. 18, Issue of May 6, pp. 13107-13114, 1994 Printed in U S A .

hsp70-Protein Complexes COMPLEX STABILITY AND CONFORMATION OF BOUND SUBSTRATE PROTEIN*

(Received for publication, December 17, 1993, and in revised form, March 1, 1994)

Daniel R. Palleros, Li Shi, Katherine L. Reid, and Anthony L. Fink* From the Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064

The presence of bound substrate protein increases the thermostability of hsp70 molecular chaperones (heat shock proteins of molecular mass 70 ma). Complexes between hsp70 and unfolded substrate proteins were isolated by size-exclusion high performance liquid chromatography. The isolated complexes were observed to dissociate at a significant rate even in the absence of ATP. The presence of ADP caused a substantial increase in the stability of the complex. Both ADP and inorganic phosphate were found to inhibit the ATP-induced dissociation of complex. ADP was also observed to increase both the rate of complex formation and its stability as a function of temperature, suggesting an important regulatory role for nucleotides during heat shock. Circular dichroism and fluorescence studies of the complex between DnaK and a thermally unstable mutant of staphylococcal nuclease indicate that the bound substrate protein is significantly unfolded. A model for the hsp70 cycle of complex formation and dissociation, which accounts for the regulatory role of nucleotides, is proposed.

Cells respond to most environmental insults by overproduc- ing a group of proteins referred to as stress or heat shock proteins (hsp).’ The family of hsp70, proteins of molecular mass 70 kDa, has been evolutionarily conserved and is present in prokaryotes, yeast, and higher eukaryotes (for reviews see McKay, 1993; Hendrick and Hartl, 1993; Gething and Sam- brook, 1992). Although there is only one member of the hsp7O family in Escherichia coli, namely DnaK, constitutive and inducible forms exist in eukaryotes (Hightower and White, 1981). Among the best studied hsp7O are bovine brain hsp73, also known as hsc70, a constitutive cytosolic form of hsp70 (Welch and Feramisco, 1985); human hep72, a highly inducible cytosolic homologue of hsp73 (Brown et al., 1993); BiP or grp78 (for glucose regulated protein of molecular mass 78 m a ) , present in the endoplasmic reticulum (Munro and Pelham, 1986) and DnaK (Zylicz et al., 1983). hsp7O also belongs to a group of proteins involved in protein folding, assembly, and transloca- tion in the cell known as molecular chaperones (reviewed by Ellis and van der Vies, 1991 and Hendrick and Hartl, 1993).

* This work was supported by National Institutes of Health Grant GM45316. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. Tel.: 408-459-2744;

The abbreviations used are: hsp, heat shock protein(s); HPLC, high performance liquid chromatography; NCA-SNase, staphylococcal nucle-

has been substituted for the tetrapeptide Tyr-Lys-Gly-Gln at positions ase A mutant where the pentapeptide sequence Ser-Gly-Asn-Gly-Ser

27-30; PAGE, polyacrylamide gel electrophoresis; RCMLA, reduced carboxymethylated a-lactalbumin; SEC, size-exclusion chromatography.

Fax: 408-459-2935.

hsp7O family members consist of two functional domains: a highly conserved N-terminal domain (44 kDa) which contains a nucleotide binding site and a less conserved C-terminal domain involved in binding to substrate (unfolded) proteins. The three- dimensional structure of the N-terminal fragment of bovine brain hsp73 is known at 2.2-A resolution (Flaherty et al., 1990). hsp7O has been shown to interact with unfolded proteins (Pal- leros et al . , 1991, 1992, 1993a; Liberek et al., 1991b; Bole et al . , 1986; Gething et al., 19861, nascent polypeptide chains (Dorner et al., 1987; Beckmann et al., 19901, and short peptides (Flynn et al., 1989, 1991; Lam and Caldenvood, 1992). hsp70 binds tightly to ATP and ADP (Welch and Feramisco, 1985; Schmid et al., 1985; Palleros et al., 1991; Gao et al., 1993) and has weak endogenous ATPase activity (McCarty and Walker, 1991; Sadis and Hightower, 1992; Blond-Elguindi et al., 1993; Palleros et al . , 1993~) which is typically increased 3-5-fold by the presence of peptides and unfolded proteins (Flynn et al . , 1989; Sadis and Hightower, 1992; Blond-Elguindi et al . , 1993; Palleros et al., 1993a). Binding to ATP causes a conformational change in DnaK, probably related to substrate-protein dissociation (Pal- leros et al., 1992,1993a). I t has recently been shown in in vitro experiments that DnaK in conjunction with other hsp, namely DnaJ, GrpE, and GroEL, assists the refolding of rhodanese (Langer et al., 1992).

Despite the wealth of knowledge on hsp function, little is known about the mechanisms of interaction between hsp70 and unfolded proteins; in particular, the link between protein- stimulated hsp70 ATPase activity and hsp70-protein interaction is not yet understood on a molecular level. From NMR experiments it has been shown that a peptide composed of 13 residues is in an extended conformation when bound to DnaK and in a helical conformation when bound to GroEL (Landry et al . , 1992). Circular dichroism (CD) studies suggest that there is a conformational change in hsp73 upon binding to a decapep- tide (Park et al . , 1993). We previously reported that binding of reduced carboxymethylated a-lactalbumin, RCMLA, to hsp73 is accompanied by a conformational change detectable by far-UV CD (Palleros et al . , 1991). In this paper we investigate the interaction of hsp70 family members (human hsp72, DnaK from E. coli) with two classes of unfolded proteins: a permanently unfolded protein, RCMLA, and a “foldable” protein, namely NCA-SNase, a thermally unstable mutant of staphylococcal nuclease which has the advantage that it can be bound to hsp7O a t a temperature at which it is unfolded (e.g. 37 “C) and released from the complex at a lower temperature (e.g. 10 “C) where it is native (Hynes et al., 1989; Antonino et al., 1991; Eftink, 1991). A mechanism that relates hsp70-protein interaction and ATP hydrolysis is proposed.

EXPERIMENTAL PROCEDURES Materials-E. coli DnaK was prepared as described previously (Pal-

leros et al., 1993~); human hsp72 from 293 cells was obtained as described elsewhere (Brown et al., 1993); all other chemicals were from the same sources as reported previously (Palleros et al., 1991, 1992).

13107

13108 Protein-hsp70 Complexes Mg-ATP refers to an equimolar solution of MgCI, and ATP (disodium salt) in the specified buffer; Mg-ADP refers to an equimolar solution of MgCI, and ADP (monosodium salt) in the specified buffer.

Methods-Far-W CD, fluorescence spectroscopy, SEC-HPLC experiments were performed as described previously (Palleros et al., 1992, 1993a). A 1-mm path length cuvette was used for the CD experiments; a 1-ml cuvette was used for fluorescence experiments. Unless otherwise stated, SEC-HPLC was run on a Bio-Si1 SEC-250 silica column (600 x 7.8 mm; Bio-Rad) using 20 m~ sodium phosphate, 200 m~ KC], pH 6.5, as a mobile phase; a Bio-SEP 3000 silica column (600 x 7.8 mm; Phe- nomenex, Torrance, CA) was used to determine Stokes radii; flow rate was 1 mumin; detection was by absorbance at 215 nm. SDS-PAGE were run on a Pharmacia Phast system; densitometry was performed as

RCMLA and DnaK. described previously (Palleros et al., 1993c) using standard curves for

Interaction between NCA-SNase and D m K followed by Fluorescence (Fig. 2)-DnaK stock solution (40 pl, 43.9 in 100 m~ Tris-HC1,5 m~ P-mercaptoethanol, pH 7.2) was mixed with NCA-SNase stock solution (30 pl, 116 p~ in 100 m~ Tris-HC1, pH 7.8), KC1 stock solution (60 pl, 3 M in 100 mM Tris-HC1, pH 7.8), and 670 pl of 100 m~ Tris-HC1, pH 7.8. The mixture was equilibrated at 4 "C for 30 min and the spectrum recorded. The temperature was increased to 37 "C over a period of 1 h; the mixture was equilibrated at that temperature for about 30 min. The temperature was decreased to 4 "C over a period of 1 h, the solution was allowed to equilibrate for 30 min, and the spectrum was recorded. Mg-ATP solution was added (40 pl, 20 m~ Mg-ATP in 100 m~ Tris-HC1, pH 7.2), the change in fluorescence signal a t 330 nm followed as a function of time (Fig. 5) , and the spectrum recorded after equilibrium was attained; the spectrum was corrected to compensate for the dilution introduced by Mg-ATP.

Interaction between NCA-SNase and DnaK followed by CD (Fig. 3)-An aliquot (60 pl) of DnaK stock solution (39.3 p~ in 100 m~ Tris- HC1, 10% glycerol, pH 7.5) was mixed with 5.8 pl of NCA-SNase stock solution (509.7 p in 20 m~ Tris-HC1, pH 7.1) and 404.2 pl of 20 m~ Tris-HC1 pH 7.1; this mixture was divided into two portions. The first portion (230 111) was placed into a 250-pl cuvette (1-mm path length), equilibrated at 10 "C for 20 min, and then the far-W CD recorded; the cuvette was removed from the CD instrument, incubated at 37 "C for 30 min, placed back into the cuvette compartment at 10 "C, and equilibrated for 20 min before the spectrum was recorded; 30 pl of Mg-ATP stock solution (7.5 m~ MgCl,, 7.5 m~ Na&TP, 0.71 M KC], 20 m~ Tris-HC1, pH 7.1) were added, mixed, equilibrated for 20 min and the spectrum scanned. The second portion (230 p1) was mixed with 30 pl of the Mg-ATP stock solution, equilibrated at 10 "C for 20 min, and the spectrum recorded. Each spectrum was the average of five scans with averaging time at each wavelength of 1 s. In each case, the equilibration time was enough to ensure that a constant ellipticity value was reached.

RESULTS Effect of Substrate Protein on the Thermal Stability of

hsp70-Fig. L4 shows the thermal unfolding of DnaK, RCMLA (a permanently unfolded protein), and the DnaK.RCMIA complex, followed by far-UV CD. The DnaK.RCMLA complex was formed by incubating DnaK with a molar excess of RCMLA at 37 "C for 40 min. Before the unfolding was studied, the mixture was equilibrated at 20 "C for 10 min; under these conditions more than 70% of DnaK is conjugated with RCMLA as determined by SEC-HPLC (not shown). The stoichiometry of the DnaK.RCMLA complex (1:l) was determined by isolation of the complex by HPLC, concentration using Centricon 3, and densitometry of SDS-PAGE with Coomassie Blue staining (Palle- ros et al., 1993a, 1993~). Fig. L4 indicates that although there are no detectable changes in 0,,, for RCMLA as the temperature is increased, DnaK unfolding with and without RCMLA proceeds in two steps; in the absence of RCMLA the midpoint of the first transition is T,,, = 40.5 "C; the second transition has a midpoint of 72 "C; these values are in agreement with previously published data and reflect the formation of a molten globule-like intermediate in the first transition (Palleros et al., 1992). In the thermal unfolding of the DnaK.RCMLA complex an increase in the midpoint of the first transition was observed (T, = 49.0 "C), but no significant change in the second transition midpoint was detected (T , = 71 "C). The difference in the

2 5

2 0

1 5

1 0

5

0

2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 Temperature ("C)

0 1 0 2 0 3 0 . 4 0 5 0 6 0 7 0 Temperature ("C)

FIG. 1. Thermal stability of DnaK and its complexes followed by CD. Change in ellipticity at 222 nm as a function of temperature. A (top): trace a , [DnaKI = 4.9 p ~ ; trace b, [RCMLA] = 25 p; trace c, [DnaKl = 5.1 p; [RCMLA] = 31.6 p.t; the mixture was incubated at 37 "C for 40 min and equilibrated at 20 "C for 10 min, before the unfolding was studied; ellipticities (222 n m ) at 20 "C for traces a, 6 , and c were: -45.9, -18.1, and -73.0 millidegrees, respectively. B (bottom): trace a , [DnaKl = 4.9 p a ; trace b, [NCA-SNasel = 6.3 p; trace c, [DnaKl = 5.0 pa; [NCA-SNasel = 6.3 p; the mixture was incubated at 37 "C for 1 h, equilibrated at 2 "C for 1 h, and then the unfolding studied; ellipticity (222 n m ) was: trace a , -45.9 millidegrees (20 "C); trace b, -5.7 millidegrees (2 "C); and trace c, -65.4 millidegrees (2 "C). In all cases temperature was increased at a rate of 0.33 "Chin; data were collected every 60 s with a time constant of 5 s. Solutions were in 18 m~ Tris-HC1, 45 m~ KC], pH 7.2

T,,, of the first transition translates into AAG = 1.9 kcallmol, calculated according to the method of Becktel and Schellman (1987). The effect of Mg-ADP on the T,,, of DnaK was also investigated in the presence of RCMLA. We previously reported an increase in the midpoint of the first transition of DnaK (to T, = 59 "C) caused by an excess of Mg-ADP in the absence of RCMLA (Palleros et al., 1992); the addition of RCMLA has no effect on the first transition when Mg-ADP is present (T , = 59 "C; data not shown).

The thermal unfolding of the complex between DnaK and a foldable protein, NCA-SNase, was studied by CD. NCA-SNase, a thermally unstable protein, is known to form complexes with hsp70 family members (Palleros et al., 1991, 1993a); this protein is unfolded at 37 "C and refolds to the native state upon lowering the temperature; its thermal unfolding is shown in Fig. lB (trace b) . As can be observed, this staphylococcal nuclease mutant shows a transition midpoint centered at 30 "C, which agrees with previously published results (Antonino et al., 1991; Eftink et al., 1991). The complex was formed by incubating a mixture of DnaK with a slight molar excess of NCA-SNase at 37 "C for 60 min. Before the thermal unfolding was studied, the mixture was equilibrated at 2 "C for 1 h to allow refolding of free NCA-SNase (no change in ellipticity at 222 nm was observed after 15 min). The thermal stability of the DnaK.NCA-SNase complex is shown in Fig. 1B (trace c ) . Two transitions were observed, one with T,,, = 26 "C, corresponding

Protein-hsp70 Complexes 13109

2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 Wavelength (nm)

FIG. 2. Interaction between NCA-SNase and DnaK followed by fluorescence. Fluorescence spectrum of NCA-SNase + DnaK trace a, at 4 "C (both proteins native); trace b, at 37 "C (DnaK native, NCA- SNase unfolded); trace c, at 4 "C after incubation at 37 "C for 30 min (complex between NCA-SNase and DnaK formed); trace d, same as trace c + Mg-ATP equilibrated for 30 min (intensity corrected for dilution; ATP quenches DnaK and NCA-SNase fluorescence). For a-c: [DnaKl = 2.2 w; INCA-SNasel = 4.4 w; [KC11 = 220 m. For trace d: [DnaK] = 2.1 w; [NCA-SNasel = 4.2 w; [KC11 = 210 m; [Mg-ATP] = 1 m. All stock solutions were in 100 II~M Tris-HC1, final pH 7.4. Excitation was at 280 nm; excitation slit, 4 nm; and emission slit, 10 nm.

to NCA-SNase in the complex, and the other with T,,, = 49.5 "C, corresponding to DnaK in the complex. The difference in the transition midpoints for the unfolding of free DnaK (T, = 40.5 "C) and the preformed complex (T,,, = 49.5 "C) corresponds to a AAG = 2 kcal/mol. The thermal unfolding of a mixture of NCA-SNase and DnaK in the presence of an excess of Mg-ADP (1 111~) was investigated in the range 10-70 "C; it showed a noncooperative transition between 10 and 40 "C and a cooperative transition with T,,, of 57 "C (data not shown).

Conformation of the Bound Substrate Protein-The conformation of NCA-SNase when bound to DnaK was investigated by fluorescence and CD. An excess of NCA-SNase was mixed with DnaK at low temperatures (4 or 10 "C), and the fluorescence and CD spectra were collected (Fig. 2, truce a, and Fig. 3, truce a , respectively); under these conditions both proteins are in their native states' (Palleros et ul., 1992; Eftink, 1991) and they do not form a complex (Palleros et al., 1991); the temperature was raised to 37 "C and maintained at this value for 30 min to allow complex formation (Palleros et ul., 1991); under these conditions free DnaK is in its native state (Palleros et al., 1992) and free NCA-SNase is denatured (Antonino et al., 1991; Eftink, 1991); the spectra were obtained (Fig. 2, truce b, and Fig. 3, truce b) , and the temperature was lowered (to 4 or 10 "C); the system was allowed to equilibrate until no change was detected in the fluorescence or CD signal, and the spectra were collected (Fig. 2, truce c , and Fig. 3, truce c ) . As is shown in the figures, there is a significant difference in the spectra at low temperature (4 or 10 "C) obtained before and after incubation at 37 "C (compare truce u with truce c in Figs. 2 and 3); the difference fluorescence and CD spectra are shown in Fig. 4, A and B, respectively. In contrast to the results obtained with NCA-SNase, the CD spectra of a mixture of RCMLA (13 w) and DnaK (10 w) obtained at 10 "C before and after incubation at 37 "C for 30 min were completely superimposable (data not shown). For comparison, Fig. 4A also shows the fluorescence difference spectrum of NCA-SNase from spectra obtained at 4 and 40 "C; before subtraction of the spectrum at 40 "C, this was multiplied by a factor of 1.5 to normalize the fluorescence intensity to 4 "C; this correction factor accounts for the temperature effect on fluorescence intensity and was obtained from the

* Under the conditions of this study, Tris-HC1 buffer, pH 7.2, at 22 "C, NCA-SNase does not show cold denaturation below 18 "C as has been reported (Antonino et al., 1991).

1 " " " " ' " " ' " " ' " " " " " J

2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 8 0 Wavelength (nm)

FIG. 3. Interaction between NCA-SNase and DnaK followed by falcW CD. Far-UV CD spectrum of NCA-SNase + DnaK trace a, at 10 "C; trace 6, a t 37 "C; trace c, at 10 "C after incubation at 37 "C for 30 min; INCA-SNasel = 6.3 PM, [DnaKl = 5.0 w. See "Experimental Pro- cedures'' for details.

5 -

4 -

3 -

2 -

1 -

0 -

2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 Wavelength (nm)

200 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 Wavelength (nm)

FIG. 4. Difference spectra for DnaKaNCA-SNase complex. A (top): trace a , difference fluorescence spectrum of those shown in Fig. 2,

of the unfolded (40 "C, extrapolated at 4 "C) and native states (4 "C) of traces a and c; trace b, difference fluorescence spectrum between those

NCA-SNase; [NCA-SNasel = 1 in 100 r m Tris-HC1, pH 7.4; excitation was at 280 n m ; excitation and emission slits were 4 and 10 nm, respectively. B (bottom), difference far-UV CD spectrum at 10 "C of NCA-SNaseDnaK mixtures before and after incubation at 37 "C. Fig. 3, trace c, was subtracted from trace a.

unfolding curve of NCA-SNase followed by fluorescence intensity at 330 nm by extrapolating the base line for the unfolded state (>40 "C) to 4 "C.

Stokes radii, R,, were determined by SEC-HPLC as described previously (Palleros et al., 1993b) using a Phenomenex Bio-SEP 3000 column. A plot of the partition coefficient, Kd (Palleros et ul., 1993b), versus 108, for standard proteins gave a good linear correlation (Kd = 0.979-0.466 logR,; r = 0.985). This equation w?s used to estimate the following Rs values (in A; error: *2 A): DnaK, 41; DnaK.RCMLA complex, 51; DnaK.NCA-SNase, 51; RCMLA, 27 and NCA-SNase, 22.

Mg-ATP was added to the DnaK.NCA-SNase complex to in- duce its dissociation (Palleros et al., 1993a), and the change in fluorescence intensity at 330 followed as a function of time (Fig.

13110 Protein-hsp70 Complexes

0 5 1 0 1 5 2 0 2 5 3 0 time (min)

FIG. 5. ATP-induced dissociation of DnaK-NCA-SNase complex followed by fluorescence. Increase in fluorescence intensity a t 330 nm as a function of time after addition of Mg-ATP to a solution of NCA-SNase and DnaK a t 4 "C previously incubated at 37 "C for 30 min. See Fig. 2 for experimental details.

5); the observed first order rate constant associated with the fluorescence intensity increase was (4.21 0.05) x s-'. Attempts to follow changes in the ellipticity at 222 nm as a function of time after addition of Mg-ATP were unsuccessful due to the rapidity of the process; most of the CD signal was recovered within the dead time after addition of Mg-ATP, indicating that secondary structure was attained faster than ter- tiary structure. After equilibrium was reached, the fluorescence (Fig. 2, truce d ) and CD spectra (not shown) were taken; they coincided with the spectra of mixtures subjected to identical treatment except that the incubation step at 37 "C had been omitted (data not shown).

Effect of ADP on Complex Formation and Dissociation-The effect of temperature and Mg-ADP on complex formation between DnaK and RCMLA was studied by SEC-HPLC (Fig. 6). Conditions were chosen so that less than 100% complex was formed (incubation at 37 "C for 15 min). As can be seen, the amount of complex formed is accelerated by Mg-ADP; in the absence of nucleotide the maximum amount of complex is observed around 40 "C; above this temperature there is a marked drop in the amount of complex detected. In the presence of an excess of Mg-ADP the maximum rate of complex formation occurs over a wider temperature range (3545 "C). The kinetics of complex formation between DnaK and RCMLA at 37 "C (data not shown) was followed by SEC-HPLC; equilibrium was reached after about 1 h of incubation, corresponding to 77% of DnaK in complex. The dissociation constant can be estimated as Kd < 8 p. This figure should be considered an upper limit since during HPLC analysis the complex partially dissociates due to mass action; also, the dilution introduced by the HPLC analysis will cause the complex to dissociate; if this effect is taken into consideration by assuming a dilution factor of 1/40 (this is a conservative number based on the dilution of 25 p1 of injected sample into a 1-ml band at the end of the run), the upper limit to the dissociation constant can be estimated as Kd

Dissociation of the hsp72.RCMLA complex was studied by SEC-HPLC (see Fig. 7) as a function of time. hsp72.RCMLA complex was formed at 37 "C (with or without Mg-ADP present), isolated by SEC-HPLC, incubated at 22 "C, and reinjected at intervals into the HPLC equilibrated at 22 "C with or without Mg-ADP in the mobile phase. It was observed that the complex dissociated as a function of time; the dissociation process was slowed down by lowering the incubation temperature from 22 to 4 "C (data not shown) and by the presence of Mg- ADP; similar results were obtained with the DnaK.RCMLA complex (data not shown). The hsp72.RCMLA complex dissociation constants with and without Mg-ADP can be estimated

< 0.2 p i .

1 0 2 0 3 0 4 0 5 0 6 0 7 0 Temperature ( O C )

FIG. 6. Effect of Mg-ADP and temperature on DnaK.RCMLA complex formation. a, DnaK (1.4 p d and RCMLA (18 p ~ ) were incubated in 18 m~ Tris-HC1, pH 7.2, for 15 min at the temperatures indicated in the figure; 6, DnaK (1.4 p), RCMLA (18 p f ) , and ADP (1 m) were incubated for 15 min at the indicated temperatures in the same buffer as in a + 1 m~ MgCl, and 1 m~ sodium phosphate. Analysis was by SEC-HPLC. Percent complex represent the fraction of DnaK in the complex, based on peak areas.

' ~ ' ' l ' ' ' ' " ~ "

ADP in mobile phase

0 - A 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0

time (mln.) FIG. 7. hsp72.RCMLA complex dissociation followed by SEC-

HPLC. a , hsp72 (8.8 p ~ ) was incubated with RCMLA (37 p) in 8 m~ sodium phosphate, 80 m~ KCl, 10 m~ Tris-HC1, 10 m~ NaCl, 50 pf EDTA, 2.5 m~ p-mercaptoethanol, 2.5 m MgCl,, pH 7, for 35 min at 37 "C and injected (100 pl) into the HPLC. The fraction corresponding to the complex was collected (-1 ml) kept a t 22 "C and reinjected (100 pl) at different time intervals as indicated in the figure; time was computed from the moment the peak of complex first eluted. Percent complex represents the fraction of hsp72 in the complex, based on peak areas; b, same as above except that DnaK and RCMLA were incubated in the presence of 1 m~ Mg-ADP in the same buffer; HPLC analysis was

ADP, pH 6.5. performed using 20 m~ sodium phosphate, 200 m KCI, 100 p~ Mg-

from the data presented in Fig. 7. Considering that only 70% of hsp72 was recovered in the complex after the first HPLC injection and assuming that the amount of hsp72 in the complexes was 60 and 10% after equilibrium was reached (200 min, Fig. 7), with and without Mg-ADP, respectively, the following dissociation constants are estimated: X, = 0.5 p~ (no Mg-ADP) and Kd = 0.02 p~ (Mg-ADP); an HPLC dilution factor of 1/10 was used, since 100-pl aliquots were injected and the complex eluted in an approximately 1-ml peak.

The effect of ADP and phosphate on complex dissociation triggered by Mg-ATP was studied by SEC-HPLC as a function of ADP and phosphate concentrations (Figs. 8 and 9). The complex between DnaK and RCMLA was formed by incubation at 37 "C for 1 h, Mg-ADP or sodium phosphate were added a t various concentrations, the mixtures were allowed to equilibrate for 10 min at room temperature, Mg-ATP (100 p i ) was added, and the mixtures were immediately analyzed by SEC- HPLC. In the absence ofADP, less than 10% of DnaK remained complexed with RCMLA after addition of Mg-ATP; as the concentration of ADP increases so does the fraction of complex: at [Mg-ADP] = 180 PM, about 70% of DnaK remains in the complex, similar to the percent of complex detected after the same


RCYLA

NucIaollda complex

e. DnaK + RCMLA

d. DnaK + RCMLA +ADP

1 0 1 5 2 0 2 5 Volume (ml)

FIG. 8. Effect of ADP on ATP-induced DnaK.RCMLA complex dissociation followed by SEC-HPLC. DnaK or DnaK + RCMLA was incubated at 37 "C for 1 h, then Mg-ADP was added (traces a, c , and d ) ,

min before HPLC injection; for traces a, b, and c, Mg-ATP was added, and the mixture was allowed to equilibrate at room temperature for 10

concentrations were: DnaK, 2.7 p ~ , RCMLA, 30.8 p ~ , Mg-ADP, 175 p ~ , and the samples were injected after 1 min at room temperature. Final

Mg-ATP, 100 p ~ . All nucleotide stock solutions were in 100 nm Tris-HC1, pH 7.5; DnaK stock solution (27 p ~ ) was in 25 rn Hepes-KOH, 50 nm KCI, 10% glycerol, 1 m~ P-mercaptoethanol, pH 7.4; RCMLA stock solution (308 p ~ ) was in HC1, pH 2.

incubation period in the absence of nucleotides (Fig. 8, c and e) . The addition of phosphate after the complex was formed has little effect on complex dissociation upon addition of Mg-ATP (Fig. 9c); however, if phosphate is present during the initial incubation of DnaK and RCMLA, a higher percent of complex remains after addition of Mg-ATP (40% at [phosphate] = 50 mM, Fig. 9b).

DISCUSSION

Effect of Substrate Protein on the Thermal Stability of hsp70-As indicated in Fig. 1, A and B , the formation of complexes between DnaK and substrate proteins results in an increase in the thermal stability of hsp7O as reflected by a difference of about 8-9 "C in the transition midpoints, from 40.5 "C in the absence of substrate protein to 49.0 and 49.5 "C with RCMLA and NCA-SNase, respectively; RCMLA does not show any significant thermal transition in this temperature range, in agreement with its unfolded nature (Ikeguchi and Sugai, 1989). No change in the midpoint of the final transition (T, = 71 2 1 "C) was observed due to complex formation, indicating that the complexes with substrate proteins dissociate before this transition; this is in agreement with results presented in Fig. 6 as discussed later.

We have previously shown that the first thermal unfolding transition of hsp7O is most likely due to the formation of a molten globule (Palleros et al., 1992); this is also in agreement with our recent findings that the guanidine-induced unfolding of DnaK proceeds via the formation of a molten globule-like intermediate (Palleros et al., 1993b). It has been proposed recently, based on calorimetric determinations, that the first thermal unfolding transition of DnaK is due to the unfolding of the N-terminal domain of the protein rather than the denaturation of the protein as a whole (Montgomery et al., 1993); the increase in the T , of the first transition of about 9 "C when the complexes with RCMLA and NCA-SNase are formed argues against this interpretation; it is known that protein binding takes place in the C-terminal domain of hsp7O (Chappell et al . , 1987), therefore, little effect should be expected in the stability of the N-terminal domain upon complex formation, and the

[phorphate]/rnM 8 0 ' ~ ' ~ I " ' ~ " " ' ~ ' ~ " ~ ' " '

10 *20 30 40

0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0

[ADP]/pM

MLA-DnaK complex by Mg-ATP. DnaK + RCMLA mixtures were FIG. 9. Effect of ADP and phosphate on the dissociation of RC-

incubated at 37 "C for 1 h, then Mg-ADP (trace a ) or sodium phosphate (truce c ) was added, and the mixtures were allowed to equilibrate at room temperature for 10 min. Mg-ATP was added, and samples were injected into the HPLC after 1 min; trace b, the procedure was the same as above with the difference that sodium phosphate was present during the initial incubation (30 min) of DnaK and RCMLA at 37 "C. Stock solutions and final concentrations are as described in the legend to Fig. 8, except for Mg-ADP and sodium phosphate whose concentrations are indicated in the figure, pH 7.4.

major effect would be visible in the second unfolding transition attributed to C-terminal domain if the independent domain unfolding takes place (Montgomery et al., 1993). However, our results indicate the opposite effects, an increase in the T , of the first transition and no difference in the midpoints of the second transition. If we accept the hypothesis of independent domain unfolding, with the N-terminal domain unfolding first, our results would imply that complex formation, which involves the C-terminal domain, translates into a significant increase in the stability of the N-terminal domain and has little or no effect in the structure (and stability) of the C-terminal domain as determined by far-W CD signal (T , = 71 "C). Montgomery et al. (1993) have observed a mid-temperature transition ( T , = 57 "C) that they attributed to the C-terminal domain; this transition is not observable by far-UV CD, and therefore the effect of the substrate protein on this transition is unknown at the moment. "he effect of temperature on complex formation, as indicated in Fig. 6, also argues against the independent domain unfolding interpretation. The amount of complex decreases dramatically as the temperature increases from 40 to 50 "C, i.e. as the first transition takes place. The formation of a molten globule-like intermediate, involving the partial unfolding of both N- and C-terminal domains, can easily account for this observation. On the other hand, if the first transition were solely due to the unfolding of the N-terminal domain, it must at least involve a conformational change in the C-terminal domain significant enough to cause complex dissociation to explain the observed decrease in the amount of complex.

As Fig. 1B indicates, the thermal unfolding of NCA-SNase was also affected by the formation of complex with DnaK, before the unfolding of DnaK takes place (T, = 49.5 "C) a smaller and less cooperative transition is observed with T , = 26 "C. This transition must be due to the unfolding of NCA-SNase bound to DnaK, since such transition was not observed in the unfolding of DnaK alone or the DnaK.RCMLA complex; the decrease of 4 "C in the transition midpoint as compared with free NCA-SNase (T , = 30 "C) indicates that the substrate protein is destabilized and hence probably partially unfolded when bound to the chaperone; additional experiments were con-


ducted to confirm this observation and they are discussed under "Conformation of the Bound Substrate Protein."

Assuming that the molten globule-like states of DnaK in the presence and absence of substrate protein are energetically identical and that the DnaK-protein complex is strong enough to undergo unfolding without dissociation, the AAG between the thermal transitions with and without substrate can be considered an estimate of the energy gain brought about by complex formation; on the basis of studies on protease-inhibitor and antibody-antigen complexes, a AG of 0.6-1 kcaVmol per amino acid pair can be estimated for protein-protein complex formation (Janin and Chothia, 1990); thus a AAG of 2 kcaVmo1, as observed in the case of DnaK with RCMLA and NCA-SNase, seems too low for a protein-protein interaction that is suspected to involve at least seven amino acid pairs (Flynn et al., 1991). This implies that the binding between DnaK and the substrate protein is not strong enough to survive a temperature increase (in the range 40-45 "C) without dissociation. This is also confirmed by the results presented in Fig. 6, where a sharp decrease in the amount of complex is observed at temperatures higher than 40 "C and by the fact, as already discussed, that no difference in the second transition midpoint for DnaK (T, = 71 2 1 "C) is observed with and without the substrate protein. The simultaneous presence of RCMLA or NCA-SNase and Mg-ADP does not result in a potentiation effect; the T, in the presence of substrate protein and Mg-ADP is identical to the T, obtained when only Mg-ADP is present (T, = 58 2 1 "C); this can be explained by the fact that between 50 and 60 "C, the hsp- substrate protein complex in the presence of Mg-ADP dissociates as the results in Fig. 6b, indicate.

Conformation of the Bound Substrate Protein-Confor- mational changes associated with complex formation and dissociation were investigated with NCA-SNase as substrate protein. Figs. 2 and 3 show that in the complex with DnaK, NCA- SNase is in a partially unfolded conformation as evidenced by the difference in the CD and fluorescence spectra, obtained at 4 or 10 "C, of a mixture of NCA-SNase and DnaK before and after incubation at 37 "C. The difference CD spectrum, Fig. 4B, with a strong signal around 220 nm indicates a loss of a-helix in the complex. Contrary to the results obtained with NCA- SNase, no difference in the CD spectra was detected when RCMLA was used as substrate protein; this agrees with the permanently unfolded nature of RCMLA, regardless of the temperature, but more importantly, it indicates that no major changes in the secondary structure of DnaK occur upon complex formation. If binding of RCMLA to DnaK does not cause significant changes in the secondary structure of DnaK, we can assume with a high degree of confidence that the changes observed in the binding of NCA-SNase are due to the substrate protein.

The difference CD spectrum can be used to calculate the degree of unfolding of the substrate protein in the complex. From our estimates of the dissociation constants between hsp7O and substrate proteins (ICd < 0.2 w), it can be predicted that at the level of protein concentration used for the experi- ment shown in Fig. 3 at least 70% of NCA-SNase is in the complex and the rest is free (this was also confirmed by SEC- HPLC results, data not shown). From the thermal unfolding curve of NCA-SNase (Fig. lB) it can be deduced that the conformational changes between the unfolded and the native states of NCA-SNase translate into a change in specific ellipticity (e) of A8,,, = 0.9 millidegredpdmm (this is the extrapolated value at 10 "C); therefore, a change in ellipticity at 222 nm (ezzz) of 2.5 millidegrees, Fig. 4B, assuming that 70% of NCA-SNase is in the complex, gives a change in specific ellipticity of Ae,,, = 2.5/(0.70 x 6.3) = 0.6 millidegree/pM mm. Since the total unfolding of the substrate protein in the absence of

DnaK is accompanied by a specific change of 0.9 millidegree/pM mm, the change observed when the complex is formed, 0.6 millidegredw mm, implies that NCA-SNase lacks about 70% of its secondary structure when bound to the chaperone.

Additional support for the fact that NCA-SNase is substantially unfolded in the complex with DnaK is shown in Fig. 4A where the difference fluorescence spectrum between those shown in Fig. 2, traces a and c, and the difference fluorescence spectrum for NCA-SNase between the unfolded (40 "C, extrapolated at 4 "C) and native states (4 "C) are compared. As can be observed, there is a very good match between both difference spectra, suggesting that the tryptophan residue of NCA-SNase bound to DnaK is exposed to the solvent as in the unfolded state. Our data cannot preclude, however, the possi- bility that the difference spectrum of Fig. 4A, trace a, is also due, in part, to a change in the environment of the only tryptophan residue of DnaK. An additional indication that NCA- SNase is substantially unfolded in the complex with DnaK was obtained by adding Mg-ATP to the complex, which results in its dissociation. Complex dissociation was accompanied by an increase of fluorescence intensity at 330 nm as expected for the refolding of NCA-SNase (Fig. 5). The rate of refolding at 4 "C was 4.31 2 0.05 x s-l which is comparable with the rate of refolding of NCA-SNase (monitored by tryptophan fluorescence at 330 nm) at the same temperature when pH-denatured NCA- SNase, pH 2, was allowed to refold at pH 7 (k = 3.2 x s"): indicating that DnaK has little or no effect on the rate of refolding of NCA-SNase once it is released from the complex and that the released protein is non-native.

Further evidence for the unfolded nature of NCA-SNase in the complex with hsp70 was obtained from the estimation of Stokes radii by SEC-HPLC. The Stokes radius of native DnaK determined ynder the experimental conditions of this study was 41 2 2 A, which agrees with previously published data determined by dynamic light scattering and SEC-HPLC (Pal- leros et al., 1993b). The DnaK Stokes radius is larger than expected for a protein of molecular mass 70 kDa. A correlation of RS3 uersus molecular mass for nine standard globular proteins (Corbett and Roche, 1984) ranging in molecular mass from 3 to 81 kDa gave a good linear correlation ( R 2 = -1707 + 0.609 molecular mass; r 0.989); using this correlation, a Stokes radius of about 34 A is expected for a globular protein of molecular mass 70 kDa; the larger radius found for native DnaK is an indication that hsp7Os deviate significantly from a spherical shape; preliminary small angle x-ray scattering experiments suggest that free DnaK possess a dumbbell shape.4 If the conformation of the substrate protein in the complex is such that the whole complex could be considered a globular protein of molecular mass equal to the total mass, for the complexes with RCMLA (14 kDa) and NCA-SNase (16.8 kDa), using th? correlation mentioned above, a Stokes radius of about 37 A would be expected. Such complexes can be visualized as having the substrate protein "wrapped around" hsp7O. However, SEC- HPLC measurements of the Stokes radius for DnaK.RCMLA and DnaK.NCA-SNase complexes gave a much more larger figure: 51 2 A in both cases, which argues against the "wrapped-around" model. Given tbe fact that the measured Stokes radius for DnaK (41 2 2 A) is larger than the value expected for a globular protein of similar molecular mass (34 A), it would be possible, in principle, to argue that the large radius for the complexes (51 2 2 A) is just a reflection of the abnormally large radius of DnaK, however, the difference between the expected (34 & and observed (51 A) Stokes radius for the complexes, i.e. 17 A, is much larger than the difference of 7

D. R. Palleros and A. L. Fink, unpublished results. L. Shi, M. Kataoka, and A. Fink, unpublished results.


A detected for free DnaK and indicates that both substrate proteins bound to DnaK are substantially unfolded. This is in agreement with the permanently denatured nature of RCMLA (it shouldo be noted that the large Stokes radius for free RC- MLA, 27 A, is consistent with the protein being in an extended conformation) and further confirms that NCA-SNase in complex with DnaK is also substantially unfolded. Similar results have been obtained for the complexes between hsp73 and RC- MLA and NCA-SNase. In summary, our Stokes radius measurements allow us to rule out the "wrapped around" model and any other model that involves the substrate protein in a com- pact conformation when bound to hsp7O.

Effect of ADP on Complex Formation and Dissociation-The increase in hsp70 stability caused by the binding of Mg-ADP is manifested in a widening of the temperature range in which complexes with unfolded proteins are stable (Fig. 6). While less than 20% complex was detected when the incubation is performed between 45 and 50 "C in the absence of Mg-ADP, about 60% was observed in the same temperature range if an excess of Mg-ADP is present in the incubation buffer. More important from a physiological viewpoint, however, is the fact that Mg- ADP also has a noticeable effect on complex formation at temperatures below 40 "C; as much as a 50% increase in the amount of complex was detected when the incubation was performed in the presence of Mg-ADP. This is in agreement with our previous observation that Mg-ADP accelerates the formation of complex between bovine brain hsp73 and RCMLA (Palleros et al . , 1991); Mg-ADP also slows the rate of complex dissociation as discussed below.

The complexes between RCMLA and hsp7O spontaneously dissociate at room temperature as our HPLC results for human hsp72 indicate (Fig. 7); the dissociation of the RCMLA.DnaK complex was also studied and similar results were obtained (not shown). The presence of Mg-ADP in the complex incubation mixture and in the HPLC mobile phase resulted in a slower rate of complex dissociation. We have shown previously that ADP forms a ternary complex with hsp73 and RCMLA that can be isolated by HPLC (Palleros et al., 1991). The dissociation constants at 22 "C obtained from the data in Fig. 7, i.e. Kd = 0.5 p~ (no Mg-ADP) and Kd = 0.02 p~ (Mg-ADP), translate into AGO = 8.6 kcaVmol and 10.4 kcaVmo1, respectively. Considering the limit values of 0.6 and 1 kcaVmol per amino acid pair for the free energy gain upon protein-protein complex formation (Janin and Chothia, 19901, the number of amino acid pairs in the hsp7O.RCMIA complex can be estimated as 8 to 14. This number is not very different than that found to be the minimal length for maximal binding of peptides to BiP, i.e. 7 residues in the peptide (Flynn et al . , 1991).

We have recently shown that ATP-induced hsp70-substrate protein dissociation requires K+ and precedes ATP hydrolysis. A mechanism for hsp70 action under physiological concentrations of K+ and nucleotides has been proposed and is shown in Scheme I (Palleros et al . , 1993a). The first step in the mechanism involves the binding of substrate proteins to an hsp7O.ADP complex; as discussed above, binding to a binary hsp7O.ADP complex is faster than binding to free hsp70, and the resulting ternary hsp70.ADP.substrate protein complex is more stable than the binary hsp70-substrate protein complex. Binding of substrate protein to hsp7O-ADP complex most likely involves a conformational change in hsp70, step 1. This conformational change accelerates the rate of ADP dissociation from hsp70, step 2, relative to the case without substrate protein (Sadis and Hightower, 1992). Addition of Mg-ATP results in ADP-ATP exchange (step 2); binding of ATP causes a conformational change that triggers substrate protein release from the complex, step 3 (Palleros et al., 1993a). Finally, ATP is hydrolyzed to ADP, step 4 (Palleros et al . , 1993a), to afford an

I

3

h.ATP h.ATP.U

U

h: hsp7O; D: ADP; T ATP; U: substrate protein; P: phosphate SCHEME I

hsp7O.ADP complex that is ready to start a new cycle of binding.

In agreement with the model presented in Scheme I, the results in Figs. 8 and 9 clearly indicate that ADP has an in- hibitory effect on RCMLA.DnaK complex dissociation; similar results were found with bovine brain hsp73 (Palleros et al . , 1991). It is clear from Fig. 9a that when equimolar amounts (100 p ~ ) of Mg-ADP and Mg-ATP are present, no more than 50% of the complex is dissociated after Mg-ATP addition; on the other hand, in the absence of ADP, no more than 10% of complex remains after addition of a Mg-ATP (100 p~). These num- bers indicate that in the presence of the substrate protein, the dissociation constants between hsp7O and ADP and ATP are very similar. The ratio of the dissociation constants for ATP. andADP.hsp73 complexes, i.e. KdATpIKdADp, was estimated as 5 in the absence of substrate protein (Palleros et al . , 1991). It was claimed recently that our Kd values (in the micromolar range) are too high (Gao et al . , 1993). We agree that under the conditions studied by Gao et al. (1993) and perhaps under most physiological conditions, these dissociation constants are in the submicromolar range; the apparent discrepancy arises from different experimental conditions. We observed that hsp7O- nucleotide complex formation and dissociation are very sensi- tive to ionic composition; they are specially dependent on the contents of K+ and M e ; it should be noted that in our previous measurement of KdmP and Kd,Arp, M e was omitted from our buffer (Palleros et al . , 1991); the lack of M e certainly results in weaker nucleotide binding to hsp7O as we have already shown (Palleros et al . , 1992). As the results in Fig. 9 (trace b ) indicate, phosphate also inhibits ATP-induced protein dissociation, although to a much lesser extent than ADP. This is probably due to its binding to the nucleotide binding site (Flaherty et a l . , 19901, which interferes with ATP binding. The reasons for the different effects observed when phosphate was present during complex formation or added afterward are not clear at the moment.

The cycle shown in Scheme I is similar to that of G proteins (Bourne et al . , 1991) in which two different forms, GTP- and GDP-bound, exist. However, in the case of hsp70, contrary to


the G proteins, the ADP-bound form is the species that binds substrate proteins. Cofactors such as GrpE and DnaJ may act as exchange proteins. It is likely that GrpE catalyzes the rate of ADP/ATP exchange (step 2 ) and DnaJ the rate of ATP hydrolysis (step 4), based on observations in the absence of substrate protein (Liberek et al., 1991a).

It has been observed in vivo that the levels of ATP sharply decrease during heat shock, whereas the levels of ADP remain constant (Findly et al., 1983). According to our model this sce- nario would facilitate the rescue of denatured proteins, one of the functions ascribed to hsp7O. At a reduced ATP level more hsp7O would be free to interact with ADP, and the concentration of hsp70.ADP complexes would rise; since these complexes have higher affinity for substrate proteins than hsp70 alone, more substrate proteins would favorably interact with hsp70 and be spared further, and possible irreversible, damage caused by the heat shock. This correlates with the high levels of hsp70-protein complexes observed as a result of hyperthermia (Dubois et al., 1991; Lewis and Pelham, 1985). It is well docu- mented that after heat shock of eukaryotic cells, there is substantial protein aggregation in which hsp7O is a major compo- nent of the aggregate; these aggregates are localized in the nucleus and nucleolus (Welch and Feramisco, 1984; Pelhman, 1984; Littlewood et al., 1987; Dubois et al., 1991). As the cell recovers from the heat shock, the increasing amounts of ATP would then permit ADP/ATP exchange and release of the proteins and the cell would resume its normal functioning; it has been observed that on return of the cell to normal conditions, there is a slow recovery of soluble and active proteins (Dubois, 1991) and a redistribution of hsp70 within the cell (Pelham, 1984; Welch and Feramisco, 1984). It is likely that the substrate protein is released from the complex with hsp7O in a non-native state, as our results with NCA-SNase suggest, and then it would become renatured (possibly through the involve- ment of hsp6O); this would explain the observation that there is very little actual turnover of proteins during and after heat shock (Carlson et al., 1987).

Acknowledgments-We thank Professor R. 0. Fox (Yale University) for a gift of NCA-SNase and Professor W. J. Welch for hsp72.

REFERENCES

Antonino, L. C., Kautz, R. A,, Nakano, T., Fox, R. O., and Fink, A. L. (1991) Proc.

Beckmann, R. I?, Mizzen, L. A,, and Welch, W. J. (1990) Science 248,850454 Becktel, W. J., and Schellman, J. A. (1987) Biopolymers 26, 1859-1877 Blond-Elguindi, S. , Fourie, A. M., Sambrook, J. E, and Gething, M.J. H. (1993) J.

Bole, D. G., Hendershot, L. M., and Kearny, J. F. (1986) J. Cell B i d . 102, 1558-

Natl. Amd. Sci. U. S. A. 88, 7715-7718

Bid. Chem. 268, 12730-12735

1566

Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127 Brown, C. R., Martin, R. L., Hansen, W. J., Beckmann, R. P., and Welch, W. J.

Chappell, T. G., Konforti, B. B., Schmid. S . L., and Rothman, J. E. (1987) J. Bid. Carlson, N., Rogers, S . , and Rechsteiner, M. (1987) J. Cell Bid. 104,547555

Dorner, A. J., Bole, D. G., and Kaufman, R. J. (1987) J. Cell Biol. 106,2665-2674 Corbett, R. J. T., and Roche, R. S . (1984) Biochemistry 23, 1888-1894

Dubois, M. E , Hovanessian, A. G., and Bensaude, 0. (1991) J. Biol. Chem. 266,

Efiink, M. R., Gryczynski, I., Wiczk, W., Laczko, G., and Lakowicz, J. R. (1991)

Findly, R. C., Gillies, R. J., and Shulman, R. G. (1983) Science 219, 1223-1225 Ellis, R. J., and van der Vies, S . M. (1991) Annu. Reu. Biochem. 60,321-347

Flaherty, K. M., DeLuca-Flaherty, C., and McKay, D. B. (1990) Nature 346,623-

Flynn, G. C., Chappell, T. G., and Rothman, J. E. (1989) Science 245,385-390 Flynn, G. C., Pohl, J., Flocco, M. T., andRothman, J. E. (199l)Nature 353,72&730 Gao, B., Emoto, Y., Greene, L., and Eisenberg, E. (1993) J. Biol. Chem. 268,

Gething, M.-J, and Sambrook, J. (1992) Nature 355,3345 Gething, M.-J., McCammon, K., and Sambrook, J. (1986) Cell 46, 939-950 Hendrick, J. P., and Hartl, F.-U. (1993)Annu. Rev. Biochem, 62,349-384

Hynes, T. R., Kautz, R. A,, Goodman, M. A,. Gill, J. F., and Fox, R. 0. (1989)Nature Hightower, L. E., and White, F. I? (1981) J. Cell. Physiol. 108,261-275

Ikeguchi, M., and Sugai, S . (1989) Int. J. Pept. Protein Res. 33, 289-297 Janin, J., and Chothia, C. (1990) J. Biol. Chem. 266,16027-16030 Lam, K. T., and Caldenvood, S . K. (1992) Biochem. Biophys. Res. Commun. 184,

Landry, S . J., Jordan, R., McMacken, R., and Gierasch, L. M. (1992) Nature 366,

Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer, M. K., and Hartl, F.-U. (1992)

Lewis, M. J., and Pelham, H. R. B. (1985) EMBO J. 4,31374143 Liberek, K, Marszalek, J., Ang, D., Georgopoulos, C., and Zylicz, M. (1991a) Proc.

Liberek, K., Skowyra, D., Zylicz, M., Johnson, C., and Georgopoulos, C. (1991b) J.

McCarty, J. S., and Walker, G. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,9513- Littlewood, T. D., Hancock, D. C., and Evan, G. I. (1987) J. Cell Sci. 88, 65-72

McKay, D. (1993) Adu. Protein Chem. 4467-97 Montgomery, D., Jordan, R., McMacken, R., and Freire, E. (1993)J. Mol. Biol. 232,

Munro, S. , and Pelham, H. R. B. (1986) Cell 46,291400 Palleros, D. R., Welch, W. J., and Fink, A. L. (1991) Proc. Null. Acad. Sci. U. S. A.

Palleros, D. R., Reid, K. L., McCarty, J. S. , Walker, G. C., and Fink, A. L. (1992) J.

Palleros, D. R., Reid, K. L., Shi, L., Welch, W. J., and Fink, A. L. (1993a) Nature

Palleros, D. R., Shi, L., Reid, K. L., and Fink, A. L. (1993b) Biochemistry 32,

Palleros, D. R., Reid, K. L., Shi, L., and Fink, A. L. (1993~) FEBS Lett. 336,124-128 Park, K., Flynn, G. C., Rothman, J. E., and Fasman, G. D. (1993) Protein Sci. 2,

Pelham, H. R. B. (1984) EMBO J. 3,3095-3100 Sadis, S., and Hightower, L. E. (1992) Biochemistry 31,9406-9412 Schmid, S. L., Braell, W. A., and Rothman, J. E. (1985) J. Biol. Chem. 260,10057-

Zylicz, M., LeBowitz, J. H., McMacken, R., and Georgopoulos. C. (1983) Proc. Natl.

Welch, W. J., and Feramisco, J. R. (1985) Mol. Cell. Bid. 6, 1229-1237 Welch, W. J., and Feramisco, J. R. (1984) J. Bid. Chem. 259,45014513

(1993) J. Cell Bid. 120,1101-1112

Chem. 262,74S751

9707-9711

Biochemistry 30,8945-8953

628

850743513

339,7%76

167-174

455-457

Nature 356,683489

Natl. Acud. Sci. U. 5'. A. 88, 2874-2878

Biol. Chem. 266,14491-14496

9517

68M92

88,57194723

Biol. Chem. 267,5279-5285

365,664-666

4314-4321

325-330

10062

Acud. Sci. U. S. A. 80,6431-6435

OF CHEMISTRY Vol. No. of May for USA. hsp70-Protein Complexes · THE JOURNAL OF BIOL~~ICAL...

Documents

Transcript of OF CHEMISTRY Vol. No. of May for USA. hsp70-Protein Complexes · THE JOURNAL OF BIOL~~ICAL...