©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Quasi-native Chaperonin-bound Intermediates in Facilitated Protein Folding (*)

(Received for publication, July 21, 1995; and in revised form, August 14, 1995)

Guoling Tian Irina E. Vainberg William D. Tap Sally A. Lewis Nicholas J. Cowan (§)

From the Department of Biochemistry, New York University Medical Center, New York, New York 10016

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Chaperonins are known to facilitate protein folding, but their mechanism of action is not well understood. The fact that target proteins are released from and rebind to different chaperonin molecules (``cycling'') during a folding reaction suggests that chaperonins function by unfolding aberrantly folded molecules, allowing them multiple opportunities to reach the native state in bulk solution. Here we show that the cycling of alpha-tubulin by cytosolic chaperonin (c-cpn) can be uncoupled from the action of cofactors required to complete the folding reaction. This results in the accumulation of folding intermediates which are chaperonin-bound, stable, and quasi-native in that they bind GTP nonexchangeably. We present evidence that these intermediates can be generated without the target protein leaving c-cpn. These data show that, in contrast to prevailing models, target proteins can maintain, and possibly acquire, significant native-like structure while chaperonin-bound.


INTRODUCTION

The final stage in the flow of genetic information from DNA to expressed proteins is the folding of each protein into the three-dimensional structure that specifies its biological activity. In principle, such folding reactions can occur spontaneously, since all the necessary information required to determine the final folded structure is contained within the primary sequence of amino acids. However, under physiological conditions, constraints of temperature and the tendency of unfolded proteins to aggregate are such that many proteins must undergo facilitated folding via interaction with protein complexes termed chaperonins(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) . These protein complexes take the form of toroidal structures that facilitate protein folding in an ATP-dependent manner. For example, the prokaryotic chaperonin GroEL facilitates the folding of a range of proteins in E. coli(6, 27) , often in conjunction with the cochaperonin GroES(1, 4) . There is evidence that GroES, which is itself a heptameric ring(12) , functions at least in part by interacting with the ends of the GroEL cylinder, such that it modulates and coordinates the hydrolysis of ATP by GroEL(11, 13, 14, 15) .

Many models depict facilitated folding occurring within the central cavity that is present in all chaperonins, thereby protecting the target protein from interaction with other proteins in the general milieu; release then occurs following acquisition of the native structure(3, 8, 9, 10, 16) . Recently, however, this concept has been challenged by evidence that target proteins can jump between different chaperonin molecules during a folding reaction (``cycling'')(11, 17) . Thus, the function of chaperonins might be to unfold and release proteins that have misfolded. In this view, protein folding occurs spontaneously in solution, while the function of the chaperonin is to ``recycle'' aberrantly folded molecules so that they can return to a potentially productive pathway. Accordingly, a given target molecule might require multiple rounds of interaction with different chaperonin molecules before partitioning to the native state.

To understand the mechanism of chaperonin-mediated folding, it is essential to know the states of folding intermediates during the cycling process. However, such intermediates are usually difficult to study because of their heterogeneity, transient existence, and pronounced tendency to aggregate. To examine the states of folding intermediates produced during chaperonin-mediated folding, we took advantage of the observation that the facilitated folding of alpha-tubulin by cytosolic chaperonin (c-cpn) (^1)cannot proceed to the native state in the absence of specific protein cofactors(18, 19) . This system allowed us to establish the existence of a novel class of chaperonin-bound quasi-folded intermediates that are generated during ATP-dependent facilitated folding.


EXPERIMENTAL PROCEDURES

Materials

[S]Methionine and alpha-[P]GTP were from DuPont NEN. c-cpn, mt-cpn, and cofactors required for the productive folding of alpha-tubulin were purified as described previously(18, 20, 21) . Hexokinase was from Boehringer Mannheim.

Folding Reactions

alpha-Tubulin folding reactions were done at 30 °C in 20 µl of folding buffer (22) containing c-cpn (3 pmol), 1 mM each of ATP and GTP, native calf brain tubulin (0.2 mg/ml), and cofactors required for the productive folding of alpha-tubulin(18) . In some experiments, ATP was quenched by the addition of glucose (to 10 mM) and hexokinase (2.5 units). The yield of various products identified on fixed, dried nondenaturing polyacrylamide gels run as described (18, 20) was quantitated using a PhosphorImager.

Sucrose Gradients

Sucrose gradients were prepared in thick-walled 1-ml Beckman polycarbonate ultracentrifuge tubes by overlaying successive layers (each of 0.13 ml) of 20, 15, 13, 11, 9, 7, and 5% sucrose in folding buffer (22) supplemented with 0.1 mM GTP. Gradients were centrifuged at 20 °C for 50 min at 166,000 times g in a Beckman TLS55 rotor, and fractions (0.13 ml) were removed in successive aliquots by careful pipetting from the top. Aliquots (20 µl) from these fractions were assayed on 4.5% nondenaturing polyacrylamide gels (18, 20) either without further treatment or following incubation for 2 min at 30 °C in the presence of cofactors and native carrier bovine brain microtubules as described(18) .

Cell-free Translation and Protease Protection Experiments

SLabeled alpha-tubulin was generated by in vitro translation in a 0.1-ml reaction using mRNA derived by transcription of a full-length alpha-tubulin cDNA (Malpha2, (23) ). The reaction mixture was cleared of particulate material by centrifugation at 150,000 times g for 20 min. GTP and carrier native bovine brain tubulin were added to the supernatant to 1 mM and 0.6 mg/ml, respectively, and the mixture was fractionated on a 1-ml Mono Q anion exchange column as described (24) . The radioactive peak containing tubulin heterodimers and eluting at 0.62 M NaCl was concentrated using a microcon 30 ultrafiltration device. Native tubulin generated by in vitro translation or target proteinbulletc-cpn binary complexes prepared as described (22) were incubated at a concentration of 0.2 µM with 74 nM proteinase K in folding buffer at room temperature. The final protein concentration in each reaction was 0.23 mg/ml (including 0.1 mg/ml hexokinase, added as a carrier protein and (in some cases) as an ATP quenching reagent). In control reactions containing translated alpha-tubulin or unfolded alpha-tubulin target protein diluted into folding buffer, unlabeled native tubulin was included as an additional carrier to adjust the total protein concentration to 0.23 mg/ml. At various times, aliquots were withdrawn and the proteolytic reaction was quenched by the addition of phenylmethylsulfonyl fluoride to 5 mM.

GTP Incorporation in alpha-Tubulin Folding Reactions

Unlabeled, unfolded alpha-tubulin target protein (22) (1 pmol) was diluted into 20 µl of folding buffer containing 4 pmol of c-cpn and incubated at 30 °C for 30 min to allow binary complex formation. The reaction was diluted 10-fold with folding buffer containing either 0.2 µM c-cpn or 0.2 µM mitochondrial chaperonin plus purified rabbit hemoglobin (22) as a carrier protein. 1 mM ATP and 2.0 µM [alpha-P]GTP (specific activity 800 Ci/mmol) were added, the incubation was continued at 30 °C for 45 min, and the label was quenched by the addition of unlabeled GTP to 2 mM. Cofactors and carrier native brain microtubules (18) were added, and the incubation was continued for an additional 2 min. Reaction products were analyzed on 4.5% nondenaturing polyacrylamide gels containing 0.1 mM GTP as described previously(18) , except that, following electrophoresis, the gels were rinsed in several changes of running buffer and dried directly without fixation or staining before exposure to autoradiographic film.


RESULTS AND DISCUSSION

The facilitated folding of beta-actin requires ATP-dependent interaction with c-cpn, the eukaryotic cytosolic homolog of GroEL(20) . In contrast, the facilitated folding of alpha- and beta-tubulin requires interaction with both c-cpn and additional protein cofactors(18, 19) . To see whether it might be possible to uncouple the ATP-driven c-cpn-mediated reaction from the action of these cofactors, an alpha-tubulin folding reaction was done in which alpha-tubulinbulletc-cpn binary complexes were incubated with ATP and GTP alone. After 45 min, the ATP-dependent reaction was quenched by the addition of hexokinase and glucose. Cofactors and carrier native tubulin were then added, and the reaction was allowed to continue at 30 °C. We found that the ATP-driven reaction is almost as efficient when uncoupled from the action of cofactors, since the amount of native tubulin product was very similar in a parallel reaction that contained cofactors at the outset of the incubation with ATP and GTP (Fig. 1a, lanes 1 and 2). A control folding reaction in which glucose and hexokinase were included at the outset resulted in no detectable product, demonstrating the effectiveness of the ATP quench in arresting c-cpn-mediated folding (Fig. 1a, lane 3). The ATP-dependent reaction that generates the species upon which cofactors act is slow, requiring about 45 min to reach equilibrium (Fig. 1, b and c), while the action of cofactors on this intermediate seems to be very fast, requiring at most only a few seconds at 30 °C (Fig. 1d).


Figure 1: Accumulation of intermediates in the c-cpn-mediated folding of alpha-tubulin. a, the nucleotide-driven c-cpn-mediated alpha-tubulin folding reaction can be uncoupled from the action of cofactors required to generate native product. Analysis of the products of alpha-tubulin folding reactions containing c-cpn, ATP, and GTP. Lane 1, control folding reaction supplemented with cofactors and native carrier tubulin (18, 19) and incubated for 45 min at 30 °C; lane 2, folding reaction incubated for 45 min at 30 °C, quenched with hexokinase, and incubated for an additional 2 min in the presence of cofactors and native carrier tubulin; lane 3, reaction identical with that shown in lane 1, but quenched with hexokinase immediately following presentation of the target protein. b, the intermediates that are the substrates upon which cofactors act accumulate slowly during ATP exchange and hydrolysis. alpha-Tubulinbulletc-cpn binary complexes were incubated at 30 °C for the times shown in the presence of ATP and GTP, quenched with hexokinase, and incubated for an additional 2 min in the presence of cofactors and carrier native tubulin. c, quantitative analysis of the data shown in b (averaged from four experiments and with the plateau level of native tubulin rationalized to 100%). d, cofactors act rapidly on intermediates accumulated as a result of incubation with ATP and GTP. alpha-Tubulin/c-cpn binary complexes were incubated in the presence of ATP and GTP for 45 min at 30 °C. The reaction was quenched with hexokinase, supplemented with cofactors and carrier native tubulin, and aliquots were withdrawn from the reaction at the times shown. e, half-life of accumulated alpha-tubulin folding intermediates. alpha-Tubulin folding reactions were incubated for 45 min at 30 °C in the presence of ATP and GTP. The reactions were quenched with hexokinase, and the incubation continued at 30 °C. At the times shown, aliquots were withdrawn, supplemented with cofactors and carrier native tubulin, and incubated for an additional 2 min. f, semi-log plot of the data shown in e (averaged from four experiments and with the initial yield of native tubulin rationalized to 100%); arrow shows t. Upper and lower arrows in a, b, d, and e show the location of the alpha-tubulinbulletc-cpn binary complex and native tubulin, respectively.



To determine the stability of the subset of ATP-generated intermediates that can be converted to native alpha-tubulin molecules by the action of cofactors, we incubated alpha-tubulinbulletc-cpn binary complexes with ATP and GTP for 45 min to generate these intermediates, quenched the ATP-dependent reaction with hexokinase and glucose, and continued the incubation at 30 °C for increasing times before completing the reaction by adding cofactors and native carrier tubulin. We found that the subset of intermediates that can be converted to native molecules by the action of cofactors is quite stable, with a half-life of about 50 min at 30 °C (Fig. 1, e and f). We define this subpopulation of end states as I(Q) (for intermediates, quasi-native (see below)). The existence of these stable intermediates provides a unique opportunity to study the mechanism of chaperonin-mediated folding.

To see whether I(Q) intermediates exist bound to c-cpn or free in solution, an alpha-tubulin folding reaction containing c-cpn, ATP, and GTP was incubated for 45 min at 30 °C, quenched with hexokinase, and applied to a sucrose gradient (Fig. 2a). Following centrifugation, fractions from the gradient were analyzed in two ways: without further incubation and following incubation with cofactors and native carrier tubulin. We found that correctly folded tubulin was generated only by addition of cofactors to those fractions that contain alpha-tubulin-chaperonin binary complex (Fig. 2b). Similarly, when the products of a quenched alpha-tubulin folding reaction done without cofactors were applied to a Superose 6 gel filtration column, only fractions that co-eluted with c-cpn were active in the generation of native tubulin upon incubation with cofactors (data not shown). These results demonstrate that there is a tight association of c-cpn and I(Q) alpha-tubulin end states upon which cofactors act.


Figure 2: Characterization of quasi-folded alpha-tubulin folding intermediates. a, sucrose gradients used for the size fractionation of I(Q) intermediates. Numbers denote the percentage of sucrose contained in successive layers. b, analysis on 4.5% nondenaturing polyacrylamide gels (18, 20) of the products of reactions done using fractions recovered from sucrose gradients. alpha-Tubulin folding reactions done with c-cpn in the presence of Mg-ATP and Mg-GTP were quenched by the addition of glucose and hexokinase, and the products were applied to sucrose gradients (as shown in a). Following centrifugation, fractions were incubated in two different reactions: alone(-) or supplemented with cofactors and carrier native tubulin (18, 20) (+). Upper and lower arrows denote the locations of alpha-tubulinbulletc-cpn binary complexes and native tubulin, respectively. c, I(Q) alpha-tubulin folding intermediates do not cycle in the presence of ATP. Quantitative analysis of the yield of alpha-tubulinbulletc-cpn binary complexes (solid bars) and native tubulin (hatched bars) produced in folding reactions in which I(Q) was formed at 30 °C for 45 min and diluted in the presence of ATP and GTP to varying extents so as to preclude efficient cycling(22) . Following incubation at 30 °C for an additional 30 min, cofactors and native carrier tubulin (18) were added and the incubation continued for an additional 2 min.



Two kinds of experiments showed that alpha-tubulin I(Q) intermediates are not cycled by c-cpn in the presence of ATP. First, they survive incubation under dilute conditions where there is a concomitant loss of counts from the binary complex (Fig. 2c). Under these conditions, there is insufficient chaperonin in the reaction to capture released molecules before they aggregate or adhere to the reaction vessel walls(22) . The loss of label from the binary complex is a result of the release of cycling intermediates under these dilute conditions, and not the disintegration of chaperonin, since we found no loss of radioactivity when the alpha-tubulinbulletc-cpn binary complex was diluted and incubated with ADP. Secondly, we found no effect on the yield of native alpha-tubulin produced when stable alpha-tubulin end states were incubated with ATP in the presence of a large molar excess of a mitochondrial chaperonin trap for the capture of non-native forms (22) before completing the reaction by addition of cofactors (data not shown).

To investigate the extent of native-like structure in alpha-tubulin target molecules acquired as a consequence of their interaction with c-cpn, we compared the extent of target protein protease resistance in alpha-tubulinbulletc-cpn binary complexes that had been incubated with or without ATP and GTP. As controls, we first established the resistance to proteolysis of urea-unfolded alpha-tubulin diluted into buffer alone, as well as the resistance of native tubulin synthesized by in vitro translation. Under the conditions used in our experiments, no intact tubulin survived beyond the initial addition of protease when the target protein was diluted into buffer (Fig. 3a). In contrast, there was no significant loss of intact alpha-tubulin in a parallel experiment done with native tubulin (Fig. 3b). In experiments to measure the proteolytic sensitivity of target proteinbulletc-cpn binary complexes, we found that resistance to proteolysis of c-cpn-bound alpha-tubulin increased significantly upon incubation with ATP and more so upon incubation with both ATP and GTP (Fig. 3, c-e). These data imply that alpha-tubulin end states generated as a result of ATP-dependent cycling with c-cpn are more extensively folded than molecules that have not been cycled.


Figure 3: Resistance to proteolysis of alpha-tubulin folding intermediates. a and b, resistance to proteolysis of unfolded alpha-tubulin diluted directly into folding buffer (a) or alpha-tubulin synthesized by translation in vitro and incorporated into tubulin heterodimers (b). c-e, resistance to proteolysis of alpha-tubulinbulletc-cpn binary complexes incubated for 45 min at 30 °C with ADP and GTP (c), ATP alone (d), or ATP and GTP (e). Reactions were quenched with hexokinase, and aliquots were incubated with proteinase K for the times shown (in minutes). Reaction products were analyzed on 10% Tricine-SDS-polyacrylamide gels(26) . Arrows mark the location of intact alpha-tubulin. f and g, quantitation of data shown in a and b (f) and c-e (g), in each case averaged from three experiments; radioactivity contained in target protein at t = 0 is normalized to 100%. , unfolded target protein diluted into folding buffer; circle, alpha-tubulin synthesized by translation in vitro; bullet, , , alpha-tubulin target protein bound to c-cpn and incubated with ADP alone, ATP alone, or ATP and GTP, respectively.



The alpha/beta-tubulin heterodimer binds two molecules of GTP, one of which is exchangeable and is located on the beta-subunit (the E-site), and a second that is nonexchangeable, located on the alpha-subunit (the N-site)(25) . Our observation that alpha-tubulinbulletc-cpn binary complexes become more resistant to proteolysis upon incubation with both ATP and GTP (compared to incubation with ATP alone) (Fig. 3, d and e) implies that at least one function of GTP binding is to stabilize tubulin molecules during their facilitated folding. To probe the state of alpha-tubulin I(Q) intermediates in terms of their nonexchangeable (N-site) GTP binding properties(25) , we performed c-cpn-mediated folding reactions in the presence of ATP and alpha-[P]GTP, using unlabeled unfolded alpha-tubulin as target protein. The incorporation of bound, labeled GTP into c-cpnbulletalpha-tubulin binary complex is ATP- and target protein-dependent (Fig. 4a). When these binary complexes were incubated with cofactors and native carrier tubulin in the presence of excess unlabeled GTP, we found nonexchangeable label in association with both c-cpn and native tubulin (Fig. 4b, lanes 1 and 2). This GTP-labeled tubulin was native as shown by its ability to copolymerize with authentic brain tubulin. These experiments demonstrate that labeled GTP is bound to the N-site of I(Q) (which gives rise to labeled native tubulin), as well as to the N-site of other intermediates which remain c-cpn-associated in the presence of cofactors. We conclude that I(Q) and other alpha-tubulin folding intermediates are so extensively native-like that they contain the GTP binding pocket.


Figure 4: alpha-Tubulin folding intermediates contain nonexchangeably bound GTP. Incorporation of nonexchangeable GTP into alpha-tubulin folding intermediates and native alpha-tubulin. a, incorporation of GTP into alpha-tubulinbulletc-cpn binary complex. Lane 1, control reaction in which purified c-cpn without added target protein was incubated with ATP and alpha-[P]GTP for 45 min at 30 °C. Lanes 2 and 3, c-cpn-mediated folding reactions in which unlabeled alpha-tubulinbulletc-cpn binary complexes were incubated for 45 min at 30 °C with either alpha-[P]GTP alone (lane 2) or alpha-[P]GTP and ATP (lane 3). b, incorporation of nonexchangeable GTP into native alpha-tubulin. c-cpn-mediated folding reactions in which c-cpn alone (lanes 1, 3, and 5) or unlabeled alpha-tubulinbulletc-cpn binary complexes (lanes 2, 4, and 6) were diluted 10-fold into folding buffer containing ATP and alpha-[P]GTP and supplemented with either 0.2 µM c-cpn (lanes 1 and 2) or a 10-fold molar excess of trap for the capture of non-native cycling intermediates (22) (lanes 3 and 4). Following incubation at 30 °C, native tubulin was discharged by the addition of cofactors(18) . Upper and lower arrows show the location of alpha-tubulinbulletc-cpn binary complexes and native tubulin, respectively.



When c-cpn is diluted sufficiently to preclude efficient cycling and in the presence of a mitochondrial chaperonin (mt-cpn) trap for the capture of released non-native target protein(22) , labeled GTP can still become incorporated into alpha-tubulin intermediates (Fig. 4b, lanes 4-6). These data suggest that the nonexchangeable GTP binding site can form while the target protein is chaperonin-bound. GTP is not acquired before binary complex formation, since the complex is formed in the absence of GTP. Nor is GTP (labeled or unlabeled) acquired after the addition of cofactors: when folding reactions containing alpha-[P]GTP and unfolded [S]methionine-labeled alpha-tubulin were quenched with unlabeled GTP before the addition of cofactors, each mole of labeled native tubulin produced contained 1 mol of nonexchangeably bound labeled GTP.

Like alpha-tubulin, the GroEL-mediated folding of rhodanese (3) and the c-cpn-mediated folding of beta-tubulin (^2)also proceed via the formation of chaperonin-bound protease-resistant intermediates. These data suggest that chaperonin-bound quasi-native intermediates are an important feature of the mechanism of facilitated folding (although there is normally no accumulation of such intermediates). The existence of such highly structured c-cpn-bound intermediates is surprising in view of current models of GroEL-mediated protein folding(11, 17) , but is consistent with our observation that different chaperonins produce distinct spectra of folding intermediates (22) . Furthermore, our evidence that alpha-tubulin can acquire its GTP binding pocket via a single cycle of interaction with c-cpn suggests that, in contrast to the prevailing view, some target protein folding occurs either on the chaperonin surface or in its central cavity, rather than in bulk solution.


FOOTNOTES

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

§
To whom correspondence should be addressed. Tel.: 212-263-5809; Fax: 212-263-8166.

(^1)
The abbreviations used are: c-cpn, cytosolic chaperonin; mt-cpn, mitochondrial chaperonin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

(^2)
I. E. Vainberg and N. J. Cowan, unpublished observations.


REFERENCES

  1. Goloubinoff, P., Christeller, J. T., Gatenby, A. A., and Lorimer, G. H. (1989) Nature 342,884-889 [CrossRef][Medline] [Order article via Infotrieve]
  2. Ostermann, J., Horwich, A., Neupert, W., and Hartl, F.-U. (1989) Nature 341,125-130 [CrossRef][Medline] [Order article via Infotrieve]
  3. Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L., and Hartl, F.-U. (1991) Nature 352,36-42 [CrossRef][Medline] [Order article via Infotrieve]
  4. Bochkareva, E. S., Lissin, N. M., Flynn, G. C., Rothman, J. E., and Girshovich, A. S. (1992) J. Biol. Chem. 267,6796-6800 [Abstract/Free Full Text]
  5. Gething, M.-J., and Sambrook, J. (1992) Nature 355,33-44 [CrossRef][Medline] [Order article via Infotrieve]
  6. Horwich, A. L., Brooks Low, K., Fenton, W. A., Hirshfield, I. N., and Furtak, K. (1993) Cell 74,909-917 [Medline] [Order article via Infotrieve]
  7. Braig, K., Otwinkowski, Z., Hegde, R., Boisvert, D., Joachimiak, A., Horwich, A. L., and Sigler, P. (1994) Nature 371,578-586 [CrossRef][Medline] [Order article via Infotrieve]
  8. Creighton, T. E. (1991) Nature 352,17-18 [CrossRef][Medline] [Order article via Infotrieve]
  9. Agard, D. A. (1993) Science 260,1903-1904 [Medline] [Order article via Infotrieve]
  10. Ellis, R. J. (1993) Nature 366,213-214 [Medline] [Order article via Infotrieve]
  11. Todd, J. M., Viitanen, P. V., and Lorimer, G. H. (1994) Science 265,659-666 [Medline] [Order article via Infotrieve]
  12. Chandrasekhar, G. N., Tilly, K., Woolford, C., Hendrix, R., and Georgopoulos, C. (1986) J. Biol. Chem. 261,12424-12419
  13. Gray, T. E., and Fersht, A. (1991) FEBS Lett. 292,254-258 [CrossRef][Medline] [Order article via Infotrieve]
  14. Langer, T., Pfeifer, G., Martin, J., Baumeister, W., and Hartl, F.-U. (1992) EMBO J. 11,4757-4765 [Abstract]
  15. Todd, J. M., Viitanen, P. V., and Lorimer, G. H. (1993) Biochemistry 32,8560-8567 [Medline] [Order article via Infotrieve]
  16. Frydman, J., Nimmesgern, E., Erdjument-Bromage, H., Wall, J. S., Tempst, P., and Hartl, F.-U. (1992) EMBO J. 11,4767-4778 [Abstract]
  17. Weissman, J. S., Kashi, Y., Fenton, W. A., and Horwich, A. L. (1994) Cell 78,693-702 [Medline] [Order article via Infotrieve]
  18. Gao, Y., Vainberg, I. E., Chow, R. L., and Cowan, N. (1993) J. Mol. Cell Biol. 13,2478-2485
  19. Rommelaere, H., Van Troys, M., Gao, Y., Melki, R., Cowan, N. J., Vandekerckhove, J., and Ampe, C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,11975-11979 [Abstract]
  20. Gao, Y., Thomas, J. O., Chow, R. L., Lee, G-H., and Cowan, N. J. (1992) Cell 69,1043-1050 [Medline] [Order article via Infotrieve]
  21. Viitanen, P. V., Lorimer, G. H., Seetheram, R., Gupta, R. S., Oppenheim, J. L., Thomas, J. O., and Cowan, N. J. (1992) J. Biol. Chem. 267,695-698 [Abstract/Free Full Text]
  22. Tian, G., Vainberg, I. E., Tap, W. D., Lewis, S. A., and Cowan, N. J. (1995) Nature 375,250-253 [CrossRef][Medline] [Order article via Infotrieve]
  23. Villasante, A., Wang, D., Dolph, P., Lewis, S. A., and Cowan, N. J. (1986) Mol. Cell Biol. 6,2409-2419 [Medline] [Order article via Infotrieve]
  24. Sternlicht, H., Farr. G., Sternlicht, M., Driscoll, J. K., Willison, K., and Yaffe, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,9422-9426 [Abstract]
  25. Spiegelman, B. M., Penningroth, S. M., and Kirschner, M. W. (1977) Cell 12,587-600 [Medline] [Order article via Infotrieve]
  26. Schagger, H., and Von Jagow, G. (1987) Anal. Biochem. 166,368-379 [Medline] [Order article via Infotrieve]
  27. Viitanen, P. V., Gatenby, A. A., and Lorimer, G. H. (1992) Protein Sci. 1,363-369 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.