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Molecular Chaperones in Cellular Protein Folding: the Birth of a Field

Molecular Chaperones in Cellular Protein Folding: the Birth of a Field

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Molecular Chaperones in Cellular Folding: The Birth of a Field

Arthur L. Horwich1,* 1Howard Hughes Medical Institute and Department of , Yale University School of Medicine, Boyer Center, 295 Congress Avenue, New Haven, CT 06510, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2014.03.029

The early decades of Cell witnessed key discoveries that coalesced into the field of chaperones, protein folding, and protein quality control.

In January 1974, at the front of the first the term got repurposed to machines that On the other hand, a class of issue of Cell, Benjamin Lewin provided bind nonnative proteins—I think with known as heat shock proteins was an opening announcement entitled ‘‘A Ron’s blessing, as indicated by his willing- becoming the subject of considerable Journal of Exciting Biology,’’ establishing ness to attend early meetings of the field, scrutiny. In 1962, regions of Drosophila the goal of publishing the elucidation of speak about nuclear biology, and strum a salivary gland chromosomes were ob- systems responsible for cellular function few songs in the beer frame. As concerns served to become ‘‘puffed’’ during heat and phenotype. For those reading across protein folding at the launch of Cell, Chris- shock (Figure 2). induced under all or part of the 40 year span currently be- tian Anfinsen had recently received the these conditions were shown by in situ ing celebrated, there can be no question Nobel Prize in Chemistry (1972) for work hybridization to be produced from these that the goal has been met beyond all showing that the primary structure of a regions (Spradling et al., 1975; McKenzie expectation. I can think of so many aston- protein contains all of the information et al., 1975). It became clear with molecu- ishing revelations that were first brought necessary for folding to the , lar cloning of the abundant heat-induced to light in Cell. The Cell paper from which lies at an energetic minimum (Anfin- RNA that one of these regions encoded Chow et al. (1977) describing splicing— sen, 1973). Who could have thought at a 70 kDa heat-shock-induced protein ‘‘An amazing sequence arrangement at that point that thermodynamics would (Schedl et al., 1978). At about the same the 50 ends of adenovirus 2 messenger not be enough to produce the native time, it was observed that a characteristic RNA’’—stands out to me as the most active form of proteins inside of the cell? set of heat-inducible proteins, including dazzling early paper (coinciding with the Who would have imagined that kinetic a 70 kDa protein, was manifest in both equally stunning paper of Berget et al., assistance by a dedicated group of pro- E.coli (Lemaux et al., 1978; Yamamori 1977). In looking back and taking stock tein machines, in most cases utilizing et al., 1978; Bardwell and Craig, 1984) of an area close to my own heart, I would ATP, would be essential for the proper and metazoan fibroblasts (Kelley and say that, as a collective, the papers inves- folding of a large cohort of proteins? Schlesinger, 1978). It seemed likely that tigating the molecular machines that That realization emerged from two con- these inducible proteins would be pro- govern the folded state of proteins inside temporaneous but initially disconnected tective to the cell under stress. Was there of the cell—the chaperones—are equally sets of observations. On one hand, it a link between heat-shock-induced pro- distinguished in describing biology that became clear that many proteins could teins and the kinetic challenges of in vivo was unexpected and exciting. Here, I’ll not spontaneously refold in a test tube in protein folding? discuss how a number of diverse lines the same way as ribonuclease in Anfin- The work of Pelham was particularly of inquiry, published during the first two sen’s early experiments, lodging instead telling with respect to heat shock. He ob- decades of Cell’s history, coalesced into in insoluble aggregates that could be served that Drosophila expressed the field of chaperones, protein folding, sedimented to the bottom of the tube. In in mouse L cells or monkey COS cells and protein quality control as we now addition, in the cellular context, as ex- enabled rapid recovery of nucleolar dam- know it. pression of mammalian proteins in E.coli age following heat shock (Pelham, 1984). The term ‘‘molecular ’’ was undertaken in the late 1970s and He subsequently analyzed release of hadn’t been coined at the time Cell was early 1980s, it became clear that many Hsp70 from the nuclei isolated from launched. That had to wait until 1978, expressed proteins were subject to mis- heat-shocked cells, observing tight bind- when Ron Laskey used the term to folding, aggregation, and localization into ing of Hsp70 to the nuclei relative to the describe nucleoplasmin, a protein that terminal (Williams et al., nonshocked cells and rapid and complete binds and conveys histones into the nu- 1982; Marston, 1986; Haase-Pettingell release upon the addition of ATP (Lewis clear compartment, shielding positive and King, 1988; Figure 1). Thus, in these and Pelham, 1985). A model based on charge of the histones via its own acidic situations, there seemed to be kinetic these findings was presented in a Cell character (Laskey et al., 1978). Obviously difficulties during protein folding. Minireview (Pelham, 1986), proposing a

Cell 157, April 10, 2014 ª2014 Elsevier Inc. 285 (ER) and the cla- units of Rubisco inside of the thrin-uncoating ATPase in the cytosol. In stroma, but not with mature Rubisco, the former case, a 70 kDa protein was formed by assembly of the large subunits found to bind selectively to immuno- with small subunits imported from the heavy chains prior to their asso- cytosol (Barraclough and Ellis, 1980). ciation with light chains, indicating once John Ellis dubbed these ring complexes again a protein-protein interaction, here . The homology of GroEL potentially facilitating oligomeric assem- with Rubisco-binding protein was then bly (Haas and Wabl, 1983). In the latter appreciated upon sequencing of the case, studies of Rothman and coworkers respective coding regions (Hemmingsen (Schlossman et al., 1984; Chappell et al., et al., 1988). 1986) and of Ungewickell (1985) indi- A role for chaperonins in polypeptide cated that a 70 kDa protein was an chain folding, as distinct from oligomeric ATP-dependent mediator of uncoating assembly, soon emerged from studies clathrin cages from vesicles during endo- of a yeast mutant affecting a GroEL cytosis, releasing clathrin triskelions. This homolog in the mitochondrial matrix, amounted to an action more like that mitochondrial Hsp60 (Cheng et al., described by Pelham, in which binding 1989). In this mutant, proteins entering of the 70 kDa protein mediates disas- mitochondria failed to reach native form. sembly of a —in the Among the first proteins found to be case of clathrin, an action carried out affected in the mutant was a monomeric under normal physiologic conditions (by protein, the Rieske iron-sulfur protein. Figure 1. Evidence of Protein Misfolding In Vivo: Formation of Inclusion Bodies what we now know to be the constitutively This suggested that proper polypeptide Transmission electron micrograph showing for- expressed heat shock 70 ‘‘cognate’’ pro- folding, as opposed to oligomeric protein mation of inclusion bodies (arrowed) in E.coli tein, Hsc70 [Xing et al., 2010]). assembly of already-folded monomers, expressing a trp-proinsulin (from Shortly thereafter, cytosolic Hsp70 pro- might be the step facilitated by the chap- Williams et al., 1982). teins became implicated in transport of eronin ring assemblies. This role was protein precursors into ER and mitochon- further established by the observation cycle of action wherein Hsp70 binds to dria. The chaperone binds the protein to that monomeric DHFR imported into incipiently aggregating proteins (as pro- be transported in the cytosol, apparently mitochondria (by attachment of an duced by heat shock) and pries them preventing its hydrophobic surfaces from N-terminal mitochondrial targeting signal) apart through recurrent cycles of binding producing aggregation and holding it in associated in a nonnative form with the and release associated with ATP binding an unfolded state that could engage with Hsp60 complex and was subsequently and hydrolysis. Because Hsp70 was and pass through translocation machin- released in a native form upon addition known to strongly bind to hydrophobic ery (Chirico et al., 1988; Deshaies et al., of ATP (Ostermann et al., 1989). Hsp60 column matrices, it was proposed that 1988; Eilers and Schatz, 1986). These proved to be an essential in yeast, it recognizes hydrophobic surfaces of events were not stress related, indicating indicating a requirement for its action the misfolding proteins and prevents a constitutive need for the action of under all conditions (Cheng et al., 1989; them from driving aggregation. It was a 70 kDa class chaperone proteins. Reading et al., 1989). prescient model. Indeed, recognition by The apparently disparate worlds of pro- Mechanistic insights were enabled by molecular chaperones generally involves tein folding and molecular chaperones reconstitution experiments. The the binding of hydrophobic surfaces converged in the 1980s, with the charac- first reconstitution experiment was car- specifically exposed in nonnative pro- terization of a separate class of heat- ried out with the dimeric Rubisco from teins by hydrophobic surfaces proffered inducible ATP-hydrolyzing proteins: olig- R. rubrum. Denatured subunit diluted by the chaperones themselves, each omeric double-ring protein complexes from denaturant became bound to GroEL, chaperone family offering a different composed of 60 kDa subunits, the and subsequent addition of ATP and geometry of binding surface (Bukau and Hsp60s. These complexes were first cochaperonin GroES (a single ring Horwich, 1998). Subsequent binding of implicated as playing a role in oligomeric composed of 10 kDa ‘‘small’’ subunits) ATP then produces allosterically medi- assembly. Genetic defects in the bacterial led to production of native, active Rubisco ated movement of the binding surface GroE operon (Georgopoulos et al., 1972; (Goloubinoff et al., 1989). Further in vitro that releases the protein substrate (Zhur- Takano and Kakefuda, 1972) were refolding experiments with monomeric avleva et al., 2012; Kityk et al., 2012; Clare observed to affect the ability of propa- DHFR and rhodanese confirmed that et al., 2012). gating phages to assemble, but they GroEL/GroES could mediate refolding to Pelham’s observations concerning the also affected bacterial cell growth. Like- the native state of these proteins following action of Hsp70 fit well with concurrent wise, a role in oligomeric assembly was their dilution from chaotrope, whereas data that emerged from studies of two ascribed to the similarly sized Rubisco quantitative aggregation occurred upon other 70 kDa proteins, the immunoglob- subunit-binding protein, which could dilution into buffer (Martin et al., ulin-binding protein (BiP) inside of the associate with newly translated large sub- 1991). Apparently the system

286 Cell 157, April 10, 2014 ª2014 Elsevier Inc. apparently necessary due to ongoing mis- Chappell, T.G., Welch, W.J., Schlossman, D.M., steps of protein folding even at physio- Palter, K.B., Schlesinger, M.J., and Rothman, J.E. 45 logic in a milieu that has a (1986). Cell , 3–13. large concentration of solute. Not only Cheng, M.-Y., Hartl, F.-U., Martin, J., Pollock, R.A., could the chaperones prevent aggrega- Kalousek, F., Neupert, W., Hallberg, E.M., Hall- berg, R.L., and Horwich, A.L. (1989). Nature 337, tion from occurring, but they could also 620–625. help proteins to maintain unfolded confor- Chirico, W.J., , M.G., and Blobel, G. (1988). mations when necessary, e.g., when Nature 332, 805–810. emerging from or when pas- Chow, L.T., Gelinas, R.E., Broker, T.R., and Rob- sage through membranes required an erts, R.J. (1977). Cell 12, 1–8. unfolded state. Finally, in the case of the Clare, D.K., Vasishtan, D., Stagg, S., Quispe, J., chaperonins, they directly promote the Farr, G.W., Topf, M., Horwich, A.L., and Saibil, native state of proteins via folding inside H.R. (2012). Cell 149, 113–123. of an encapsulated chamber. Thus, the Deshaies, R.J., Koch, B.D., Werner-Washburne, existence of a diverse and dedicated M., Craig, E.A., and Schekman, R. (1988). Nature machinery for protein ‘‘management’’ in 332, 800–805. the cell had been uncovered. Eilers, M., and Schatz, G. (1986). Nature 322, 228–232. Georgopoulos, C.P., Hendrix, R.W., Kaiser, A.D., Postscript and Wood, W.B. (1972). Nat. New Biol. 239, 38–41. A host of other chaperone machines have Goloubinoff, P., Christeller, J.T., Gatenby, A.A., Figure 2. A Transcriptional Response to been identified in a variety of compart- and Lorimer, G.H. (1989). Nature 342, 884–889. Heat Stress ments—too many to list here. In addition, Haas, I.G., and Wabl, M. (1983). Nature 306, Drosophila busckii salivary gland chromosome the field has expanded to include the 387–389. spreads, showing ‘‘puffing’’ of two regions follow- characterization of stress-sensing path- ing temperature shift of larvae from 25C (top) to Haase-Pettingell, C.A., and King, J. (1988). J. Biol. 30C for 30 min (bottom) (from Ritossa, 1962). ways that provide exquisite regulation of Chem. 263, 4977–4983. the chaperone systems. It is now also Hemmingsen, S.M., Woolford, C., van der Vies, apparent that the chaperone systems S.M., Tilly, K., Dennis, D.T., Georgopoulos, C.P., link to the - and auto- Hendrix, R.W., and Ellis, R.J. (1988). Nature 333, could prevent or remove proteins from phagy systems as part of a global quality 330–334. kinetically trapped states and allow them control network. Unexpected discoveries Horwich, A.L., and Saibil, H.R. (2011). The GroEL/ to reach native form. The chaperonin re- are surely still to come. GroES chaperonin machine. In Molecular Ma- action could be experimentally broken chines in Biology, Joachim Frank, ed. (New York: into steps: binding to GroEL prevented Cambridge University Press), pp. 191–207. wholesale aggregation of the protein ACKNOWLEDGMENTS Kelley, P.M., and Schlesinger, M.J. (1978). Cell 15, substrate, and subsequent addition of 1277–1286. I’d like to particularly thank Walter Neupert and GroES/ATP produced the native state Kityk, R., Kopp, J., Sinning, I., and Mayer, M.P. Ulrich Hartl for a well-timed phone call that trig- over a period of minutes, with GroES (2012). Mol. Cell 48, 863–874. gered a wonderful collaboration to understand appearing to couple the folding reaction the mitochondrial-folding-defective yeast mutant Laskey, R.A., Honda, B.M., Mills, A.D., and Finch, to the GroEL ring assembly (Goloubinoff affecting Hsp60. I thank all of the talented people J.T. (1978). Nature 275, 416–420. et al., 1989; Martin et al., 1991). We now who have worked in my group over the years, as Lemaux, P.G., Herendeen, S.L., Bloch, P.L., and know that substrate proteins bind to a well as my collaborators, with whom I’ve enjoyed Neidhardt, F.C. (1978). Cell 13, 427–434. watching our field grow and develop. I thank the surrounding hydrophobic surface in the Lewis, M.J., and Pelham, H.R.B. (1985). EMBO J. NIH and Howard Hughes Medical Institute for cavity of an open GroEL ring and are, 4, 3137–3143. generous support of the chaperonin work. And 240 upon ATP/GroES binding, ejected into a I thank Ben Lewin and his successors for their Marston, F.A.O. (1986). Biochem. J. , 1–12. now hydrophilic GroES-encapsulated ongoing interest in our field, which I think has lived Martin, J., Langer, T., Boteva, R., Schramel, A., chamber where they proceed to fold in up to providing ‘‘exciting biology.’’ Horwich, A.L., and Hartl, F.-U. (1991). Nature isolation, without the chance of aggrega- 352, 36–42. tion (Horwich and Saibil, 2011). 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