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

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Molecular Chaperones in Cellular Protein Folding: the Birth of a Field Leading Edge Commentary Molecular Chaperones in Cellular Protein Folding: The Birth of a Field Arthur L. Horwich1,* 1Howard Hughes Medical Institute and Department of Genetics, 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 proteins 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). RNAs 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 native state, 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 Hsp70 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 chaperone’’ 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 inclusion bodies (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 endoplasmic reticulum (ER) and the cla- units of Rubisco inside of the chloroplast 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 globulin 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 chaperonins. 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 protein complex—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 fusion protein (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 gene 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 in vitro 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.
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