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REVIEW

Protein quality control: triage by chaperones and

Susan Gottesman/'^ Sue Wickner/ and Michael R. Maurizi^ Laboratories of ^ and ^Cell Biology, National Cancer Institute, Bethesda, Maryland 20892-4255 USA

Proteases and chaperones together serve to maintain destruction of the target rather than reactivation. quahty control of cellular . Both types of en­ Therefore, we have named these -associated zymes have as their substrates the variety of misfolded chaperone activities charonins (van Melderen et al. 1996; and partially folded proteins that arise from slow rates of Maurizi et al. 1997). Chaperone activities have been folding or assembly, chemical or thermal stress, intrinsic demonstrated for three different families of ATP-depen­ structural instability, and biosynthetic errors. The pri­ dent proteases: The Clp family, the AAA proteases such mary function of classical chaperones, such as the Esch­ as FtsH, and Lon proteases (summarized in Table 1; Hor- erichia coh DnaK/ and its cochaperones, DnaJ and wich 1995; Gottesman 1996a; Langer and Neupert 1996; GrpE, and GroEL/Hsp60 and its cochaperone, GroES, is Schirmer et al. 1996). These protease families are found to modulate pathways, thereby prevent­ universally throughout both prokaryotes and eukary- ing misfolding and aggregation, promoting refolding and otes, primarily in the cytoplasm of prokaryotes and in proper assembly. Recent work has demonstrated that organelles of both lower and higher (see ATP-dependent proteases, as well as closely related pro­ Table 1). teins, have intrinsic chaperone activity, suggesting that the initial steps in energy-dependent protein degradation may be similar to those of chaperone-dependent protein Clp family folding. The classical chaperones are also required for The clearest examples of chaperone activities of prote­ degradation of certain proteins in vivo, but we propose ases have been provided by in vitro studies of the ATPase below that generally they affect indirectly by subunits of the E. coh ClpAP and ClpXP proteases. The maintaining proteins in soluble forms that would other­ ClpAP protease, the first member of this family to be wise aggregate and become inaccessible to proteases. In identified, is composed of two types of polypeptides, the this review we present the evidence linking the activi­ ClpA ATPase and the ClpP peptidase. A number of sub­ ties of chaperones and proteases, and propose a general families of closely related ATPases have also been iden­ model for what can be thought of as a triage system for tified, including ClpX, which in combination with ClpP, handling misfolded proteins in vivo, assuring swift re­ forms a different ATP-dependent protease with a unique folding of proteins with functional potential and rapid substrate specificity. The ClpY subfamily is closely re­ degradation of irreversibly denatured or damaged pro­ lated in sequence to ClpX; however, it has been shown teins. recently that ClpY combines with a completely different peptidase, ClpQ, to form an ATP-dependent protease (called ClpYQ or HslUV) (Kessel et al. 1996; Missiakas et Chaperone activities of proteases al. 1996; Rohrwild et al. 1996). Energy-dependent proteases recognize and select their Because the bond cleavage activity lies in a protein targets in an environment of other proteins that separate polypeptide (ClpP) from the substrate recogni­ should not be degraded. They then present the proper tion component (ClpA or ClpX), no proteolytic activity substrates, and only the proper substrates, to the proteo­ is displayed by the ATPase in the absence of ClpP. lytic active site. It seems reasonable to expect that pro­ Through in vitro studies of the ATPase components, it tein unfolding is necessary to move a portion of the sub­ has been demonstrated that both ClpA and ClpX have strate into position to be cleaved. Because degradation is chaperone-like activity, and some aspects of the reaction generally quite processive, further unfolding may be re­ mechanism are understood (Fig. 1). Although it has not quired to fully degrade the protein. Such unfolding might been reported yet, it is likely that ClpY, by itself, will be be considered analogous to the usual activities of classi­ found to have chaperone activity. cal chaperones, although in this case the result is the In a purified system, ClpA activates the bacteriophage Pi replication protein, RepA, for DNA binding. ClpA converts the inactive dimer of RepA into an active ^Corresponding author. monomer, a remodeling activity that requires ATP hy­ E-MAIL [email protected]; FAX (301) 496-3875. drolysis (Wickner et al. 1994). In every respect, activa-

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Table 1. ATP-dependent proteases

Substrate

Protease Occurrence" protease chaperone Comments References''

Clp family ClpAP E. coli; E. coli MazE; RepA (in vitro) ClpP: 2 rings of 7 Wickneret al. (1994); plant chloroplasts; N-end rule p-Gal luciferase (in vitro) subunits (peptidase Kesseletal. (1995); ClpP: human fusion proteins; activity only); ClpA: 1 Gottesman (1996) mitochondria Other p-Gal ring of 6 subunitS; 2 fusions; ATP-binding PI Rep A, casein, sites/subunit and other proteins (ATP-dependent (in vitro) chaperone); complex of ClpP and ClpA has ATP-dependent protease activity ClpXP £. coli; E. coli RpoS; PI Phd; MuA; XO protein (in ClpX: homo-oligomer; 1 Horwich (1995); ClpX: human, \0 protein; vitro) ATP binding Levchenko et al. 5. ceievisiae MuA (transposase); site/subunit (1995); mitochondria MuC (repressor) (ATP-dependent Wawrzynow et al. chaperone); ClpP and (1995, 1996); ClpX together have Kruklitis et al. ATP-dependent (1996) protease activity Clp YQ E. coli casein (in vitro) not examined ClpQ: 2 rings of 6 Missiakas et al. (HslUV) subunits (peptidase (1996); Kesseletal. activity only); ClpY: 1 (1996); Rohrwildet ring of 6 or 7 subunitS; al. (1996) 1 ATP-binding site/subunit (ATPase activity); complex of ClpQ and ClpY has ATP-dependent protease and peptidase activities

ClpB E. coli; S. ceievisiae none known protein aggregates; 1 ring of 6 subunitS; no Parselletal. (1994); Hspl04 () RNA splicing known Chemoff et al. Hsp78 components; \psi*]; hetero-oligomeric (1995); Moczko et (mitochondria) luciferase complexes al. (1995); Vogelet al. (1995); Schirmer et al. (1996) AAA family FtsH (HflB) E. coli; S. ceievisiae E. coli (T^^; E. coli membrane protein ATPase and protease Tomoyasu et al. SecY; \cll complexes active sites in one (1993, 1995); polypeptide; Herman et al. homo-oligomer; (1995); Akiyamaet membrane proteins al. (1996); Shiraiet with Zn active site; al. (1996) active sites in cytosol Ytal0/Ytal2 S. ceievisiae unassembled assembly of ATP ATPase and protease Pajicetal. (1994); (Afg3)/(Rcal) mitochondria membrane proteins synthase active sites in one Tauer et al. (1994); polypeptide; Arlt et al. (1996); hetero-oligomer; Guzelin et al. mutants have (1996); Langerand respiratory defects Neupert (1996) Lon family Lon E. coli £. coli SulA; E. coli unknown multimer of identical Maurizi (1992); RcsA; XN protein; subunitS; protease and Goldberg et al. F plasmid CcdA; ATPase active sites in (1994); Gottesman abnormal proteins one polypeptide (1996a,b) Lon (PIMl) mammalian, unassembled ATP assembly of ATP mutants accumulate Suzuki etal. (1994); S. ceievisiae synthase subunits synthase in YtalO mitochondrial DNA Wagner et al. mitochondria mutants (in deletions and have (1994); Rep et al. multicopy) respiratory defects (1996)

^(Occurrence) organisms in which the protein, as well as the , have been identified and studied. The proteases found in E. coli are also found in many other , and those found in S. ceievisiae and mammals are likely to be found in all eukaryotes. ''References are not all-inclusive. Emphasis is on review articles and papers demonstrating the chaperone activity of these proteases and related proteins.

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Protein triage by chaperones and proteases

Figure 1. Chaperone activity of the Clp proteases. Substrates (red) are remodeled in ATP-dependent reactions by the ATPase components (blue), ClpA or ClpX, of the ClpAP and ClpXP proteases. In the pres­ ence of the ClpP peptidase (green), the same substrates are degraded in an ATP- dependent fashion. For ClpA and its sub­ Substrate (RepA2, strate, RepA, the initial step in the remod­ MUA4-DNA, X O2) eling reaction is the formation of stable complexes of ClpA (six-membered rings of ClpA subunits) with inactive RepA dimers (Wickner et al. 1994; Kessel et al. 1995) containing equimolar amounts of ClpA hexamers and RepA dimers (Pak and Wick­ CIpA/X-ATP CIpA/X-ATP-CIpP ner 1997) in a reaction requiring binding of Atf only. Upon ATP hydrolysis, RepA monomers are released in a form active in DNA binding. In the alternative pathway, ClpAP [a complex of ClpA hexamers and two seven-membered rings of ClpP sub- units (Kessel et al. 1995)] probably forms complexes with RepA dimers without a need for hydrolysis of the bound ATP. r Substrate-ClpA/X-ATP Substrate-ClpA/X-ATP-CIpP With ATP hydrolysis, RepA is degraded. Very likely by similar mechanisms, ClpX ATP^ V^ATP disassembles MuA tetramers bound to ^§ DNA (Levchenko et al. 1995) and disaggre­ ADP + Pj.«—/ CIpP V---^ ADP + Pj gates \0 protein (Wawrzynow et al. 1995) and ClpXP degrades both of these sub­ C\pAfX<——7 V ^^ CIpA/X-CIpP strates (Wojtkowiak et al. 1993; Levchenko et al. 1995). However, the structure of ClpX and the intermediates in protein re­ oo modeling and degradation have not been Active/Stable Protein Degraded Protein described.

tion of RepA by ClpA is identical to activation by the Two close relatives of ClpA, ClpB from E. coli and purified DnaJ/DnaK/GrpE chaperone system, which ap­ Hspl04 from , have not been pears to be the chaperone system required for activation implicated in proteolysis or shown to have any associ­ of RepA in vivo (Bukau and Walker 1989; Tilly and Yar- ated peptidase subunits but do exhibit chaperone-like molinsky 1989; Wickner et al. 1991, 1992). ClpX stimu­ activities on a variety of substrates and are necessary for lates replication of bacteriophage Mu by promoting the thermotolerance in vivo (Squires et al. 1991; Sanchez et disassociation of the MuA protein from DNA al. 1992; Schirmer et al. 1996). In vivo, Hspl04 functions (Levchenko et al. 1995; Kruklitis et al. 1996). This ClpX in the reactivation of luciferase and mRNA splicing ac­ chaperone activity also requires ATP hydrolysis. In vivo, tivity after heat inactivation (Parsell et al. 1994; Vogel et Mu replication is impaired in a clpX mutant but not in a al. 1995). In both cases, Hspl04 acts to resolubilize pro­ clpP mutant, confirming that the biologically important tein aggregates. Another Clp ATPase, yeast mitochon­ reaction dependent on ClpX is related to its chaperone drial Hsp78 (mtHSPZS), provides partial redundancy activity rather than its activity as part of the ClpXP pro­ with mtHspZO in promoting import of mitochondrial tease (Mhammedi-Alaoui et al. 1994). proteins and preventing aggregation of some misfolded ClpX and ClpA also have other in vitro activities char­ proteins (Schmitt et al. 1995). The explanation for this acteristic of chaperones. ClpA protects luciferase from may be that mtHsp78 helps to maintain function and/or irreversible heat inactivation, although it cannot pro­ solubility of the mutant mtHsp70 (Moczko et al. 1995). mote reactivation of the heat-treated enzyme (Wickner An overlap in function has also been found for Hspl04 et al. 1994). ClpX can prevent aggregation of \0 protein and some of the cytoplasmic of yeast (Sanchez et and aid in its disaggregation (Wawrzynow et al. 1995). al. 1993). Some of these activities (e.g., preventing aggregation) do not require ATP hydrolysis and hence do not appear to involve active refolding but may reflect the ability of the FtsH/AAA Family ATPase subunit to bind to regions of unfolded or mis- The FtsH/AAA family of proteases are anchored to folded proteins. membranes but contain cytoplasmic domains with

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ATPase activity and Zn^"^ metalloprotease active sites membrane proteins, a function that appears to reflect (Tomoyasu et al. 1993, 1995). In eukaryotes, these pro­ chaperone activity rather than proteolysis (Rep et al. teases are found associated with mitochondria. Muta­ 1996). As with Ytal0/Ytal2, a mutation in the protease tions in YtalO or Ytal2 (two mitochondrial FtsH-like active site of the overproduced Lon also promotes mem­ proteins, also called Afg3 and Real) block both the deg­ brane protein assembly. Membrane assembly is actually radation of abnormal proteins and membrane protein as­ enhanced in this mutant. Therefore, like the Clp and sembly (Pajic et al. 1994; Tauer et al. 1994). Evidence AAA proteases, Lon appears to have protein remodeling from in vivo studies suggests that these two related poly­ activity, judging by its in vivo role in membrane assem­ assemble to form a heterooligomeric ATP-de- bly. pendent protease with associated chaperone activities. Mutations in the protease active site of either subunit interfere with in vivo degradation of misfolded proteins Chaperone activity of proteases is important for but do not impair the function of YtalO or Ytal2 in al­ proteolysis lowing cell growth on nonfermentable carbon sources, a These examples suggest that the protein-binding do­ measure of mitochondrial function (Arlt et al. 1996; Gu- mains of the ATP-dependent proteases can interact with zelin et al. 1996). The protease active site mutants retain substrates and promote chaperone-like remodeling/re­ the ability to promote assembly of the ATP synthase, folding of proteins. Within the Clp family, the chaperone demonstrating an in vivo chaperone-like activity that is activities show specificity. Proteins closely related to independent of protease activity. ClpA, including ClpX and the even more closely related In E. coli, some aspects of membrane assembly and ClpB/Hspl04 proteins, cannot act on RepA (Wickner et protein localization depend on FtsH in a pathway that al. 1994), and, conversely, ClpA does not disassemble may be independent of its degradation activity; the re­ MuA-DNA complexes (Levchenko et al. 1995). The quirement for FtsH can be suppressed by overproduction specificities of the chaperone activities are mirrored by of chaperones (Akiyama et al. 1994; Shirai et al. 1996). the observed substrate specificities of the ClpAP and Whether this is a true chaperone activity of FtsH re­ ClpXP proteases. When ClpP is present, the chaperone mains to be shown. substrates become substrates for proteolysis; ClpAP de­ The ability of the Ytal0/Ytal2 protease complex to grades RepA but not MuA, and ClpXP degrades XO pro­ both promote assembly and degrade subunits of the un­ tein and MuA but not RepA (Gottesman et al. 1993; assembled ATP synthase leads to an interesting problem. Wojtkowiak et al. 1993; Wickner et al. 1994; Levchenko What is the molecular mechanism underlying the deci­ et al. 1995). sion to direct polypeptide chains into the assembly path­ For Lon, direct evidence that unfolding is a necessary way or to present them for proteolysis? For proteases in part of the proteolytic pathway comes from in vitro stud­ which the chaperone (i.e., ClpA or ClpX) and the proteo­ ies of Lon degradation of a known in vivo substrate, the lytic activity (i.e., ClpP) are on separate subunits, the 72-amino acid F plasmid CcdA protein and a 41-amino chaperone component may sometimes be free of the pro­ acid carboxy-terminal fragment of the same protein, teolytic component and therefore able to participate in­ CcdA41 (van Melderen et al. 1996). Native CcdA dis­ dependently in protein folding and assembly. For prote­ plays significant secondary structure (as judged by far UV ases in which the protease active site resides in the same circular dichroism measurements) and is degraded by polypeptide as the ATPase, two related possibilities can Lon in an ATP hydrolysis-dependent reaction. However, be considered. If cycles of substrate release and rebinding heat treatment results in a loss of secondary structure in occurred before commitment to the degradation path­ CcdA and diminishes the requirement for ATP hydroly­ way, only proteins unable to reach their native state of sis for degradation. The truncated CcdA41 protein is un­ folding and assembly (therefore no longer recognized as a structured even at ambient temperature and is degraded substrate) would be available to be degraded. Alterna­ without ATP hydrolysis, suggesting that ATP hydrolysis tively, there may be separate sites on the chaperone por­ is required only when unfolding is necessary (i.e., with tion of the protease for substrate binding, one leading to CcdA, not CcdA41). Similarly, degradation of small poly­ degradation and another leading to refolding. These pos­ peptides by Lon and ClpAP proteases are catalyzed with­ sible mechanisms await testing. out ATP hydrolysis, although degradation of larger pro­ tein substrates requires cleavable ATP (Waxman and Goldberg 1986; Maurizi 1987; Thompson and Maurizi Lon family 1994). These results suggest that unfolding of complex Lon protease is a homo-oligomer, each subunit of which substrates requires the ATP-dependent chaperone activ­ carries a protease active site and an ATP-binding site ity of the protease for efficient proteolysis. consensus (Goldberg et al. 1994; Gottesman 1996a,b). It is a cytoplasmic protein in prokaryotes and a mitochon­ Participation of classical chaperones in proteolysis drial matrix protein in eukaryotes. Lon participates in the proteolysis of both specific protein targets (such as Because protein unfolding is an essential step for prote­ Res A and SulA in E. coli] and abnormal proteins. Lon olysis by the energy-dependent intracellular proteases, overproduction in yeast mitochondria can substitute for can classical chaperones provide or augment this func­ the FtsH-like Ytal0/Ytal2 protease in the assembly of tion, and is this an important component of protein deg-

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Protein triage by chaperones and proteases radation in vivo? Although a coupled unfolding-degrada- HspZO, p60, and other factors and could be reactivated in tion reaction requiring both chaperones and energy-de­ an ATP-dependent reaction in reticulocyte lysates pendent proteases has not been demonstrated in a (Schneider et al. 1996). When was inhibited (by purified in vitro system, a number of observations have ansamycins), the rate of luciferase degradation increased, suggested that classical chaperones of the Hsp70 and whereas the rates of both luciferase release from Hsp90 Hsp60 families participate in protein degradation in and luciferase reactivation decreased. Degradation is pre­ vivo. Unfortunately, the pleiotropy of most chaperone sumably by the -dependent 26S protease; this mutants complicates the interpretation of many in vivo study suggests that in contrast to the proteases discussed experiments. above, the chaperone-bound polypeptide is the substrate The primary observation linking chaperone activity for either ubiquitin-tagging or proteolysis. and proteolysis is that mutations in the gene [htpR] for the general heat shock regulator of E. coli, RpoH, can lead to significantly slowed degradation of abnormal pro­ Quality control: participation of chaperones and teins (Baker et al. 1984; Goff et al. 1984). The genes for proteases many cellular proteases are under heat shock control, Under normal, wild-type conditions, the function of including the ATP-dependent Lon, ClpXP, FtsH, and chaperones is to maximize the proper folding, refolding, HslUV (ClpYQ) proteases (Gottesman 1996a; Gross and assembly of proteins, and that of proteases is to dis­ 1996). Although an htpR mutant would be expected to pose of those that cannot be folded. For such a triage have lower expression of these proteases, the defects in system to work properly, there must be stages in the proteolysis appear to be much greater than the defects in process that allow a given protein to be examined for protease synthesis. Therefore, it is unlikely that de­ folding capability so that the appropriate pathway can be creased levels of proteases provide the full explanation chosen. Below, we discuss the possible ways in which for the decreased protein degradation activity in htpR chaperones might affect proteolysis and whether these mutants. effects provide some type of quality control. Mutations in specific chaperones (DnaK/DnaJ/GrpE One could imagine that chaperones act in proteolysis and GroEL/GroES) generally result in lowering the to promote synthesis, assembly, or activation of the pro­ amount of protein degradation in cells but, in specific teases themselves. This possibility has not been tested instances, can lead to increased degradation of individual for most of the ATP-dependent proteases. In the one in­ substrates (Keller and Simon 1988; Straus et al. 1988; stance where it has been examined, for the Lon protease Kroh and Simon 1991; Kandror et al. 1994; Jubete et al. in E. coli, it is not the case, as the effect of a dnaJ mu­ 1996). As mutations in dnaj, dnaK, and grpE can lead to tation on the degradation of Lon substrates is substrate- constitutive expression of the and specific rather than protease-specific (lubete et al. 1996). consequently to increased protease gene transcription Another possibility is that chaperones act directly and (Gross 1996), the decreased degradation generally seen in intimately in the proteolytic pathway, feeding all or chaperone mutants is all the more striking. Overproduc­ many substrates to the protease. By binding to a sub­ tion of GroEL and GroES can help protect some proteins strate, the chaperone would tag the substrate for destruc­ from degradation (LaRossa and Van Dyk 1991); in other tion. The protease might recognize either features of the cases, they can stimulate proteolysis of a specific abnor­ chaperone-protein complex itself or an exposed region of mal protein (Kandror et al. 1994), suggesting that GroEL/ the substrate made accessible by chaperone binding. In GroES chaperones also function in the degradation path­ this model, the triage process for separating refoldable way for some proteins. proteins from those that should be degraded would have Several in vitro observations have also implicated to be reflected in the nature of the chaperone-substrate chaperones in ATP-dependent proteolysis. For example, complex. For instance, are complexes of chaperones with CRAG, a hybrid abnormal protein whose degradation de­ "unfoldable" proteins relatively more stable, giving pro­ pends in large part on ClpP and to some extent on Lon, teases sufficient time to interact with the chaperone- was found associated with GroEL and another protein bound protein? Or, are there different modes of chaper­ suspected to have chaperone activity, trigger factor (Kan­ one binding to refoldable and nonrefoldable substrates, dror et al. 1994, 1995). Similarly, PhoA61, a nonsecreted giving rise to structurally different complexes, only one mutant form of alkaline phosphatase that is degraded of which is recognized by the protease? rapidly in vivo, is found in cell extracts associated with Although some data support predictions of this model, both DnaK and to a lesser extent, Lon (Sherman and others do not. The decreased degradation of abnormal Goldberg 1991, 1992). The degree of association of proteins in chaperone mutants, reviewed above, might CRAG with GroEL and of PhoA61 with DnaK correlates be interpreted as evidence for protease recognition of the well with the rate of degradation of these proteins, sug­ substrate only in the context of the chaperone. Data gesting that the complexes may be intermediates in the showing increased degradation of Hsp90-bound lucifer­ degradation pathway. The yeast cytoplasmic Dnal ho- ase (discussed above) suggest that in that system the molog Ydjl is also found associated with specific un­ chaperone-bound substrate may be directly presented for stable proteins (Lee et al. 1996). proteolysis (Schneider et al. 1996). A similar increase in In a recent report, denatured luciferase was found to be degradation of some substrates in a dnaK756 mutant in complexes with the eukaryotic Hsp90 chaperone. might reflect binding that mimics that of a nonrefoldable

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Gottesman et al. protein (Sherman and Goldberg 1992). However, degra­ chaperone requirement for proteolysis be substrate spe­ dation of other substrates, including abnormal proteins, cific, and the direct participation of chaperones in prote­ is blocked rather than enhanced in the same dnaK mu­ olysis cannot be a prerequisite for all protein degrada­ tant, which is inconsistent with the targeting of protein- tion. There may be some specific substrates for which DnaK complexes for proteolysis (Straus et al. 1988). In interaction with a chaperone is essential for degradation, addition, no in vitro evidence supports the idea that but efforts to demonstrate this in vitro, for example, by binding of some (nonrefoldable) substrates to chaperones including chaperones in degradation reactions in vitro, is more stable or qualitatively different from the binding have been unsuccessful thus far (Sherman and Goldberg of other (refoldable) substrates. 1992). If chaperone-protein complexes are the targets for pro­ tein degradation, one might also expect specificity in the A kinetic model for protein triage coupling of chaperones and proteases and a requirement for the specific chaperone for efficient proteolysis by a The bulk of the available evidence supports an indirect given protease. This is certainly not true for all degrada­ role for chaperones in proteolysis, reflecting the ability tion. In vivo, dnaj is necessary for Lon-dependent degra­ of chaperones to maintain abnormal proteins, potential dation of abnormal proteins and RcsA, whereas two substrates for degradation, in a soluble state. In this other substrates, SulA and XN protein, are degraded nor­ model (outlined in Fig. 2), chaperones assist in generat­ mally in dnaJ mutants (Jubete et al. 1996). In vitro, Lon ing and stabilizing a population of soluble, misfolded, can degrade XN protein, CcdA, and SulA; ClpXP can de­ non-native forms of polypeptides by binding and releas­ grade MuA and XO protein; and FtsH can degrade u^^ ing misfolded or unassembled proteins. This pool also without the participation of chaperones (Maurizi 1987; will include non-native forms arising from new synthe­ Wojtkowiak et al. 1993; Levchenko et al. 1995; Sonezaki sis of some normal as well as abnormal proteins and et al. 1995; Tomoyasu et al. 1995; van Melderen et al. from heat or chemical denaturation. The fates of these 1996). Therefore, this model would also require that the misfolded proteins, and therefore quality control, would

Pool of soluble, non-native proteins V-«»J from: • Synthesis of normal and abnormal proteins. • Cycles of chaperone binding and release. Non-native forms of a protein I • Heat or chemical denaturation.

Protease o e o Chaperone + ATP + ATP Kinetic partitioning of non-native proteins: ADP ^

Cliaperone ^ Capture of non-native protein uAT P by protease and degradation. ^ Spontaneous aggregation of mis­ Substrate-Chaperone folded proteins and disaggregation Substrate-Protease Complex by chaperones. Complex ADP ^ Binding of non-native protein to Inactive/Aggregated ADP chaperone and release of either Protein h -i active protein or a non-native \^^ form. Native/Active/Stable Protein Degraded Protein

Figure 2. A kinetic model for protein triage. Polypeptides in unfolded and other non-native forms (shown in red) may interact with a number of other cellular proteins. The relative rates at which these occur will determine the ultimate fate of the protein, (1) They may be recognized by proteases (shown in purple and green), almost always leading to degradation; (2) they may aggregate with themselves or other proteins; reversal of aggregation may in some cases be promoted by chaperones, thus returning these polypeptides to the pool of soluble proteins; or (3) they may bind chaperones (shown in blue). Release from the chaperone may either return the protein to the soluble pool of non-native forms or may result in an active/refolded protein that is no longer a substrate for either protease or chaperone binding. In the absence of chaperones, a competition between the rates of aggregation and degradation will determine the fate of the polypeptide. Naturally unstable proteins obey similar rules; relative rates of degradation and aggregation will determine the sensitivity of degradation to chaperones. We would predict that in the eukaryotic cytoplasm, the systems play the primary recognition role akin to that of the protease shown here.

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Protein triage by chaperones and proteases reflect the relative affinities of non-native forms for pro­ drial ATP synthases that depend on the chaperone activ­ teases, chaperones, and other cellular components and ity of the AAA protein family for assembly become sub­ the relative rates of degradation, aggregation, and folding ject to degradation (by the same protease) when assembly (Fig. 2, pathways 1, 2, and 3, respectively). If the native is blocked by incorrect subunit stoichiometery (Pajic et form of a protein were reached (and the hydrophobic re­ al. 1994; Tauer et al. 1994; Guzelin et al. 1996). In these gions were buried), either spontaneously or via chaper- cases, degradation outpaces aggregation. We propose that one binding and release, the protein would no longer be in other cases of failed assembly, degradation is not ob­ recognized by chaperones or proteases as an appropriate served because aggregation outpaces degradation. target. Alternatively, some of the non-native structures might have higher affinity for specific proteases than for chaperones, thereby directing the polypeptide down the Conclusions pathway of degradation. Other non-native, misfolded The initial steps in the pathway of protein degradation forms (presumably with very large hydrophobic regions by energy-dependent proteases show many of the char­ exposed) might aggregate rapidly and become inacces­ acteristics of chaperone-dependent protein remodeling, sible to either proteases or to chaperones. The bulk of the and, in , ATP-dependent proteases have intrinsic pro­ current data supports this model of kinetic partitioning tein remodeling activity. This overlap in function prob­ of substrates. ably reflects the requirement for energy-dependent pro­ If this kinetic model is reliable, in the absence of chap­ teases to recognize and bind non-native proteins and un­ erones there should be a significant increase in aggre­ fold them sufficiently to allow them entry into the gated proteins in vivo and aggregation should correlate proteolytic active site, just as classical chaperones must with decreased degradation. These predictions seem to recognize and bind non-native proteins in the pathway of be consistent with many experimental findings. Abnor­ folding or remodeling. The recognition of the unfolding mal proteins have been found to aggregate in the absence activity of proteases raises the question of whether clas­ of DnaJ, and even newly made normal proteins appear in sical chaperones play a role in presenting substrates to aggregates in cells mutant in both the GroEL/GroES and the protease. We suggest that this role is indirect and DnaK/DnaJ/GrpE chaperone systems (Gragerov et al. that chaperones generally contribute to the rapid degra­ 1992; Jubete et al. 1996). In cells devoid of DnaJ, the Lon dation of abnormal proteins by maintaining them in a substrate RcsA aggregates and its degradation by Lon is relatively soluble and therefore protease-accessible form. slowed. Degradation of another Lon substrate, SulA, oc­ The similiarities between the early stages of energy-de­ curs at the same rate in the presence or absence of DnaJ pendent proteolysis and chaperone-mediated protein in lon'^ cells, but degradation in the absence of Lon folding suggest that our understanding of each will be shows significant dependence on DnaJ. In lon dnaj enriched by a study of the other. double mutants, SulA is found in an insoluble fraction, suggesting that the protein aggregates more rapidly than it is attacked by other proteases in the cell (Jubete et al. References 1996). In eukaryotes, mutations in mitochondrial DnaK Akiyama, Y., T. Ogura, and K. Ito. 1994. Involvement of FtsH in and DnaJ homologs (mtHsp70 and Mdjl) slow the degra­ protein assembly into and through the membrane. I. Muta­ dation of misfolded dihydrofolate reductase (DHFR), tions that reduce retention efficiency of a cytoplasmic re­ which is dependent on the Lon (PIMl) protease; the porter. /. Biol. Chem. 269: 5218-5224. DHFR is found in an aggregated state in the chaperone Akiyama, Y., A. Kihara, H. Tokuda, and K. Ito. 1996. FtsH (HflB) mutants (Wagner et al. 1994). Thus, in several cases in is an ATP-dependent protease selectively acting on SecY and which chaperone mutants have been shown to slow deg­ some other membrane proteins. /. Biol. Chem. 271: 31196- radation, the undegraded substrates are found to be ag­ 31201. gregated. Arlt, H., R. Tauer, H. Feldmann, W. Neupert, and T. Langer. 1996. The YtalO-12 complex, an AAA protease with chap- An important additional in vivo activity of classical erone-like activity in the inner membrane of mitochondria. chaperones that can have profound effects on protein Cell 85: 875-885. degradation is the participation of chaperones in assem­ Baker, T.A., A.D. Grossman, and C.A. Gross. 1984. A gene regu­ bly of multiprotein complexes. In many of these cases, lating the heat shock response in E. coli affects proteolysis. the components of the multimeric complexes are totally Proc. Natl. Acad. Sci. 81: 6779-6783. stable when properly assembled but are subject to rapid Bukau, B. and G.C. Walker. 1989. AdnaK52 mutants of Esche­ degradation when unassembled, either because compo­ richia coli have defects in segregation and plasmid mainte­ nents are not present in the proper stoichiometry (which nance at normal temperatures. /. Bacteriol. 171: 6030-6038. we have referred to as "proteins without partners" (Got- Chemoff, Y.O., S.L. Lindquist, B. Ono, S.G. Inge-Vechtomov, tesman 1989; Gottesman and Maurizi 1992) or because and S.W. Liebman. 1995. Role of the chaperone protein defects in chaperone activity slow the assembly process Hspl04 in propagation of the yeast -like facter \psi*]. Science 268: 881-S84. and therefore allow a switch in the kinetic hierarchy, as Goff, S.A., L.P. Casson, and A.L. Goldberg. 1984. Heat shock shown in Figure 2. For instance, degradation of the SecY regulatory gene htpR influences rates of protein degradation protein in E. coli by FtsH is observed when the proteins and expression of the lon gene in . Pioc. with which it normally assembles are not available Natl. Acad. Sci. 81: 6647-6651. (Kihara et al. 1995). Similarly, subunits of the mitochon­ Goldberg, A., R.P. Morschell, C.H. Chung, and M.R. Maurizi.

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Gottesman et al.

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Protein quality control: triage by chaperones and proteases.

S Gottesman, S Wickner and M R Maurizi

Genes Dev. 1997, 11: Access the most recent version at doi:10.1101/gad.11.7.815

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