The Ins and Outs of GroEL-Mediated Protein Folding

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sequences in the hopes of detecting additional, new relationships among this diverse array of proteins. Suzanne Pfeffer Department of Biochemistry Stanford University School of Medicine Stanford, California 94305

Pfeffer, S.R. (1999). Transport vesicle targeting: tethers before SNARES. Nat. Cell Biol. 1, E17–E22. Sacher, M., Barrowman, J., Wang, W., Horecka, J., Zhang, Y., Pypaert, M., and Ferro-Novick, S. (2001). TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport. Mol. Cell 7, 433–442. Sato, T.K., Rehling, P., Peterson, M.R., and Emr, S.D. (2000). Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion. Mol. Cell. 6, 661–671.

Chen, Y.A., and Scheller, R.H. (2001). SNARE-mediated membrane fusion. Nat. Rev. Mol. Cell. Biol. 2, 98–106.

Schaffer, A.A., Aravind, L., Madden, T.L., Shavirin, S., Spouge, J.L., Wolf, Y.I., Koonin, E.V., and Altschul, S.F. (2001). Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res. 29, 2994–3005.

Conibear, E., and Stevens, T.H. (2000). Vps52p, Vps53p and Vps54p form a novel multisubunit complex required for protein sorting at the yeast late Golgi. Mol. Biol. Cell 11, 305–323.

TerBush, D.R., Maurice, T., Roth, D., and Novick, P. (1996). The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15, 6483–6494.

Guo, W., Sacher, M., Barrowman, J., Ferro-Novick, S., and Novick, P. (2000). Protein complexes in transport vesicle targeting. Trends Cell Biol. 10, 251–255.

Whyte, J.R.C., and Munro, S. (2001). Characterization of the Sec34/ 35 Golgi transport complex reveals it to be a relative of the exocyst, defining a family of protein complexes involved in multiple steps of membrane traffic. Dev. Cell 1, 527–537.

Selected Reading

Peterson, M.R., and Emr, S.D. (2001). The class C vps complex functions at multiple stages of the vacuolar transport pathway. Traffic 2, 476–486.

The Ins and Outs of GroEL-Mediated Protein Folding Two papers recently published in Cell investigate the role of protein encapsulation by GroEL in assisting folding. The first shows how encapsulation can “smooth” the folding landscape, accelerating folding of some proteins. The second defines a confinementindependent pathway, which allows GroEL to assist folding of substrates too large to be encapsulated. Chaperonins, such as the Escherichia coli protein GroEL, are large, double-ring structures, which form a central cavity that provides a protected environment in which proteins can fold. GroEL-mediated folding in general requires ATP hydrolysis as well as the co-chaperonin GroES, which acts as a lid to the GroEL box. A convergence of structural and mechanistic studies has revealed how ATP, GroES, and polypeptides interact in a chaperonin reaction cycle (Figure 1; Sigler et al., 1998; Rye et al., 1999). For the present discussion, the critical intermediate in this reaction cycle is a cis ternary complex in which polypeptide and GroES bind to the same GroEL ring. In this cis complex, polypeptide is sequestered within the cage formed by the GroEL-GroES complex. This sequestration provides the substrate with a relatively polar environment that favors protein folding while preventing inappropriate interactions with other misfolded proteins. The importance of protein encapsulation has been established by a number of mechanistic studies (Sigler et al., 1998). For example, earlier work had shown that for the typical intermediate sized GroEL-GroES substrate ornithine transcarbamylase, chaperonin-mediated folding required formation of the cis complex (Weissman et al., 1995). Moreover, by preventing release of GroES or substrate, it was proven that polypeptides could com-

Zerial, M., and McBride, H. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell. Biol. 2, 107–117.

plete folding while they remained sequestered within the GroEL-GroES complex (Mayhew et al., 1996; Weissman et al., 1996). While those studies demonstrated that encapsulation was a critical step in the reaction cycle, the extent to which substrate folding might also occur outside of the GroEL-GroES box was less well understood. This is an important issue since it is known that during GroELGroES folding reactions, substrates are continually being released in a nonnative form into the bulk solution (Todd et al. 1994; Weissman et al., 1994), raising the possibility that significant folding might also occur after release. Using an elegant strategy in which substrate rebinding to GroEL is rapidly inhibited by binding of streptavidin to biotin covalently attached to the GroEL apical substrate binding domain, Brinker and coworkers (2001) were able to directly evaluate whether folding required continued association between polypeptide and GroEL. The results are striking: under nonpermissive conditions (i.e., conditions in which the substrates do not fold in the absence of GroEL and GroES), folding of rhodanese and RuBisCo halted as soon as reassociation between substrate and GroEL is prevented. Even more remarkable, under conditions where RuBisCo was able to fold on its own but GroEL-GroES accelerated folding, inhibiting the reassociation between RuBisCo and GroEL caused an immediate shift from the fast, assisted rate to the slower spontaneous rate. Thus both the ability to rescue folding under nonpermissive conditions and the ability to accelerate folding under permissive conditions required confinement of the unfolded protein in the GroEL-GroES cavity. Moreover, as suggested by some theoretical studies (e.g., Zhou and Dill, 2001), confinement of the folding reaction within the limited space in the GroEL-GroES complex seemed to play an active role in altering, and in the case of RuBisCo accelerating (Todd et al., 1994), the rate of folding. If folding by GroEL-GroES requires encapsulation within the GroEL central cavity, what then is the fate of sub-

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Figure 1. Schematic of the Standard “cis” GroEL-GroES Reaction Cycle See Chaudhuri et al., 2001, for a description of the novel “trans” reaction used for folding of substrate too large to be encapsulated. (i) The nonnative substrate (yellow line figure) binds to the open trans ring of the GroEL-GroES-ADP (D) asymmetric complex. (ii) ATP (T) and GroES (orange figure) bind to the same ring occupied by the substrate, forming a cis ternary complex in which the polypeptide is encapsulated within the GroEL-GroES structure. Binding of GroES induces a large conformational change in GroEL that leads to an approximate doubling of the volume of the central cavity and obscures GroEL’s hydrophobic polypeptide recognition regions. As a consequence, the substrate polypeptide in the cis complex is encapsulated within a relatively polar environment that favors folding. (iii) ATP bound to the cis complex acts as a timer, giving the substrate at least 8–10 s to fold inside the cavity after which ATP hydrolysis primes GroES for release from the cis ring. (iv) ATP hydrolysis in the cis ring also induces a conformational change in the trans (bottom) ring allowing it to rapidly ATP and a nonnative polypeptide. (v) Binding of ATP induces dissociation of the cis ligands (GroES and polypeptide). This dissociation is accelerated 30- to 50-fold by binding of nonnative substrates to the trans ring. Note that polypeptide can be released in either a native form (N), a form committed to reaching the native state in the bulk solution (Ic), or an uncommitted nonnative state (Iuc), which is either recaptured by another chaperone or eventually targeted for proteolysis.

strates too large (greater than ⵑ60 kDa) to be encapsulated? The simple answer might be that larger proteins cannot use the GroEL-GroES system. However, a recent study showed that in vivo loss of the mitochondrial GroEL or GroES homologs, Hsp60 or Hsp10, led to the misfolding of an 82 kDa protein, aconitase (Dubaquie et al., 1998). Chaudhuri et al. (2001) examined the basis for this chaperonin dependence of aconitase folding and in doing so have defined a novel and quite unexpected chaperonin folding cycle in which GroES assists GroEL-mediated folding without encapsulating the substrate. Chaudhuri et al. first established that GroEL-GroES could directly mediate the folding of aconitase and then confirmed that aconitase was in fact too large to be encapsulated within the cis GroEL-GroES complex. Finally, using a single ring mutant of GroEL, as well as a mixed double ring complex in which one of the rings contains mutations that prevented both substrate and GroES binding—the same tools that were instrumental in defining the standard cis reaction cycle—Chaudhuri et al. demonstrated that folding of aconitase required ATP and GroES binding to the trans ring. Thus in contrast to the cis folding reaction, here GroES binding to one GroEL ring induces a conformational change in the second ring allowing productive folding of larger substrates. Thanks to these two papers, we now know that encapsulation changes the energy landscape of a folding reac-

tion in a way that is critical for the assistance of folding of typical intermediate sized substrates. In addition, we know that there is an alternate pathway used by larger substrates that does not involve encapsulation. Several questions are of immediate interest. How does encapsulation lead to a smoothing of the energetic landscape, thus accelerating folding? How many other substrates use the alternate trans reaction? How does binding of GroES to one ring induce a conformational change that is transmitted over 140 A˚ to allow folding of substrate from the opposite GroEL ring? Finally, does folding of larger substrates occur while the polypeptide is bound to the GroEL ring or after release into solution? Jonathan S. Weissman Howard Hughes Medical Institute Department of Cellular and Molecular Pharmacology University of California, San Francisco San Francisco, California 94143 Selected Reading Brinker, A., Pfeifer, G., Kerner, M.J., Naylor, D.J., Hartl, F.U., and Hayer-Hartl, M. (2001). Cell 107, in press. Chaudhuri, T.K., Farr, G.W., Fenton, W.A., Rospert, S., and Horwich, A.L. (2001). Cell 107, in press. Dubaquie, Y., Looser, R., Funfschilling, U., Jeno, P., and Rospert, S. (1998). EMBO J. 17, 5868–5876.

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Mayhew, M., da Silva, A.C.R., Martin, J., Erdjument-Bromage, H., Tempst, P., and Hartl, F.-U. (1996). Nature 379, 420–426.

Weissman, J.S., Kashi, Y., Fenton, W.A., and Horwich, A.L. (1994). Cell 78, 693–702.

Rye, H.S., Roseman, A.M., Chen, S., Furtak, K., Fenton, W.A., Saibil, H.R., and Horwich, A.L. (1999). Cell 97, 325–328.

Weissman, J.S., Hohl, C.M., Kovalenko, O., Kashi, Y., Chen, S., Braig, K., Saibil, H.R., Fenton, W.A., and Horwich, A.L. (1995). Cell 83, 577–588.

Sigler, P.B., Xu, Z., Rye, H.S., Burston, S.G., Fenton, W.A., and Horwich, A.L. (1998). Annu. Rev. Biochem. 67, 581–608. Todd, M.J., Viitanen, P.V., and Lorimer, G.H. (1994). Science 265, 659–666.

New Twists in Understanding the Fate of Antisense Oligodeoxynucleotide mRNA Targets In this issue of Molecular Cell, Thoma et al. show that after a target mRNA is cleaved, upon treatment with an antisense oligodeoxynucleotide, the 3ⴕ cleavage product persists and is translated to produce an N-terminally truncated version of the protein encoded by the target mRNA. Antisense oligodeoxynucleotides are commonly used for therapeutic and research purposes to disrupt gene function. Antisense oligodeoxynucleotides against specific mRNAs can interfere with mRNA function through many different mechanisms; however, a common mechanism is endonucleolytic cleavage of the target mRNA by an endogenous RNase H activity (reviewed in Baker and Monia, 1999). Cleavage of the target mRNA leads to a decreased level of the full-length target mRNA and corresponding protein. Therefore, the phenotypic effects of antisense oligodeoxynucleotides on cells are often attributed to loss of the targeted gene product (Crooke, 2000). The findings of Thoma et al. (2001), described in this issue of Molecular Cell, indicate that antisense oligodeoxynucleotide treatment may cause biologically significant effects by creating stable fragments of target mRNAs that produce truncated versions of the encoded protein. One surprising finding presented by Thoma et al. is that the 3⬘ cleavage products generated from antisense oligodeoxynucleotide treatment of a target mRNA are stable. Northern blot analysis of the antisense oligodeoxynucleotide cleavage products revealed that the 5⬘ fragment is not detectable, presumably because it is unstable. In contrast, the 3⬘ fragment was readily observed. The accumulation of 3⬘ fragments appears to be a general outcome of antisense oligodeoxynucleotide treatment, since Thoma et al. observe the same results with oligonucleotides specific for different regions within a target mRNA, different mRNA targets, and in both human and avian cells. RNase H cleavage of a target mRNA would generate a 5⬘ fragment lacking a poly(A) tail and a 3⬘ fragment lacking a 5⬘ cap. Thoma et al. demonstrate that the 3⬘ fragment is polyadenylated, and at least some of the fragment is uncapped. The stability

Weissman, J.S., Rye, H.S., Fenton, W.A., Beechem, J.M., and Horwich, A.L. (1996). Cell 84, 481–490. Zhou, H.X., and Dill, K.A. (2001). Biochemistry 40, 1289–1291.

of the 3⬘ fragment is unexpected given what is known about eukaryotic mRNA decay. The primary mRNA decay mechanism in yeast involves removal of the 3⬘ poly(A) tail, which leads to decapping and rapid 5⬘ to 3⬘ exonucleolytic digestion (Decker and Parker, 1993; Muhlrad et al., 1994). The second decay pathway, which appears to degrade mRNAs more slowly then the primary pathway, initiates with deadenylation followed by 3⬘ to 5⬘ exonucleolytic decay that is catalyzed by the exosome, a large complex of exonucleases (JacobsAnderson and Parker, 1998). The 5⬘ cleavage product is expected to be unstable, since it would be subject to decapping and subsequent 5⬘ to 3⬘ decay or to 3⬘ to 5⬘ exonucleolytic digestion. If the 3⬘ fragment is indeed stable in the absence of a cap structure, then this finding would suggest that 5⬘ to 3⬘ exonucleolytic digestion is not a major decay mechanism in human and avian cells. Degradation by the exosome, which is conserved in eukaryotes, may instead be the predominant mRNA decay pathway in multicellular organisms. A second surprising finding of Thoma et al. (2001) is that the 3⬘ fragment is translated, producing proteins that are truncated at the N terminus. Normally, eukaryotic translation initiation requires the 5⬘ cap. Translation initiation factor eIF4E binds to the cap and associates with eIF4G, which in turn mediates the interaction between the mRNA and the 40S subunit of the ribosome (reviewed in Gingras et al., 1999). Alternatively, some mRNAs utilize internal ribosome entry sites (IRES) rather than the cap to recruit the 40S subunit. The 3⬘ poly(A) tail can stimulate translation initiation through interactions with poly(A) binding protein (PABP), which associates with eIF4G (Tarun and Sachs, 1996). Thoma et al. speculate that translation of the 3⬘ cleavage fragments is dependent on the poly(A) tail, since these mRNAs do not contain any defined IRES elements. The poly(A) tail alone can mediate translation initiation of some uncapped polyadenylated viral mRNAs in vivo (Gallie, 1998) and, under certain conditions, in vitro (Tarun and Sachs, 1995). The idea that uncapped mRNA fragments are stable and translated is so unexpected that it is important to consider if the 3⬘ fragment is recapped following RNase H cleavage. The evidence that the 3⬘ fragment is uncapped is based on the presence of a PCR product that can be amplified only after the 5⬘ and 3⬘ termini of the 3⬘ fragment are ligated together. Uncapped mRNAs can be ligated, whereas the 5⬘ cap inhibits RNA ligation, and thus capped mRNAs are not amplified. A limitation of this approach is that it is not quantitative, therefore the fraction of 3⬘ fragment which is uncapped cannot be