The GroEL–GroES chaperonin system, which is responsible for the folding of a subset of newly synthesized proteins, resembles a cylinder with two cavities (GroEL) and a lid (GroES). Substrates enter this box, are folded and then released. But do all proteins that use chaperonins really need to cram themselves entirely into the box, and, if so, what could be happening to them while they are inside?

The enzymology of the chaperonin system is very well understood. In the classic cis cycle, the folding substrate binds to one cavity of GroEL — operationally defined as the cis cavity — and is shortly followed by ATP and GroES. ATP hydrolysis in the cis cavity causes ATP to bind to the trans cavity, which in turn induces the release of GroES and the substrate from the cis cavity.

What is less understood is the principle underlying the chaperonin-mediated folding process. In particular, there is much debate about whether the GroEL–GroES system acts as a passive cage that simply protects the substrate from aggregation, which would occur if the protein were to attempt folding in solution, or whether it actively unfolds misfolded proteins and guides them in their efforts towards correct folding. New data reported in Cell by the Hartl and Horwich groups indicate that, as so often happens, the truth might lie somewhere in the middle.

To directly test whether encapsulation in the chaperonin cage is essential for the folding reaction, Brinker and colleagues inhibited the rebinding of substrate to GroEL — and hence, its encapsulation — by binding streptavidin to biotinylated GroEL. Under conditions that favour their aggregation in solution, RuBisCo and rhodanese — two obligate chaperonin substrates — could fold only in the presence of ATP, GroES and nonbiotinylated GroEL. This confirmed that one function of encapsulation is to protect the substrate from aggregation.

To their surprise, the authors found that encapsulation also has a second function: it actively assists folding. Indeed, under conditions in which it could fold freely in solution, RuBisCo folded four times faster if a fully functional chaperonin system was present. But the accelerated folding was substrate specific, as it was not observed for rhodanese. The authors propose that confinement in the narrow space of the cage “smooths the energy landscape” of the folding reaction, either by preventing the formation of trapped intermediates or by facilitating progression towards the folded state. The observed difference might depend on the size of the substrate — RuBisCo is 50 kDa, whereas rhodanese is only 33 kDa — or on whether the substrate has a tendency to form kinetically trapped intermediates, as RuBisCo has.

But, if substrate size is an issue, what happens to substrates that are too large to be encapsulated? Chaudhuri and colleagues studied folding of mitochondrial aconitase, an 82-kDa monomeric enzyme that is known to aggregate in chaperonin-deficient mitochondria. They found that both GroEL and GroES are required for aconitase folding in vivo and in vitro. However, this does not involve encapsulation by the chaperonin system, as aconitase did not become resistant to protease digestion during folding. So, how could GroES — the lid of the box — assist protein folding if not by encapsulation? Using single-ring and mixed-ring GroEL mutants that cannot bind GroES in trans, the authors deduced that a novel 'trans cycle' assists folding of larger substrates. In this alternative pathway, instead of binding to the cis ring, GroES binds to the trans ring and is required, in addition to ATP, to release the non-native substrate from the cis ring.

So, it seems that there is more than one way in which the chaperonin system can assist folding. Depending on their size and propensity to form aggregates, folding substrates are either encapsulated by GroEL–GroES — simply to protect them from aggregation or, in some cases, also to facilitate the progression towards the folded state — or they only bind to GroEL and undergo a trans cycle, which somehow assists folding. The fine details of these different folding reactions still elude us but these two studies should bring us a step closer to the light.