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Cells Keep Bad Guys In A Cage Until They Are Fit To Socialize

Researchers at the Max Planck Institute for Biochemistry in Martinsried/Germany found that the cage structure of the chaperonin GroEL is essential for cellular protein folding (Nature Structural Biology, Vol. 5, Number 11, November 1998). Enclosure of an unfolded protein in the cage provided by the oligomeric GroEL protein efficiently prevents protein aggregation during folding. This is particularly critical for aggregation sensitive proteins (the bad guys) that expose significant hydrophobic surfaces to the solvent. In contrast to earlier proposals, repeated unfolding of partially folded polypeptides is not an essential part of this mechanism.

In the November issue of Nature Structural Biology (page 977-985), the authors Frank Weber, Manjit Hayer-Hartl and Franz-Ulrich Hartl report a study showing that "The oligomeric structure of GroEL-GroES is required for biologically significant chaperonin function in protein folding". This study includes in vivo complementation studies performed by the co-authors France Keppel and Costa Georgopoulos from the University of Geneva, Centre Medical Universitaire (Switzerland).

Chaperonins have been found in all cells investigated to date including organelles like mitochondria and chloroplasts. The Escherichia coli chaperonin GroEL is encoded by an essential gene and consists of 14 identical subunits. These subunits are arranged in two heptameric rings which are stacked back-to-back. Each ring encloses a central cavity in which substrate polypeptide is bound by interaction with the apical domains of the GroEL monomers, which form the opening of the cylinder. After substrate is bound inside the cavity, GroES, a dome-shaped heptameric complex, closes the opening of the ring and causes the release of unfolded substrate polypeptide into the central cavity. In this enclosed environment, the substrate can fold to its native state. It is subsequently released into the cytosol upon ATP-dependent dissociation of GroES from GroEL. Substrate polypeptide which failed to reach its native state rebinds to GroEL for another round of folding.

Although this folding cycle has been well established during recent years, the mechanism by which GroEL contributes to the folding event has remained unclear. Two mechanisms are being considered for chaperonin-assisted protein folding in E. coli : i) GroEL/ GroES act primarily by enclosing substrate polypeptide in a folding cage in which aggregation is prevented during folding; ii) GroEL mediates the repetitive unfolding of misfolded polypeptides, thereby returning them onto a productive folding track. The present research evaluates the contribution of these - not mutually exclusive - models for the mechanism of GroEL assisted protein folding.

A single ring version of GroEL, SRI, has previously been characterized to be deficient in GroES release. Denatured substrate stays encapsulated inside a stable SRI/GroES complex without continuous GroES binding and release. Cycling of GroES is considered to be essential in a previously suggested model of GroEL function. In this model, GroEL functions by an iterative annealing mechanism in which misfolded intermediates are continuously unfolded by binding and release from GroEL. However, SRI/GroES-assisted refolding of denatured rhodanese gave the same yields and kinetics in the absence of cycling as a GroEL/GroES-promoted reaction, demonstrating that continuous unfolding is not necessary for the folding of the chaperonin substrate rhodanese.

Based on refolding studies with the monomeric apical polypeptide binding domain of GroEL, it has been suggested recently that unfolding of polypeptide is the principle mechanism of GroEL function, while enclosure of substrate is unnecessary. This hypothesis has been tested in the present study by analyzing several versions of the monomeric apical domain for their ability to assist the refolding of aggregation-sensitive chaperonin substrates. Although an interaction of unfolded polypeptide with the isolated apical domain could be established, refolding of aggregation-sensitive polypeptides (rhodanese, malate dehydrogenase, citrate synthase) was inefficient. Furthermore, the apical domain did not stabilize folding intermediates of rhodanese for subsequent folding by GroEL/GroES. In contrast, highly efficient refolding of all three substrates has been achieved with GroEL/GroES.

These results indicate that unfolding of substrate by the monomeric apical domain, if it occurs, is not sufficient to promote folding because, in the absence of complete oligomeric GroEL, aggregation out-competes productive folding. Aggregation is efficiently prevented by encapsulation of substrate inside the GroEL cavity, supporting the view that sequestration of aggregation-prone intermediates in a folding cage is an essential element of the chaperonin mechanism.

These results are supported by in vivo studies with intact E. coli cells showing that constructs of the monomeric apical domain are not able to replace the groEL gene or to complement various defects of groEL mutants.

It thus appears that the chaperonin cage has evolved as an elegant solution of the aggregation problem during protein folding in the cell.

Max-Planck-Gesellschaft. November 1998.

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