It has been suggested that newly synthesized proteins are maintained in their unfolded state by cellular ATP-driven factors which may prevent or reverse the formation of misfolded structures or promote the correct assembly of oligomeric proteins or post-translational secretion. Using a photocross-linking approach, we have identified the 20S heat-shock GroEL protein as the major cytosolic component which forms a complex with the unfolded newly synthesized pre-beta-lactamase or chloramphenicol acetyltransferase in Escherichia coli. Dissociation of these complexes is ATP-dependent. The unfolded state of pre-beta-lactamase, maintained by the transient interaction with GroEL, may be essential for the secretion of this protein.

Hydrophobic signal sequences direct the translocation of nascent secretory proteins and many membrane proteins across the membrane of the endoplasmic reticulum. Initiation of this process involves the signal recognition particle (SRP), which consists of six polypeptide chains and a 7S RNA and interacts with ribosomes carrying nascent secretory polypeptide chains. In the case of aminoterminal, cleavable signal sequences, in the absence of microsomal membranes it exerts a site-specific translational arrest in vitro. The size of the arrested fragment (60-70 amino-acid residues) suggests that elongation stops when the signal sequence has emerged fully from the ribosome. However, a direct interaction between the signal sequence and SRP has not previously been demonstrated and has even been questioned recently. We now show for the first time a direct interaction between the signal sequence of a secretory protein and a component of SRP, the 45K polypeptide (relative molecular mass (Mr) 54,000). This was achieved by means of a new method of affinity labelling which involves the translational incorporation of an amino acid, carrying a photoreactive group, into nascent polypeptides.

The important Escherichia coli heat-shock protein GroEL of relative molecular mass 57,259 is a typical molecular chaperone. It possesses ATPase activity and interacts in ATP-driven reactions with non-folded proteins to stimulate their correct folding and/or assembly by preventing the formation of improper protein structures or aggregates. As GroEL is isolated and functions as a 20-25S tetradecameric particle (GroELp), the question arises--what is the mechanism of its own assembly? Here we show the (Mg-ATP)-dependent self-stimulation ('self-chaperoning') in vitro of GroELp reassembly from its monomeric state.

No abstract

The universally distributed heat-shock proteins (HSPs) are divided into classes based on molecular weight and sequence conservation. The members of at least two of these classes, the HSP60s and the HSP70s, have chaperone activity. Most HSP60s and many HSP70s feature a striking motif at or near the carboxyl terminus which consists of a string of repeated glycine and methionine residues. We have altered the groEL gene (encoding the essential Escherichia coli HSP60 chaperonin) so that the protein produced lacks its 16 final (including nine gly, and five met) residues. This truncated product behaves like the intact protein in several in vitro tests, the only discernible difference between the two proteins being in the rate at which ATP is hydrolysed. GroELtr can substitute for GroEL in vivo although cells dependent for survival on the truncated protein survive slightly less well during the stationary phase of growth. Elevated levels of the wild-type protein can suppress a number of temperature-sensitive mutations; the truncated protein lacks this ability.