ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal.

Protein unfolding is a key step in several cellular processes, including protein translocation across some membranes and protein degradation by ATP-dependent proteases. ClpAP protease and the proteasome can actively unfold proteins in a process that hydrolyzes ATP. Here we show that these proteases seem to catalyze unfolding by processively unraveling their substrates from the attachment point of the degradation signal. As a consequence, the ability of a protein to be degraded depends on its structure as well as its stability. In multidomain proteins, independently stable domains are unfolded sequentially. We show that these results can explain the limited degradation by the proteasome that occurs in the processing of the precursor of the transcription factor NF-kappaB.

Protein unfolding by mitochondria. The Hsp70 import motor.

Protein unfolding is a key step in the import of some proteins into mitochondria and chloroplasts and in the degradation of regulatory proteins by ATP-dependent proteases. In contrast to protein folding, the reverse process has remained largely uninvestigated until now. This review discusses recent discoveries on the mechanism of protein unfolding during translocation into mitochondria. The mitochondria can actively unfold preproteins by unraveling them from the N-terminus. The central component of the mitochondrial import motor, the matrix heat shock protein 70, functions by both pulling and holding the preproteins.

Mitochondria unfold precursor proteins by unraveling them from their N-termini.

Protein unfolding is a key step in the life cycle of many proteins, including certain proteins that are degraded by ATP-dependent proteases or translocated across membranes. The detailed mechanisms of these unfolding processes are not understood. Precursor proteins are unfolded and imported into mitochondria by a macromolecular machine that spans two membranes and contains at least nine different proteins. Here we examine import of a model precursor protein derived from the ribonuclease barnase and show that mitochondria unfold this protein by unraveling it from its N-terminus. Because barnase in free-solution unfolds by a different pathway, our results demonstrate that mitochondria catalyze unfolding in the way that enzymes catalyze reactions, namely by changing reaction pathways. The effectiveness of this mechanism depends on the structure of the N-terminal part of the precursor protein.

Hsp60-independent protein folding in the matrix of yeast mitochondria.

Proteins that are imported from the cytosol into mitochondria cross the mitochondrial membranes in an unfolded conformation and then fold in the matrix. Some of these proteins require the chaperonin hsp60 for folding. To test whether hsp60 is required for the folding of all imported matrix proteins, we monitored the folding of four monomeric proteins after import into mitochondria from wild-type yeast or from a mutant strain in which hsp60 had been inactivated. The four precursors included two authentic matrix proteins (rhodanese and the mitochondrial cyclophilin Cpr3p) and two artificial precursors (matrix-targeted variants of dihydrofolate reductase and barnase). Only rhodanese formed a tight complex with hsp60 and required hsp60 for folding. The three other proteins folded efficiently without, and showed no detectable binding to, hsp60. Thus, the mitochondrial chaperonin system is not essential for the folding of all matrix proteins. These data agree well with earlier in vitro studies, which had demonstrated that only a subset of proteins require chaperones for efficient folding.

Movement of the position of the transition state in protein folding.

Hammond behavior, in which two neighboring states move closer to each other along the reaction coordinate as the energy difference between them becomes smaller, has previously been observed for the transition state of unfolding of barnase. Here, we report Hammond behavior for the small protein chymotrypsin inhibitor 2 (CI2), which folds and unfolds via a single rate-determining transition state and simple two-state kinetics. Mutants have been generated along the entire sequence of the protein and the kinetics of folding and unfolding measured as a function of concentration of denaturant. The transition state was found to move progressively closer to the folded state on destabilization of the protein by mutation. Different regions of CI2 all show a similar sensitivity to changes in the energy of the transition state. This is in contrast to the behavior of barnase on mutation for which the position of the transition state for its unfolding is sensitive to mutation in some regions, especially in its major alpha-helix, but not in others. The transition state for the folding and unfolding of CI2 resembles an expanded version of the folded state and is formed in a concerted manner, in contrast to that for barnase, in which some regions of structure are fully formed and others fully unfolded. The reason for the general sensitivity of the position of the transition state of CI2 to mutation is presumably the relatively uniform degree of structure formation in the transition state and the concerted nature of its formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclophilin catalyzes protein folding in yeast mitochondria.

Cyclophilins are a family of ubiquitous proteins that are the intracellular target of the immunosuppressant drug cyclosporin A. Although cyclophilins catalyze peptidylprolyl cis-trans isomerization in vitro, it has remained open whether they also perform this function in vivo. Here we show that Cpr3p, a cyclophilin in the matrix of yeast mitochondria, accelerates the refolding of a fusion protein that was synthesized in a reticulocyte lysate and imported into the matrix of isolated yeast mitochondria. The fusion protein consisted of the matrix-targeting sequence of subunit 9 of F1F0-ATPase fused to mouse dihydrofolate reductase. Refolding of the dihydrofolate reductase moiety in the matrix was monitored by acquisition of resistance to proteinase K. The rate of refolding was reduced by a factor of 2-6 by 2.5 microM cyclosporin A. This reduced rate of folding was also observed with mitochondria lacking Cpr3p. In these mitochondria, protein folding was insensitive to cyclosporin A. The rate of protein import was not affected by cyclosporin A or by deletion of Cpr3p.