Active unfolding of precursor proteins during mitochondrial protein import.

Precursor proteins made in the cytoplasm must be in an unfolded conformation during import into mitochondria. Some precursor proteins have tightly folded domains but are imported faster than they unfold spontaneously, implying that mitochondria can unfold proteins. We measured the import rates of artificial precursors containing presequences of varying length fused to either mouse dihydrofolate reductase or bacterial barnase, and found that unfolding of a precursor at the mitochondrial surface is dramatically accelerated when its presequence is long enough to span both membranes and to interact with mhsp70 in the mitochondrial matrix. If the presequence is too short, import is slow but can be strongly accelerated by urea-induced unfolding, suggesting that import of these 'short' precursors is limited by spontaneous unfolding at the mitochondrial surface. With precursors that have sufficiently long presequences, unfolding by the inner membrane import machinery can be orders of magnitude faster than spontaneous unfolding, suggesting that mhsp70 can act as an ATP-driven force-generating motor during protein import.     

The folding of an enzyme. II. Substructure of barnase and the contribution of different interactions to protein stability.

Barnase is described anatomically in terms of its substructures and their mode of packing. The surface area of hydrophobic residues buried on formation and packing of the structural elements has been calculated. Changes in stability have been measured for 64 mutations, 41 constructed in this study, strategically located over the protein. The purpose is to provide: (1) information on the magnitudes of changes in stabilization energy for mutations of residues that are important in maintaining the structure; and (2) probes for the folding pathway to be used in subsequent studies. The majority of mutations delete functional moieties of side-chains or make isosteric changes. The energetics of the interactions are variable and context-dependent. The following general conclusions may be drawn, however, from this study about the classes of interactions that stabilize the protein. (1) Truncation of buried hydrophobic side-chains has, in general, the greatest effect on stability. For fully buried residues, this averages at 1.5 kcal mol-1 per methylene group with a standard deviation of +/- 0.6 kcal mol-1. Truncation of partly exposed leucine, isoleucine or valine residues that are in the range of 50 to 80 A2 of solvent-accessible area (30 to 50% of the total solvent-accessible area on a Gly-X-Gly tripeptide, i.e. those packed against the surface) has a smaller, but relatively constant effect on stability, at 0.81 kcal mol-1 per methylene group with a statistical standard deviation of +/- 0.18 kcal mol-1. (2) There is a very poor correlation between hydrophobic surface area buried and the free energy change for an extensive data set of hydrophobic mutants. The best correlation is found to be between the free energy change and the number of methylene groups within a 6 A radius of the hydrophobic groups deleted. (3) Burial of the hydroxyl group of threonine in a pocket that is intended for a gamma-methyl group of valine costs 2.5 kcal mol-1, in the range expected for the loss of two hydrogen bonds.(ABSTRACT TRUNCATED AT 400 WORDS)     

The structure of precursor proteins during import into mitochondria

Precursor proteins must be at least partially unfolded during import into mitochondria, but their actual conformation during translocation is not known. Are proteins fully unfolded and threaded through the import machinery amino acid by amino acid, or do they retain some partial structure? The folding pathway of most proteins in vitro contains a partially folded intermediate known as the molten globule state, and it has been suggested that proteins are in the molten globule state during translocation across membranes. Here we show that precursors are normally fully unfolded during import into mitochondria. However, precursors containing residual structure can be imported, if less efficiently.     

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.     

Protein unfolding by the mitochondrial membrane potential

Mitochondria can unfold importing precursor proteins by unraveling them from their N-termini. However, how this unraveling is induced is not known. Two candidates for the unfolding activity are the electrical potential across the inner mitochondrial membrane and mitochondrial Hsp70 in the matrix. Here, we propose that many precursors are unfolded by the electrical potential acting directly on positively charged amino acid side chains in the targeting sequences. Only precursor proteins with targeting sequences that are long enough to reach the matrix at the initial interaction with the import machinery are unfolded by mitochondrial Hsp70, and this unfolding occurs even in the absence of a membrane potential.     

Die Adventivknospen an den Wedeln vonCystopteris bulbifera (L.) Bernhardi

Ohne Zusammenfassung     

Ubiquitin‐like domains can target to the proteasome but proteolysis requires a disordered region

Ubiquitin and some of its homologues target proteins to the proteasome for degradation. Other ubiquitin‐like domains are involved in cellular processes unrelated to the proteasome, and proteins containing these domains remain stable in the cell. We find that the 10 yeast ubiquitin‐like domains tested bind to the proteasome, and that all 11 identified domains can target proteins for degradation. Their apparent proteasome affinities are not directly related to their stabilities or functions. That is, ubiquitin‐like domains in proteins not part of the ubiquitin proteasome system may bind the proteasome more tightly than domains in proteins that are bona fide components. We propose that proteins with ubiquitin‐like domains have properties other than proteasome binding that confer stability. We show that one of these properties is the absence of accessible disordered regions that allow the proteasome to initiate degradation. In support of this model, we find that Mdy2 is degraded in yeast when a disordered region in the protein becomes exposed and that the attachment of a disordered region to Ubp6 leads to its degradation.