| Proteins must fold into well-defined three-dimensional structures to be active. However, regulated unfolding is also critically important in several processes in the cell, such as translocation across mitochondrial and chloroplast membranes, as well as degradation by ATP-dependent proteases. Approximately half of all proteins synthesized in the eukaryotic cell are transported into or across a membrane, and the protein translocation machineries are well defined biologically. In this study, we used a biochemical approach to characterize protein unfolding during translocation.; One of the best-studied translocation processes is protein import into mitochondria. By examining import of a model precursor protein, we showed that mitochondria catalyze protein unfolding during import by changing protein unfolding pathways, namely, by unraveling proteins from their N-termini. The effectiveness of this mechanism depends on the structure of the N-termini of precursor proteins. In order to determine whether the import machinery at the outer mitochondrial membrane destabilizes precursor proteins, we studied the structure and energetics of the model precursor protein at the mitochondrial surface immediately before import. By measuring contributions of individual interactions to protein stability and binding constants of a protein-protein complex, we found that the mitochondrial surface does not destabilize importing precursor proteins in its vicinity, and it does not preferentially destabilize any type of interaction (e.g., hydrogen bonds, nonpolar, etc.) within the protein. Finally, we investigated the roles of different components of the mitochondrial import machinery in protein unfolding. We found that the electrochemical potential across the inner mitochondrial membrane unfolds precursor proteins during import by acting directly on the positively charged amino acid side chains of targeting sequences. Precursor proteins with targeting sequences that are long enough to reach the mitochondrial matrix at the initial interaction with the import machinery are actively unfolded even in the absence of an electrochemical potential, presumably by mitochondrial Hsp70. |
