Protein unfolding during import into mitochondria and degradation by ATP-dependent proteases.

摘要

Protein unfolding is essential for several cellular processes including mitochondrial import of proteins into the matrix and protein degradation by ATP-dependent proteases. Mitochondria and ATP-dependent proteases can actively unfold some proteins by unraveling them from their terminal targeting sequences. Here, I examine the unfolding mechanisms of both mitochondria and the bacterial ATP-dependent protease ClpAP. I propose that the efficiency of the mitochondrial unraveling mechanism and thus the efficiency of import depends on the local structure of the N-terminal amino acids of the precursor's mature domain. I have found that precursor proteins with mature domains containing N-terminal alpha-helices are imported more efficiently than precursors with mature domains containing N-terminal beta-strands. Using single molecule atomic force microscopy, I show that the local terminal structures of proteins also influence their resistance to mechanical unfolding. Together, these results suggest that the efficiency of precursor protein unfolding during mitochondrial import correlates with the mature domain's resistance to mechanical unfolding, and thus also suggest a mechanism of mitochondrial-catalyzed unfolding of proteins driven by mechanical forces and movements. I propose that the bacterial protease ClpAP similarly generates a mechanical force in order to actively unfold its substrates. I hope to be able to measure this force by tethering a ClpAP complex and its substrate between two beads within an optical trap and quantifying the role of force generation by ClpAP during substrate degradation. Here, I show that I can produce stable ClpAP-substrate complexes, bound to Protein G-beads, in the presence of an ATP analog, AMPPNP, which I have successfully degraded by chasing with excess amounts of ATP. I give evidence that I have established an experimental system, which will be able to be used to analyze the mechanism of substrate unfolding and translocation in protease degradation.