PROTEIN UNFOLDING/REFOLDING DURING MITOCHONDRIAL IMPORT

New Mexico State University Las Cruces, Las Cruces NM

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Abstract

Proteins must unfold prior to transport into most organelles. In the mitochondrial import mechanism, molecular chaperones begin the unfolding process for most proteins and then pass the partially denatured protein to the import machinery in the mitochondrial membrane. It is unclear if and to what extent proteins in the transport machinery of the mitochondrial outer membrane (TOM) posses intrinsic unfolding activity that they use to help unfold importing proteins. If TOM proteins do contain unfolding activity, it would mean that mitochondria themselves participate in unfolding during import rather than leaving the entire job to the cytoplasmic chaperones. To determine if TOMs posses unfolding activity, two members of the human TOM complex (Tom20 and Tom22) will be tested for their ability to unfold the test protein dihydrofolate reductase (DHFR). Hydrogen exchange mass spectrometry, a method very sensitive to changes in protein structure and unfolding, will be used to compare the deuterium exchange into DHFR alone versus DHFR incubated with either Tom20 or Tom22. The results are expected to demonstrate how much unfolding activity Tom20 and Tom22 have, the magnitude of resulting unfolding and the location of the unfolding in DHFR. With such data, the unfolding that occurs at several individual steps in the transport process will be elucidated, information that is key to understanding the fundamental biological process of unfolding during import.