9418257 Verner This proposal addresses the topic of cotranslational import of proteins into mitochondria. This is a question of fundamental importance, since the "textbook" view is that protein import into mitochondria occurs via post-translation import (release of peptide from polysome unattached to the targeted membrane). This view has resulted from high profile research which places perhaps an unwarranted degree of reliance on in vitro import reactions vs the in vivo situation. This high profile research has shown that post translational import can occur in vitro, whether it does occur in vivo, to the exclusion of the co-translational mechanisms, is the question addressed by this proposal. The experiments proposed examine the properties of mitochondrial precursor proteins that may influence the coupling of their synthesis and import and explore the function of cytosolic chaperone proteins, as well as other cytosolic components in the import reaction. In experiments related to the structure of precursor proteins, gene fusion technology will be employed to generate various artificial proteins that will be studied in both in vitro and in vivo import reacitons. The effect of mitochondrial presequences and the relative stability of overall precursor structure will be examined in order to determine the characteristics of precursor proteins that influence their ability to be imported by either a cotranslational or posttranslational mechanism. In addition, in vitro import reactions will be performed to evaluate the involvement of other cytosolic components in mitochondrial biogenesis. Such components will be identified and isolated using standard biochemical procedures. %%% Results obtained from the proposed studies are anticipated to add significantly to our understanding of protein import into mitochondria and other organelles. The bulk of the proposed experiments are designed to explore the integration of mitochondrial biogenesis with other cellular phenomena such as cytosolic protein synthesis, translation rates, and interactions between precursor proieins and cytosolic components. This is an important step in viewing the import process in the context of the overall complexity of the living cytosol and will be complementary to other work in the field in which the process tends to be studied in great detail but often in isolation of other cellular reactions. ***
