The goal of this application is to study membrane sorting and insertion of functionally important proteins. Three voltage dependent anion channels (VDACs) and one of the monoamine oxidases (MAO A) will be the subjects of this investigation. These VDACs are believed to be part of the peripheral benzodiazepine and gamma-aminobutyric acid (GABA)(A) receptor complexes, present either in the plasma membrane or the mitochondrial membrane. MAO A, an enzyme bound to mitochondrial membrane, is known for its functional role in regulating monoamine levels in the nervous system. The investigator emphasizes the notion that faulty assembly of these proteins with the membrane could result in serious disturbances in neuronal function. Amino acid sequences in the three VDACs that lead to their sorting and insertion into either the outer membrane of the mitochondria or the plasma membrane will first be identified. To that end, point mutations in human cDNAs will be obtained for the three proteins and in a yeast gene for one of the proteins. By evaluating the effects of mutations on the ability of these proteins to assemble into the membranes in vitro and in vivo, the sequences that are required for proper assembly can be derived. Once these features are known, their roles in the sorting and insertion of MAO A will be examined. The first three specific aims outlined in the proposal are designed, stepwise, to achieve these goals.
The fourth aim of the project is to seek fundamental information about protein sorting and insertion into membranes. It is hypothesized that if VDACs and MAO A share common sequences for assembly into membranes, these may be general sorting and insertion motifs for other proteins. To test the hypothesis, mutants from a soluble cytoplasmic protein will be generated to include these features. The effects of these mutations on their ability to insert into the membranes will then be evaluated.
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