Proteolytic processing of imported precursors is a crucial step in the biogenesis of mitochondrial enzymes. It is our aim to study the proteolytic removal of amino-terminal octapeptides from the mitochondrial intermediate proteins. This cleavage event is unique to a sub-group of mitochondrial enzymes and is specifically catalyzed by a mitochondrial intermediate peptidase (MIP), likely to represent the first member of a new family of endopeptidases. The main objective of this research proposal is the characterization of MIP. The experiments proposed are designed: 1) to determine the structural features of the MIP family; 2) to establish the mechanism of action of MIP and define the elements that direct recognition and cleavage of the mitochondrial intermediate proteins; 3) to investigate the importance of MIP function for mitochondrial homeostasis and cell viability. The experimental design will require expression of the MIP gene in vitro and in vivo, and reconstitution of MIP biogenesis and activity. The functional studies will involve analysis of biological and biochemical aspects of MIP inactivation in Saccharomyces cerevisiae. Specific techniques employed will include: cloning, sequencing and manipulation of cDNA; genetic manipulation in S. cerevisiae; isolation of mitochondria from rat liver and S. cerevisiae; over-expression of recombinant proteins in E. coli; PCR amplification; DNA and protein blotting. This study may provide information at the level of a) components of the mitochondrial protein import apparatus; b) novel mechanisms of endoproteolytic activity; c) new pathways of mitochondrial disfunction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM048076-01
Application #
3307515
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1992-09-30
Project End
1995-08-31
Budget Start
1992-09-30
Budget End
1993-08-31
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520