RESEARCH 2002
RESEARCH 2001
RESEARCH 2000

Katie O’Sullivan 
University of Notre Dame
Notre Dame, IN
Mentor: Dr. Rosemary A. Stuart

Proteins of the inner mitochondrial membrane play many important roles in metabolic processes of the cell including the generation of energy for the cell in the form of ATP via oxidative phosphorylation. Integral membrane proteins are sorted and inserted into the inner membrane from their origin of synthesis through a variety of distinct mechanisms, one of which involves the polytopic inner membrane protein Oxa1 in yeast. In addition to the insertion of nuclear-encoded gene products, Oxa1 has been specifically identified as a mediator of the co-translational insertion of mitochondrially-encoded proteins. 

Previous chemical cross-linking studies of mitochondrial proteins have identified a close association between matrix-exposed elements of Oxa1 and Mrp20, a protein on the large subunit of the mitochondrial ribosome that flanks the peptide exit tunnel. The close proximity of these subunits suggests that their interaction enhances the insertion of the nascent polypeptide chain into the inner membrane immediately following translation. In order to maximize the probability of interaction, these cross-linking experiments were performed such that Oxa1 was over-expressed in yeast cells. Hence mitochondria isolated from these cells contain elevated levels of the Oxa1 protein. However, it was uncertain whether the noted association was specific or whether it was due to electrostatic interactions between the many positively-charged Oxa1 proteins and negatively-charged rRNA of the ribosome. 
 

My experiments aimed to investigate the specificity of the association between Oxa1 and Mrp20. A his-tagged derivative of Oxa1 was over-expressed in yeast mitochondria deficient in a specific integral membrane protein, such as Pnt1, Cox18, Mss2, Pet111, or Pet309. These proteins are required for the synthesis or insertion of mitochondrially-encoded polytopic proteins. A high level of Oxa1His expression was achieved by transforming the DNA with the Yeast Integrating Plasmid (Yip) under the control of the galactose-inducible promoter, Gal10. The expression of Oxa1His was verified through Western Blot analysis of isolated mitochondria. Finally, chemical cross-linking experiments using an amino-specific chemical showed the coupling of over-expressed Oxa1to Mrp20 in two of the transformed yeast null mutants, _cox18 and _pnt1. The interaction between the integral membrane protein Oxa1 and the ribosomal protein Mrp20 was observed to occur in a Cox18 and Pnt1 independent fashion.