Mutational and Cross-Linking Analysis of F₁F_o ATP Synthase Subunit e in Saccharomyces cerevisiae Mitochondria

Brendan Corcoran
St. Mary’s University of Minnesota
Winona, MN
Mentor: Dr. Rosemary A. Stuart

Subunit e (Su e) is one of a series of polypeptides that make up a mitochondrially located protein complex known as F₁F_o ATP Synthase. This structure is comprised of two main regions, F₁ and F_o, which are imperative for the ATP production process known as oxidative phosphorylation. As a component of the Fo complex, Su e and a protein called Subunit g (Su g) have been demonstrated to be important factors involved in the dimerization of F₁F_o ATP Synthase complexes as well as in the formation of inner membrane structures called cristae.

To evaluate the importance of specific amino acids on Su e function and morphology, single site mutations were made on conserved amino acid residues of the transmembrane region of Su e using mutant primers and PCR, and transformed into Su e null mutant yeast (?su e) using the yeast integrating vector, Yip351 (LEU2), plasmid . Expression of Su e, as well as the functional integrity of F₁F_o ATP Synthase in the Su e mutant mitochondria were evaluated by Western blotting and oxidative phosphorylation activity, respectively. Mitochondrial phenotypes will further be evaluated by inserting a GFP-labeled CoxIV gene into the yeast Su e mutant strains and evaluating the mitochondrial cristae morphology with fluorescent microscopy.

Su e has been suspected of interacting with other proteins or molecules to maintain mitochondrial structure. Thus it is important to identify what proteins share an environment with Su e. An unknown 32 kDa protein is capable of DTNB cross-linking with Su e in Wild Type yeast. In order to identify this protein, DTNB cross-linking was performed in mitochondria isolated from null mutant yeast strains deficient in the gene encoding different candidate 30-33 kDa mitochondrial membrane proteins. The cross-linking profiles of Su e, in the absence of candidate proteins, were studied following SDS-PAGE and Western blotting. 
 
 
 

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