RESEARCH 2003
RESEARCH 2002
RESEARCH 2001
RESEARCH 2000

Jeremy Bushman
Marquette University
Summer Mentor: Dr. Rosemary Stuart

The electron transport chain is a series of protein complexes in the inner mitochondrial membrane that functions as the major cellular energy production pathway of the cell. The proteins of the electron transport chain are encoded by nuclear and mitochondrial DNA – proteins from both origins require a means of transportation into the inner mitochondrial membrane where they function. Oxa 1 (named for Oxidase Assembly 1) is an inner mitochondrial membrane protein in yeast that has been shown in previous studies to help facilitate the movement of different proteins from the matrix into the inner mitochondrial membrane; however, the actual mechanisms of Oxa1 are largely unknown. 

Oxa1 is a protein that spans the mitochondrial inner membrane 5 times in a N_out, – C_in orientation. Further study revealed that transmembrane domain one (TM 1) of Oxa1 is longer than the average transmembrane domain by about 10 amino acids. A blastp search encompassing TM1 resulted in a close match to the p-loop region in potassium channels. This p-loop region can move in and out of a membrane to open and close a channel. We tested the “p-loop like” region in Oxa1 and found it also displayed movement into the membrane when certain stimuli were present. Furthermore, the “p-loop like” region is highly conserved in Oxa1 homologs. 

These results have prompted our lab to investigate the N- terminal region of Oxa1. I am making mutations in the region just upstream of TM 1 and the “p-loop like” region that has shown a high degree of conservation, and in addition has glycine and proline residues that might be responsible for the bending of the protein structure. We hope to observe a phenotype from one or more of these mutations in an attempt to learn more about the function of Oxa1. 

I used a PCR based mutagenesis to create three mutations: mutation 1 is a deletion of amino acids I¹⁰⁰, G¹⁰¹, and L¹⁰², mutation 2 is a deletion of amino acids Y107, W108, and P109 and also a change from S¹¹⁰ to C¹¹⁰, and mutation 3 is a change of S¹¹⁰ to C¹¹⁰. The mutations were constructed in the OXA1 open reading frame, which had been cloned in pGEM together with 228bp of the 5’ promoter region of OXA1. I verified positive results for all three mutations by comparing the mutated DNA sequence to the wild type sequence. 

The mutated Oxa1 constructs were sub cloned into a yeast expression shuttle vector (Yip 351 [LEU2]). We then transformed the vector into wild type and null oxa1 yeast cells. After the growth was observed on - leu media we streaked colonies on glycerol plates. oxa1 null mutants have defective oxidative phosphorylation and hence cannot grow on a non-fermentable carbon source such as glycerol. The functional capacity of the oxa1 deletion mutants #1-3 is tested by their ability to complement the oxa1 null growth phenotype on glycerol. Mutants #2 and #3 both complimented the oxa1 null mutants at 30 and 37º C, indicating that amino acids 107-110 are not essential for Oxa1’s function. The ability of mutation #1 to complement the oxa1 null mutant is currently being tested.

In summary, we generated three different Oxa1 mutations that have been verified through DNA sequencing. According to our preliminary tests, mutants 2 and 3 can complement the oxa1 null mutants, suggesting that amino acids 107-110 are not essential for Oxa1 function. However, it is still possible that Oxa1 function is compromised in the mutants #2 and #3; as we know, the yeast do not display a visible growth defect unless the level of cytochrome oxidase complex is reduced below 30 % of wild type levels. In the next stage of this project we will isolate mitochondria from the mutant strains and analyze Oxa1 function directly.