Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Allison McDonald

Advisor Role

Supervisor

Abstract

Alternative oxidase (AOX) is an inner mitochondrial membrane protein that introduces a branch point at ubiquinone within the respiratory electron transport system (ETS). The AOX protein bypasses two sites of proton translocation within the ETS and as a result the yield of ATP per oxygen consumed is significantly reduced. Although AOX appears to be energetically wasteful, recent studies have revealed that AOX has a wide taxonomic distribution. AOX multigene families, transcripts, protein levels, and enzymatic activity have been most thoroughly characterized in many angiosperm (flowering) plants. Given the data available for angiosperm AOXs, evidence of non-angiosperm AOXs in the primary literature is scarce and therefore is a logical starting point for comparative studies. The bioinformatics results suggest that AOX is an ancient character in the Viridiplantae, as it is present within algae, a liverwort, a moss, lycopods, ferns, and many gymnosperms. Most interestingly, it appears that the moss Physcomitrella patens possesses a single AOX gene; a characteristic unobserved in all angiosperm AOXs studied to date. This finding has been validated by sequence analysis of a cloned P. patens AOX cDNA that was amplified by PCR using specific genomic primers. Reverse transcriptase PCR has demonstrated that AOX is expressed in P. patens and sequencing analysis suggests that AOX has all the residues to be catalytically active. At the protein level, moss AOX was heterologously expressed in the yeast Saccharomyces cerevisiae and protein expression was analyzed by performing mitochondrial isolations, SDS-PAGE, and immunoblots. Preliminary studies using respirometry and site-directed mutagenesis of key residues were undertaken, and it is anticipated that the optimization of these procedures will yield useful data in future studies. A greater understanding of the AOX pathway in non-angiosperm plants is of importance as it will contribute to understanding the evolutionary history of AOX and may help determine its physiological function(s).

Convocation Year

2013

Convocation Season

Fall

Download

Share

COinS