Expression of alternative oxidase (AOX) in the filamentous fungus Neurospora crassa.

Abstract

When chemical inhibitors or mutations impair the standard mitochondrial electron transport chain (sETC) in Neurospora crassa, alternative oxidase (AOX) is induced. AOX transfers electrons directly from coenzyme Q to oxygen, thus bypassing the later steps of the sETC. In N. crassa AOX is encoded by the nuclear aod-1 gene. Over the years, some genes playing a role in the expression of AOX have been identified using traditional genetic screens. More recently, the gene knockout (KO) library of N. crassa was screened for mutants unable to grow in the presence of antimycin A (AA), an inhibitor of Complex III of the sETC. AOX levels in such mutants were determined using western blotting following growth in the presence of chloramphenicol (Cm, inhibitor of mitochondrial translation). Eight new mutants with severe AOX deficiencies were identified. As part of this thesis, in an attempt to further characterize two of these strains, it was found that the AOX deficiency in them could not be rescued by transformation with the wild-type gene thought to be affected in each strain. This led to a detailed analysis of all eight AOX deficient KO mutants. PCR amplification analysis, Southern blotting and crossing the KO mutants with wild-type strains suggested the presence of additional secondary random mutation(s), in the genome of seven of the eight KO mutant strains tested. Sequencing the aod-1 gene from the remaining seven strains suggested the presence of an identical frame-shift mutation in the coding region of the aod-1 gene in two of these KO strains. This frame-shift leads to production of a truncated AOX protein due to the occurrence of a premature stop codon. The other five KO strains have a wild-type aod-1 gene. Thus, they likely carry secondary mutation(s) in a regulatory gene for AOX production. The KO mutant library screen also identified strains that showed very poor or no growth in the presence of AA but had virtually normal levels of AOX protein under Cm inducing conditions. The proteins affected in these mutants were thought to play a role in assembly of AOX into a functional protein or its insertion into the MIM. As part of this thesis, five such KO strains were tested by carbonate extraction to determine the possibility of aberrant membrane association of AOX in these mutants. No conclusions could be reached based on these experiments.