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Access Type

WSU Access

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biochemistry and Molecular Biology

First Advisor

Sharon Ackerman

Abstract

The F1 domain is the catalytic subunit of the mitochondrial ATP synthase. Studies with respiratory-deficient yeast identified ATP1 and ATP2 as nuclear genes encoding the alpha and beta subunits, respectively, of the mitochondrial F1-ATPase. The mutations in the atp1 and atp2 genes were cloned and sequenced, and they appear to affect the ATP synthase. Most yeast strains with mutations in the or the subunit primarily show an F1 assembly defective phenotype. This feature is similar to the assembly-defective mutants missing the chaperones required for assembly of the F1 oligomer or either the alpha/beta subunits.

Some of the atp2 and atp1 yeast mutants are interesting because they show evidence of a soluble F1 oligomer with "new" phenotypic characteristics. The yeast strains E892 and E793 with a mutation in the P-loop are capable of assembling the F1 in vivo, but extraction of the F1Fo from the inner mitochondrial membrane using detergent renders it unstable forming oligomeric structures. The yeast mutant E323 has a phenotypic characteristic that resembles F1 assembly defective mutants. However, the defect is not because the mutation affects the structural stability of the protein but due to the inability of the dimers to assemble a soluble F1. The yeast mutant N15 presents two mutations (G227D, D469N) in the beta subunit with impaired catalytic activity. Work in our lab has shown that atp2 yeast mutants with the G227D mutation are incapable of assembling the F1Fo. We suggest that the D469N mutation rescues the deleterious phenotypic effect of the G227D mutation.

The F1- and subunits are assembled into a soluble hexamer with the aid of two nuclear-encoded chaperones Atp12p and Atp11p respectively. Chaperones maintain the activity of proteins that are destabilized by mutations. Prokaryotes show increased levels of chaperones to alleviate the deleterious effects of mutations. To explore this possibility, we overexpressed the ATP11 and ATP12 genes to determine if it rescues the mutant phenotype. Our efforts so far have proved unsuccessful.

Thus, to summarize, we biochemically evaluated the effect of mutations in the atp1 and atp2 genes of the F1-ATPase. The work presented here will give valuable insight into the role of individual amino acids in the functioning of the ATP synthase. Mutational studies combined with structural data will allow us to completely understand the mechanism of the ATP synthase

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