Identification and characterization of suppressors of the non-fermentable carbon source defects of coa1Δ
Type of Degreedissertation
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The assembly of cytochrome c oxidase (COX) in yeast mitochondria is dependent on the assembly factor Coa1. ATP1 was identified as a multi-copy suppressor of the coa1∆ respiration defect. ATP1 is additive with previously discovered suppressors, MSS51, COX10, MDJ1, suggesting ATP1 functions via a different pathway. Using biochemical and microscopic approaches we found ATP1 rescues a previously unrecognized mitochondrial biogenesis defect in coa1∆ that result in low total mitochondrial numbers and a morphology defect. ATP1 overexpression did not rescue the closely related assembly factor mutant shy1∆. Mitochondrial morphology and biogenesis is dependent on intact mitochondrial ultrastructure. ATP1 and COA1 were found to rescue a defect in aim37∆, a member of the mitochondrial inner membrane organization and structure (MINOS) complex. We propose the rescue of respiration in coa1∆ by multi-copy ATP1 is due to increased mitochondrial biogenesis perhaps due to improved inner membrane organization in coa1∆ or improved localization of COX to cristae. We undertook an approach to isolate evolved homozygous diploid strains capable of growth on non-fermentable carbon source in the absence of coa1. To identify the mutations, we used the next generation sequencing. Four haploid strains were sequenced separately and compared to a reference sequence. An optimized sequencing library preparation procedure resulted in 4.1G data for the four strains. The whole genome was covered (99.9%) and with an average coverage of 92. We identified expected and unexpected single nucleotide polymorphisms present in haploid strains. The mechanism of suppression of the coa1∆ yeast appears to be linked to a unique mutation in COX1 in the mitochondrial genome. To understand intracellular iron handling, we attempted to use a genetic strategy exploiting the hydroxyurea sensitivity of an iron deficiency (aft1△aft2△). We isolated 6 suppressor mutants of aft1△aft2△ double mutant that can tolerate HU and attempted to characterize the suppressing mutation. Our result showed that SPT3 mutation affected total cellular metals and hydroxyurea sensitivity in this background.