Impact Various Pcp1p Mutations Have on Mitochondrial Genome Maintenance and Morphology

Pcp1p is a transmembrane protease in the mitochondrial inner membrane of Saccharomyces cerevisiae . Mutations in PCP1 negatively impact cellular respiration due to defects in mitochondrial morphology and loss of mitochondrial DNA (mtDNA) that arise as a result of the improper processing of the Pcp1p substrate, Mgm1p. To understand the structural basis of Pcp1p activity, a mutagenesis screen was carried out and temperature sensitive PCP1 alleles selected based on their varied ability to utilize nonfermentable carbon (E/G). Defects in mitochondrial morphology for each of the mutants were assessed by fluorescence microscopy using mtGFP. In addition to an increase in ‘fragmented’ mitochondria, a number of the mutants had GFP targeted to the cytosol, suggesting defects in mitochondrial membrane potential. TEM will be carried out to better characterize the mitochondria ultrastructure for each of these mutants. Poor or absent growth on E/G suggests that these mutant pcp1 alleles are likely to have acquired mtDNA mutations or have lost their entire mitochondrial genome. To assay the extent of mtDNA loss, quantitative PCR was carried out to determine the relative mitochondria to nuclear genome ratio using ACT1 , COX1 , and COX3 primers. Strains that grew similarly to PCP1 had relative mitochondria to nuclear ratios of 0.8 to 1, while mutants that were unable to grow on E/G had normalized ratios that ranged from 0 to 0.3. A number of the pcp1 alleles had relative ratios that fell between 0.5 and 0.8, which correlates with biochemical data that suggests partial peptidase activity. Quantitative differences in mitochondrial genome content will be confirmed by DAPI. The data provides insight into the role different amino acids play in influencing Pcp1p peptidase activity.