Abstract

Intracellular heme trafficking is important for cell respiration, antioxidant defence, and ergosterol biosynthesis in yeast, but information about this process is lacking. In Saccharomyces cerevisiae, heme production ends up with FeII insertion in protoporphyrin IX by ferrochelatase (FECH) but no heme acceptors from FECH have been identified. Mitochondrial cytochrome c peroxidase (Ccp1) is such a candidate due to its heme-independent synthesis and reported involvement in heme trafficking. However, FECH and Ccp1 are separated by the inner mitochondrial membrane, so this thesis examines Pet9, a well-characterized ADP/ATP transporter and a putative heme carrier, as a candidate to deliver heme from FECH to Ccp1. In Chapter 2, potential tools for this study were characterized including hem15Δ S. cerevisiae cells, which were shown to be devoid of FECH activity. Commercial cells homologously expressing N-terminally GST-tagged proteins also were examined to probe their applicability for GST pull-down assays, but cleavage of the fusion proteins negates their use. Chapter 3 reports on the LC-MS/MS analysis of GST pull-downs of soluble and membrane proteins from S. cerevisiae following the addition of exogenously expressed GST-apoCcp1 bait to different amounts of cell lysate. Screening the pull-downs at a 1:5 bait-prey ratio from lysates containing 0.6% N-octylglucoside identified 123 mitochondrial proteins as Ccp1-specific interactors, including FECH and Pet9. Computer modelling predicts that Ccp1-Pet9-FECH complexation is driven by electrostatic complementarity. Further computer modelling in Chapter 4 shows how Ccp1 maturation likely controls its association with Pet9 and hence heme channeling within the Ccp1-Pet9-FECH complex. More specifically, heme transfer from Pet9 to Ccp1 is possibly synchronized with the second cleavage of Ccp1’s mitochondrial targeting sequence. The outcomes of both Chapters 3 and 4 suggest that FECH, Pet9, and Ccp1 form a functional complex for heme transfer.
Heme is an essential coenzyme for the ergosterol pathway in S. cerevisiae. To probe links between heme and ergosterol biosynthesis, in Chapter 5 the S. cerevisiae proteome was analyzed by LC-MS/MS after cells were challenged with a sublethal dose of miconazole, a known Erg11 inhibitor. Proteins of 20 biochemical pathways were altered, including ergosterol and heme biosynthesis. Overall, this thesis supports a role for Ccp1 as a “frontline” protein in heme trafficking from FECH and sheds light on the importance of heme in the adaptive response of yeast to miconazole. The results suggest future investigations to clarify pathways and mechanisms of heme trafficking and how these, and heme production, could be targeted to potentiate antifungals.