Deciphering iron/heme homeostasis using the fission yeast model.

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The long-term goal of this proposal is to decipher the molecular basis of heme acquisition in eukaryotes. Due to its essential physiological role as a cofactor for several key cellular enzymes, organisms have evolved with two different ways to obtain heme: heme biosynthesis and heme uptake from external sources. As opposed to the well-characterized enzymes responsible for heme biosynthesis, knowledge of the players and mechanisms involved in acquisition of exogenous heme is very limited and remains poorly understood. Studies in the Labbe's group are aimed at filling this gap in our knowledge. Our studies use the model organism S. pombe. This yeast, unlike S. cerevisiae, can assimilate exogenous heme, allowing the use of the power of genetics to selectively block heme biosynthesis, setting conditions to investigate the mechanisms by which external heme is taken up by the cells. Our studies have revealed the existence of genes encoding proteins that have novel functions in heme assimilation, yet little is known about them, including their mechanism of action and how they are regulated in response to changes in heme levels. During the next 5 years, we will focus our research program on the following specific aims. 1. Elucidate the molecular basis whereby Shu1, a cell surface-anchored heme receptor, cooperates with its interacting partners in heme transport. Shu1 undergoes heme-stimulated internalization that involves an interaction with Nbr1, which functions as a cargo for ESCRT proteins. We will: a) investigate the heteromeric nature of the Shu1-Nbr1 complex; b) identify a tethering factor(s) that fosters Shu1-Nbr1 association. 2. Elucidate the molecular participation of the heme transporter Str3 and the chaperone Tpx1 in the acquisition and trafficking of heme. Under microaerophilic conditions, disruption of Tpx1 results in cells being unable to use exogenous hemin. We will: a) characterize the heme-binding properties of Tpx1; b) investigate the mechanism whereby Tpx1 and Str3 interact with one another. 3. Determine the pathway whereby heme is required for inhibition of Fep1 under low Fe conditions. Frp1 is a hemeprotein. The repressor Fep1 is constitutively bound to the Frp1 promoter in the absence of heme. In contrast, when exogenous hemin is added, Fep1 dissociates from this promoter, resulting in Frp1 expression. We will: a) identify proteins that are required to signal the presence of heme to Fep1; b) use RNA- & ChIP-Seq approaches to identify a genome-wide picture of the heme regulon in fungal cells. Students trained under this program will acquire education/training in multidisciplinary approaches, including areas of yeast genetics, molecular & cellular biology, biochemistry, next-generation sequencing & mass spectrometry. Students without distinction as to minorities will develop critical skills, allowing them to gain the status of highly trained scientists for the Canadian biotech sector &/or academic research community.