Deciphering iron/heme homeostasis using the fission yeast model.

这项提案的长期目标是破译真核生物中血红素获取的分子基础。由于其作为几种关键细胞酶的辅因子的重要生理作用，生物体已经进化出两种不同的方式来获得血红素：血红素生物合成和从外部来源摄取血红素。与负责血红素生物合成的充分表征的酶相反，对参与外源血红素获取的参与者和机制的了解非常有限，并且仍然知之甚少。拉贝小组的研究旨在填补我们知识中的这一空白。 本研究以模式生物S.粟酒这种酵母菌与S.酿酒酵母可以同化外源血红素，允许使用遗传学的力量来选择性地阻断血红素生物合成，设置条件来研究细胞吸收外部血红素的机制。我们的研究揭示了编码在血红素同化中具有新功能的蛋白质的基因的存在，但对它们知之甚少，包括它们的作用机制以及它们如何响应血红素水平的变化而受到调节。 在未来5年内，我们将把研究计划集中在以下具体目标上。 1.阐明细胞表面锚定血红素受体Shu 1的分子基础， 在血红素运输中与其相互作用的伙伴合作。Shu 1经历血红素刺激的内化，涉及与Nbr 1的相互作用，Nbr 1作为ESCRT蛋白的货物发挥作用。我们将：a）研究Shu 1-Nbr 1复合物的异聚体性质; B）鉴定促进Shu 1-Nbr 1缔合的束缚因子。 2.阐明血红素转运蛋白Str 3和伴侣蛋白Tpx 1在血红素获得和运输中的分子参与。在微需氧条件下，Tpx 1的破坏导致细胞无法使用外源性氯化血红素。我们将：a）表征Tpx 1的血红素结合特性; B）研究Tpx 1和Str 3相互作用的机制。 3.确定血红素在低铁条件下抑制Fep 1所需的途径。Frp 1是一种血红素蛋白。阻遏物Fep 1在没有血红素的情况下与Frp 1启动子组成型结合。相反，当加入外源性氯化血红素时，Fep 1从该启动子解离，导致Frp 1表达。我们将：a）确定所需的信号血红素的存在Fep 1的蛋白质; B）使用RNA-“>学生不分少数民族将发展关键技能，使他们能够获得加拿大生物技术部门和/或学术研究界训练有素的科学家的地位。

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- ">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.