Genetic and Biochemical Analysis of INSIG Proteins

描述（由申请人提供）：insg蛋白是哺乳动物固醇稳态的关键调控因子，它们与具有固醇传感结构域（SSD）的蛋白的跨膜结构域相互作用，并影响SSD客户端位置或稳定性的变化。我们最近发现酵母酿酒酵母具有insg的同源物，称为Nsglp和Nsg2p，它们类似地调节酵母HMG-CoA还原酶同工酶Hmg2p的稳定性。酵母Hmg2p在其跨膜区有一个SSD，这是酵母insg相互作用和影响Hmg2p稳定性所必需的。因此，insg - ssd客户端交互是广泛保守的，允许通过使用简单的酵母方法来研究Nsglp- Hmg2p交互作为所有insg - ssd客户端交互的模型来分析insg作用的未知特征。哺乳动物的INSIG-SSD相互作用需要固醇，我们的初步证据表明，Nsg1p-Hmg2p相互作用的这一方面同样是保守的。

DESCRIPTION (provided by applicant): The INSIG proteins are key regulators of sterol homeostasis in mammals, where they interact with the transmembrane domains of proteins with a Sterol Sensing Domain (SSD) and effect changes in the location or stability of the SSD clients. We recently discovered that the yeast S. cerevisiae has homologues of INSIGs, called Nsglp and Nsg2p, that similarly regulate the stability of yeast HMG-CoA reductase isozyme Hmg2p. Yeast Hmg2p has an SSD in its transmembrane region, which is required for the interaction and effects of yeast INSIGs on Hmg2p stability. Thus, the INSIG-SSD client interaction is broadly conserved allowing analysis of unknown features of INSIG action by use of facile yeast approaches to study the Nsglp- Hmg2p interaction as a model of all INSIG-SSD client interactions. The INSIG-SSD interaction in mammals requires sterols, and our preliminary evidence indicates that this aspect of the Nsg1p-Hmg2p interaction is similarly conserved. Taken together, these results indicate that the INSIG-SSD client interaction has been extant in biology for over 1 billion years, and its study in yeast will allow understanding of the mechanism and actions of the INSIG proteins in all cases. Specifically we plan to 1) Test the hypothesis that a late sterol pathway product regulates the interaction of the Nsg1p-Hmg2p pair. We will discover the identity of this regulator using a variety of approaches, and confirm its function using a number of in vitro assays of Nsg function. 2) Examine the sequence requirements for Nsg1p-Hmg2p interaction. We will test the role of the Hmg2p SSD in Nsglp- Hmg2p interaction, and similarly explore the role of conserved residues in the Nsg protein. We will also use yeast techniques to perform unbiased screens of these portions of the proteins to complement the directed tests of conserved motifs. In this way the details of an INSIG-SSD domain interaction will be delineated for the first time. Finally, 3) we will use a combination of proteomic techniques and phenotype-based genetic screens to explore other possible functions for the Nsglp protein. Because the INSIG proteins are so important in sterol regulation, any use of them as potential clinical targets will require a detailed understanding of they mechanisms and any possible involvement in unknown aspects of cell biology.