Studies of Yeast BTN1P and Human CLN3P in Yeast

描述（由申请人提供）：神经性神经样脂褐质病（NCL）可能是儿童中最常见的进行性神经退行性疾病，发病率高达1 / 12,500活产，在美国约有44万携带者。青少年NCL/Batten病是这些疾病中最常见的，也是本提案的主题。患有该疾病的个体被发现含有1 kb的缺失，这引入了一个移码，导致181个氨基酸的预测翻译产物，其中只有前153个残基对应于正常的438个氨基酸CLN3基因产物的前153个。鉴定出与CLN3同源的酵母，并将其命名为BTN1。我们之前已经证明btn1p可能参与维持pH稳态。重要的是，CLN3能够补充缺乏btn1p的酵母模型中液泡pH的改变，这表明它们具有相似的细胞功能，如果不是相同的。我们最近的研究表明，缺乏Btnlp会导致精氨酸的液泡运输缺陷，而CLN3能够补充精氨酸运输缺陷。总的来说，我们的研究表明酵母细胞可以维持pH稳态，而btn1p是这一过程的生物学组成部分。本研究旨在研究bfn1 /l介导的酵母菌单细胞内pH稳态的破坏。通过阐明这种单细胞生物平衡细胞内pH的机制，通过揭示Btnlp和其他蛋白质在btn通路中的特定功能，我们将为理解哺乳动物细胞中的pH稳态奠定基础。我们建议进一步表征Btn1p依赖的液泡ph调节的生物化学特征。此外，通过利用与Btn1p功能相关的分析，如液泡精氨酸运输和液泡质子泵送，我们将进一步确定Btn1p/CLN3的结构要求。在这些研究的同时，我们将通过使用各种遗传筛选，如表型抑制和合成致死性，来确定BTN1途径的组成部分。最后，我们将描述转运Btnlp到液泡的途径。进一步了解酵母中的Btnlp（和ClnSp）将为巴登病的发病机制提供有价值的信息。

DESCRIPTION (provided by applicant): The neuronal ceroid-lipofuscinoses (NCL) are possibly the most common group of progressive neurodegenerative diseases in children, with an incidence as high as one in 12,500 live births, and with about 440,000 carriers in the USA. Juvenile NCL/Batten disease is the most common of these disorders and the subject of this proposal. Individuals with the disease were found to harbor a 1 kb deletion, which introduces a frameshift that leads to a predicted translation product of 181 amino acids, of which only the first 153 residues correspond to the first 153 of the normal 438 amino acid CLN3 gene product. The yeast homolog to CLN3 was identified and designated BTN1. We had previously shown that Btn1 p may be involved in maintaining pH homeostasis. Importantly, CLN3 is able to complement the alteration in vacuolar pH in the yeast model lacking Btn1 p, indicating that they have similar, if not the same cellular functions. Our more recent studies indicated that lacking Btnlp resulted in a defect in vacuolar transport of arginine, and again CLN3 is able to complement the defect in arginine transport. Overall our studies indicate that yeast cells work to maintain pH homeostasis, and that Btn1 p is an integral part of the biology of this process. This proposal sets out to investigate bfn1-/l-mediated disruption of pH homeostasis within the single cell of yeast. By elucidating the mechanisms by which this single celled organism balances intracellular pH and by uncovering the specific function of Btnlp and other proteins in the BTN-pathway we will establish a basis for understanding pH homeostasis in mammalian cells. We propose to further characterize the biochemistry of Btn1 p-dependent regulation of vacuolar pH. Moreover by exploiting assays that correlate to Btn1 p function such as vacuolar arginine transport and vacuolar proton pumping we will further define the structural requirements of Btn1p/CLN3. Concomitant to these studies we will identify components of the BTN1- pathway through use of a variety of genetic screens such as phenotypic suppression and synthetic lethality. Finally we will characterize the pathway of trafficking Btnlp to the vacuole. Further understanding of Btnlp (and ClnSp) in yeast will provide valuable information on the pathogenesis of Batten disease.