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. ? ? ?
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