pH-dependent localization of Btn1p in the yeast model for Batten disease.

Btn1p the yeast homolog of human CLN3, which is associated with juvenile Batten disease has been implicated in several cellular pathways. Yeast cells lacking BTN1 are unable to couple ATP hydrolysis and proton pumping activities by the vacuolar ATPase (V-ATPase). In this work, we demonstrate that changes in extracellular pH result in altered transcription of BTN1, as well as a change in the glycosylation state and localization of Btn1p. At high pH, Btn1p expression was increased and the protein was mainly located in vacuolar membranes. However, low pH decreased Btn1p expression and changed its location to undefined punctate membranes. Moreover, our results suggest that differential Btn1p localization may be regulated by its glycosylation state. Underlying pathogenic implications for Batten disease of altered cellular distribution of CLN3 are discussed.

Interaction between Sdo1p and Btn1p in the Saccharomyces cerevisiae model for Batten disease.

Juvenile Batten disease is an autosomal recessive pediatric neurodegenerative disorder caused by mutations in the CLN3 gene. The CLN3 protein primarily resides in the lysosomal membrane, but its function is unknown. We demonstrate that CLN3 interacts with SBDS, the protein mutated in Shwachman-Bodian-Diamond syndrome patients. We demonstrate that this protein-protein interaction is conserved between Btn1p and Sdo1p, the respective yeast Saccharomyces cerevisiae orthologs of CLN3 and SBDS. It was previously shown that deletion of BTN1 results in alterations in vacuolar pH and vacuolar (H(+))-ATPase (V-ATPase)-dependent H(+) transport and ATP hydrolysis. Here, we report that an SDO1 deletion strain has decreased vacuolar pH and V-ATPase-dependent H(+) transport and ATP hydrolysis. These alterations result from decreased V-ATPase subunit expression. Overexpression of BTN1 or the presence of ionophore carbonyl cyanide m-chlorophenil hydrazone (CCCP) causes decreased growth in yeast lacking SDO1. In fact, in normal cells, overexpression of BTN1 mirrors the effect of CCCP, with both resulting in increased vacuolar pH due to alterations in the coupling of V-ATPase-dependent H(+) transport and ATP hydrolysis. Thus, we propose that Sdo1p and SBDS work to regulate Btn1p and CLN3, respectively. This report highlights a novel mechanism for controlling vacuole/lysosome homeostasis by the ribosome maturation pathway that may contribute to the cellular abnormalities associated with juvenile Batten disease and Shwachman-Bodian-Diamond syndrome.

Absence of Btn1p in the yeast model for juvenile Batten disease may cause arginine to become toxic to yeast cells.

Lymphoblast cell lines established from individuals with juvenile Batten disease (JNCL) bearing mutations in CLN3 and yeast strains lacking Btn1p (btn1-Delta), the homolog to CLN3, have decreased intracellular levels of arginine and defective lysosomal/vacuolar transport of arginine. It is important to establish the basis for this decrease in arginine levels and whether restoration of arginine levels would be of therapeutic value for Batten disease. Previous studies have suggested that synthesis and degradation of arginine are unaltered in btn1-Delta. Using the yeast model for the Batten disease, we have determined that although btn1-Delta results in decreased intracellular arginine levels, it does not result from altered arginine uptake, arginine efflux or differences in arginine incorporation into peptides. However, expression of BTN1 is dependent on arginine and Gcn4p, the master regulator of amino acid biosynthesis. Moreover, deletion of GCN4 (gcn4-Delta), in combination with btn1-Delta, results in a very specific growth requirement for arginine. In addition, increasing the intracellular levels of arginine through overexpression of Can1p, the plasma membrane basic amino acid permease, results in increased cell volume and a severe growth defect specific to basic amino acid availability for btn1-Delta, but not wild-type cells. Therefore, elevation of intracellular levels of arginine in btn1-Delta cells is detrimental and is suggestive that btn1-Delta and perhaps mutation of CLN3 predispose cells to keep arginine levels lower than normal.