The Interrelationship between pH, Btn1p and Phospholipid Regulation

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2009.Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), or Batten disease, is a devastating pediatric neurodegenerative disorder characterized by blindness, the onset and worsening progression of seizures, and cognitive and motor decline. JNCL is ultimately fatal, with death generally occurring in the patient’s twenties. Mutations in the gene CLN3 were identified as the cause of JNCL in 1995. The protein product is an integral membrane protein with six transmembrane spanning domains that localizes predominately to components of the endo-lysosomal membrane system. CLN3 function however, remains unknown. Given that the only treatments currently available for JNCL are those intended to manage the symptoms, determination of the protein’s function would provide a significant step forward in providing a specific target for therapy. The budding yeast Saccharomyces cerevisiae possesses a highly annotated genome and is easily genetically manipulated, thus providing a useful model system in which to study the protein function of Btn1p, the yeast homolog of CLN3. The sequences of Btn1p and CLN3 are 39% identical and 59% similar. Functional homology is demonstrated by the ability of plasmid-borne human CLN3 to complement the strain that harbors a deletion in BTN1. To date, putative Btn1p functions can be categorized in three major groups: pH homeostasis, arginine metabolism, and nitric oxide production. In order to further our understanding of Btn1p function, we have investigated the relationship between Btn1p and pH homeostasis. Specifically we have demonstrated that, not only does the loss of Btn1p result in alteration to the pH homeostasis of the cell, but that changes in the pH of the cellular environment result in significantly altered expression levels of BTN1, changes to the glycosylation state of Btn1p, and finally a shift in Btn1p localization. It was previously shown that loss of Btn1p impacts the ability of the vacuolar H+-ATPase (V-ATPase) to couple its ATP hydrolysis activity with proton transport, yet Btn1p has never been demonstrated to interact with any of its many subunits. Interestingly, it has also been demonstrated that alterations in the lipid environment of the vacuole can have direct implications for V-ATPase activity. By measuring the phospholipid composition of both the vacuole and whole cells, we demonstrate that btn1- cells lack the ability to regulate their lipid levels in response to changes to extracellular pH. Further supporting the idea that Btn1p may play a role in lipid regulation is the finding that btn1-Δ is synthetically lethal with psd2-Δ. PSD2 encodes a vacuolar protein responsible for the biosynthesis of the phospholipid phosphatidylethanolamine via the decarboxylation of phosphatidylserine at the vacuolar membrane. Taken together, these results demonstrate that Btn1p may be responsible for maintaining vacuolar pH homeostasis, but that it may be occurring indirectly via regulation of vacuolar phospholipid levels. Btn1p localization is dependent upon the extracellular pH, and loss of Btn1p alters the cell’s ability to regulate its phospholipid levels in response to pH, suggesting that Btn1p localization is critical for its functioning in this pathway. Moreover, because of the synthetic lethality between btn1-Δ and psd2-, Btn1p function is likely only necessary at pH 6.0 when it co-localizes with Psd2p at the vacuole. As the cell acidifies the media, Btn1p function becomes unnecessary and BTN1 transcription is drastically reduced, while concomitantly, Btn1p is sequestered away from the vacuole