Lyu, Yaxuan; (2022) Elucidating the role of btn1, the S. pombe orthologue of the Batten disease gene CLN3, in vacuole homeostasis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Schizosaccharomyces pombe (fission yeast) can be used as a simple cell model to study disease. S. pombe is a rod-shaped unicellular eukaryote that has a well-annotated genome and is easy to manipulate genetically. It has a single CLN3 orthologue named btn1 which encodes a 396 amino acid transmembrane protein that is 30% identical and 48% similar to human CLN3. Defects in this gene have many cellular consequences. CLN3 affects the lysosome and Btn1 affects the vacuole, the equivalent organelle in yeast, as well as the Golgi complex. The aim of this study is to understand more about the role of Btn1 at the vacuole - in vacuole homeostasis and especially vacuole fragmentation. When yeast cells are placed under hypertonic (salt) stress vacuoles get smaller by budding; this is known as fragmentation. Yeast has interlinked phosphatidylinositol (PI) pathways where the addition of phosphate groups to the basic phosphatidylinositol structure is catalysed by kinases. One of the crucial kinases in yeast for vacuole fragmentation and homeostasis is Fab1, the sole kinase that phosphorylates PI(3)P to give a transient burst of PI(3,5)P2 at the vacuole. Deletion of fab1 in the budding yeast S. cerevisiae results in enlarged vacuoles and failure of vacuole fragmentation under hypertonic stress. Deletion of fab1 in S. pombe also results in larger vacuoles, as does deletion of Vps34, the kinase that phosphorylates PI to produce PI3P. This study shows that S. pombe cells deleted for fab1 cannot undergo vacuole fragmentation under hypertonic stress, and that vacuoles in btn1Δ cells are delayed in fragmentation under hypertonic stress compared to wild-type cells. Expressing Btn1 in btn1Δ cells restores vacuole fragmentation but expressing Btn1 in fab1Δ cells does not, consistent with fragmentation requiring the production of PI(3,5)P2. Expressing Fab1 in fab1Δ cells restores vacuole fragmentation, as does expressing Fab1 in btn1Δ cells. This suggests that the defect in vacuole homeostasis of btn1Δ cells (vacuole fragmentation) is due to a delay in producing PI(3,5)P2. Furthermore, a novel biosensor for PI(3,5)P2 in S. pombe was developed and applied. Results using this biosensor are consistent with a delay in producing PI(3,5)P2 in btn1Δ cells responding to hypertonic stress. S. pombe cells deleted for btn1 have a defect in responding to stress via the TORC1 pathway. The connection between the role of Btn1 in TORC1 regulated nutrient sensing and Fab1 regulated PI(3,5)P2 was investigated. The results show that btn1Δ cells have a nitrogen sensing defect, and that btn1Δ cells have less Pop3 (a subunit of TORC1) on the vacuole membrane compared to wild-type cells, which suggests that btn1Δ cells have lower TORC1 activity. By examining colocalization with Pop3, it was found that Btn1 colocalises with Pop3 on the vacuole membrane but not at pre-vacuolar compartments. fab1Δ cells have no detectable Pop3 indicating a defect in TORC1 activity. Therefore, the TORC1-dependent nitrogen sensing defect of btn1Δ cells may be caused by insufficient production of PI(3,5)P2 via Fab1.

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