Translational Impact

Abstract

SUMMARYThe function of the CLN3 protein, which is mutated in patients with the neurodegenerative lysosomal storage disorder Batten disease, has remained elusive since it was identified 13 years ago. Here, we exploited the Schizosaccharomyces pombe model to gain new insights into CLN3 function. We modelled all missense mutations of CLN3 in the orthologous protein Btn1p, as well as a series of targeted mutations, and assessed trafficking and the ability of the mutant proteins to rescue four distinct phenotypes of btn1⌬ cells. Mutating the C-terminal cysteine residues of Btn1p caused it to be internalised into the vacuole, providing further evidence that this protein functions from pre-vacuole compartments. Mutations in the lumenal regions of the multi-spanning membrane protein, especially in the third lumenal domain which contains a predicted amphipathic helix, had the most significant impact on Btn1p function, indicating that these domains of CLN3 are functionally important. Only one mutant protein was able to rescue the cell curving phenotype (p.Glu295Lys), and since this mutation is associated with a very protracted disease progression, this phenotype could be used to predict the disease severity of novel mutations in CLN3. The ability to predict disease phenotypes in S. pombe confirms this yeast as an invaluable tool to understanding Batten disease. RESEARCH ARTICLEmodelled some disease-causing mutations of CLN3 in Btn1p and found that they altered the trafficking and/or function of Btn1p (Gachet et al., 2005; Codlin et al., 2008b). In this study, we modelled the full complement of disease-causing mutations in order to build up a complete picture of the impact of mutations on protein function. To exploit the S. pombe model effectively, we required distinct phenotypes associated with the deletion of btn1 that could be assayed easily. In the absence of btn1, S. pombe cells have larger vacuoles, which are also less acidic, than those in wild-type cells. Cells with the btn1 deletion are also delayed in the final stages of cytokinesis, and more cells are in the process of septation compared with wild-type cells (Codlin et al., 2008b). The cytokinesis delay is more severe at 37°C, when growth of btn1⌬ cells is temperaturesensitive. Following 18 hours of growth at 37°C, btn1⌬ cells are swollen or lysed owing to total loss of bipolar growth. Prior to this, after 7 hours of growth at 37°C, btn1⌬ cells fail to initiate bipolar growth and remain monopolar for growth (Codlin et al., 2008a; Codlin et al., 2008b). Additionally, we report here that after 4 hours of growth at the non-permissive temperature, btn1⌬ cells lose their rod-shaped morphology and become bent or curved. Therefore, we chose the following four readily-assayable marker phenotypes of btn1⌬ cells: (1) increased vacuole size, (2) septation index during normal growth conditions, (3) cell curving and (4) monopolar growth at 37°C. Importantly, ectopic expression of Btn1p or CLN3 can rescue all of these phenotypes in btn1⌬ cells. In these phenotypic assays, w...