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First published online 15 November 2005
doi: 10.1242/jcs.02656

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btn1, the Schizosaccharomyces pombe homologue of the human Batten disease gene CLN3, regulates vacuole homeostasis

Yannick Gachet^1,*,, Sandra Codlin^2,,, Jeremy S. Hyams^1,¶ and Sara E. Mole^1,2,,**

¹ Department of Biology, University College London, Gower Street, London, WC1E 6BT, UK
² Department of Paediatrics and Child Health, University College London, Gower Street, London, WC1E 6BT, UK

View larger version (83K): [in a new window]   Fig. 1. Btn1p is the homologue of human CLN3. (A) Protein alignment between human CLN3 and its homologous proteins in mouse (Cln3) and two yeast species (Btn1), S. cerevisiae (S.cerev) and S. pombe (S.pombe). Shading indicates identical (dark) or similar (grey) residues. The position of residues mutated in S. pombe Btn1 during the course of this work that mimic those causing NCL are indicated by an asterisk (*). The likely transmembrane segments (TMS) in human CLN3 are indicated by ==. The position of TMS are based on original predictions (Janes et al., 1996), recent work (Ezaki et al., 2003; Kyttälä et al., 2003; Mao et al., 2003) and the assumption that the sequence of TMS will be conserved between mammalian and yeast species. (B) Wild-type (inset) and btn1 cells grown in YES medium at 29°C and stained with calcofluor. btn1 cells are longer at division (16±2.0 µm) than the wild-type cells from which they were derived (13±1.7 µm). Bar, 10 µm. (C) Cell-cycle length of btn1 cells (black bar) is increased compared to wild-type cells (white bar) at 29°C.

View larger version (38K): [in a new window]   Fig. 2. btn1 cells have larger vacuoles. (A) Wild-type cells (top panels) and btn1 cells (lower panels) labelled with FM4-64 and observed by fluorescence microscopy during exponential growth (left panels) or hypotonic shock (right panels). Bar, 5 µm. (B) Vacuole size distribution before (upper panel) and after (lower panel) hypotonic shock in wild-type cells (black bars) and btn1 cells (white bars). Vacuole size is increased in btn1 cells before and after fusion in response to hypotonic shock.

View larger version (40K): [in a new window]   Fig. 3. Vacuoles of btn1 cells are more alkaline. (A) Cells deleted for btn1 and prelabelled with Hoechst (blue) to stain nuclei and calcofluor (blue) to stain septa, were mixed with unlabelled wild-type cells, also prelabelled with calcofluor (blue), and incubated with CDCFDA (green, to indicate relative intravacuolar pH) for 10 minutes. A wild-type and a btn1 cell (distinguished by round nuclear staining) are indicated by arrows. Vacuoles in btn1 cell had reduced CDCFDA fluorescence indicating an increased pH. Bar, 10 µm. (B) Correlation between CDCFDA fluorescence and vacuole size for cells deleted for btn1 (open circles) and wild-type cells (filled circles). (C) Time course of hydrolysis of CDCFDA as an indicator of relative intravacuolar pH and measured by quantitative fluorescence in wild-type cells (filled squares), cells deleted for btn1 (open circle) or cells deleted for btn1 and expressing GFP-Btn1 for 18 hours (filled circles). GFP-Btn1 partially rescues the reduced CDCFDA fluorescence of btn1 cells. (D) Wild-type cells (left panel) and btn1 cells (right panel) labelled with LSDND as an indicator of relative vacuole pH. Note that btn1 cells have reduced fluorescence. Bar, 10 µm. (E) Vacuole pH estimation of wild-type and btn1 cells. The vacuoles of btn1 cells are pH 5.1 compared to those in wild-type cells which are 4.14. (F) Mean vacuole diameter of wild-type and btn1 cells exposed to media of different pH. The vacuole diameter of both strains increased as cells were exposed to media of increasing pH.

View larger version (30K): [in a new window]   Fig. 4. Localisation and effect of overexpressed Btn1p. (A) Cells deleted for btn1 and expressing GFP-Btn1 for 18 hours (green, left top panel) were labelled with FM4-64 (red, left middle panel) to show the steady state location of GFP-Btn1. At time 0 the promoter nmt42 was repressed by the addition of thiamine and the location of GFP-Btn1 followed and compared with FM4-64 staining of vacuoles at hourly intervals. GFP-Btn1 was first visible in compartments adjacent to the poles or septum of the cells, then over a 3-hour period trafficked through these small pre-vacuolar compartments to the vacuolar membrane. Bar, 10 µm. (B) In cells deleted for btn1 and expressing GFP-Btn1 (green, top panel), the uptake of FM4-64 after 3 minutes and 30 minutes (red, middle panel) was compared with the location of GFP-Btn1. GFP-Btn1 colocalised with compartments that were stained with FM4-64, indicating trafficking through the endocytic route. (C) Correlation of vacuole size with GFP-Btn1 expression levels in btn1 cells. Vacuoles were visualised by FM4-64 staining and GFP expression by densitometry. Cells having the highest levels of expression of GFP-Btn1 had the smallest vacuoles, and vice versa. (D) Time course of vacuole size and GFP-Btn1 expression in btn1 cells. Vacuole size (open circles) was visualised by FM4-64 staining and GFP expression (filled circles) by fluorimetry. As overall levels of GFP-Btn1 increased following induction of expression, the average vacuolar size decreased.

View larger version (44K): [in a new window]   Fig. 5. btn1 and vma1 show conditional synthetic lethality. (A) btn1vma1 cells were viable at 25°C but Btn1 protein was essential for survival at 30°C. (B) DAPI and calcofluor staining of cells deleted for vma1 and btn1vma1 at 36°C. btn1vma1 cells were unable to initiate septum formation and many were elongated. Bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 6. Trafficking of Btn1p to the vacuole is Ypt7 dependent. (A) Localisation of GFP-Btn1 (green, left panel) in cells deleted for ypt7 and pre-labelled with FM4-64 (red, middle panel). GFP-Btn1 does not traffic to the vacuole in ypt7 cells. Bar, 10 µm. (B) Cells deleted for ypt7 or for ypt7 and btn1 were incubated with FM4-64 for 2 hours (left panel) then submitted to hypotonic shock (right panel). Vacuoles of ypt7 cells were smaller than those of cells deleted for ypt7 and btn1, and vacuoles of both strains were unable to fuse in response to hypotonic shock. Bar, 10 µm. (C) Vacuole size analysis in wild-type (wt) and cells deleted for btn1, ypt7 or ypt7 and btn1. Absence of Btn1p in ypt7 cells resulted in larger vacuoles. (D) Cells were incubated with CDCFDA for 10 minutes, to indicate relative intravacuolar pH. Cells deleted for ypt7 and btn1 had reduced CDCFDA fluorescence, indicating vacuoles were more alkaline than those of cells deleted for ypt7. Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 7. Localisation and vacuolar effects of ectopically expressed CLN3 and Btn1p mutants. (A) Cells deleted for btn1 and expressing human CLN3 protein (GFP-CLN3) for 18 hours (green, left panel) were labelled with FM4-64 (red, middle panel). CLN3 trafficked to the vacuole in btn1 cells, as can be seen in the merged images. Bar, 10 µm. (B) Vacuole size analysis of wild-type cells (wt), cells deleted for btn1 (btn1), and cells deleted for btn1 and expressing wild-type (GFP-Btn1) protein or human CLN3 protein (GFP-CLN3), or cells deleted for btn1 and expressing mutant Btn1 protein (GFP-Btn1G136A, -Btn1E240K or -Btn1V278F). The vacuoles of cells expressing the mutant proteins remained enlarged in contrast to those expressing wild-type Btn1 or CLN3 which were reduced in size. (C) CDCFDA fluorescence determined in wild-type cells (wt), in cells deleted for btn1 and expressing GFP from vector alone (GFP), or wild-type Btn1 protein (GFP-Btn1) or human CLN3 protein (GFP-CLN3), or cells deleted for btn1 and expressing mutant Btn1 protein (GFP-Btn1G136A, -Btn1E240K or -Btn1V278F). The vacuolar pH of cells expressing GFP-Btn1G136A was similar to that of btn1 cells. The vacuolar pH of cells expressing GFP-Btn1E240K and GFP-Btn1V278F was partially rescued. (D) Expression of GFP-Btn1 mutant protein (green, top panel) was repressed in cells deleted for btn1 for 3 hours, then cells were labelled with FM4-64 (red, middle panel). GFP-Btn1G136A was retarded in the endoplasmic reticulum but GFP-Btn1E240K and GFP-Btn1V278F trafficked to the vacuolar membrane. Cells overexpressing GFP-Btn1E240K were also strikingly elongated. Bar, 5 µm.