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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-Btn1^G136A, -Btn1^E240K or -Btn1^V278F). 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-Btn1^G136A, -Btn1^E240K or -Btn1^V278F). The vacuolar pH of cells expressing GFP-Btn1^G136A was similar to that of btn1 cells. The vacuolar pH of cells expressing GFP-Btn1^E240K and GFP-Btn1^V278F 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-Btn1^G136A was retarded in the endoplasmic reticulum but GFP-Btn1^E240K and GFP-Btn1^V278F trafficked to the vacuolar membrane. Cells overexpressing GFP-Btn1^E240K were also strikingly elongated. Bar, 5 µm.

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