JCS02656 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Btn1p, the homologue of human CLN3, is not essential for vegetative growth. Southern blot showing the presence of the btn1 gene within a 6 kb EcoRI genomic DNA fragment in wild-type cells, and confirming its absence in btn1D cells.  

• Supplemental Figure 2 -

  Fig. S2. btn1D cells have larger vacuoles. (A) Time course of FM4-64 uptake by endocytosis in wild-type and btn1D cells. The rate of endocytosis was indistinguishable in the two strains. Bar, 4 mm. (B) Vacuole size distribution of wild-type (white bars), btn1D cells (black bars) and btn1D cells expressing the human CLN3 gene (red bars).  The CLN3 gene compensated for the lack of the btn1 gene and restored the vacuole size to wild-type values.

• Supplemental Figure 3 -

  Fig. S3. Vacuoles of btn1D cells are more alkaline. Ten-fold serial dilutions of cells on MM containing 1.0 mM ANP.  btn1D cells (bottom row) were more sensitive than wild-type cells (top row) to growth on ANP.

• Supplemental Figure 4 -

  Fig. S4. btn1 and vma1 show conditional synthetic lethality. (A) Cells deleted for vma1 and expressing GFP-Btn1 for 18 hours (green, left panel). GFP-Btn1p trafficked to the vacuole membrane. Bar, 10 mm.  (B) CDCFDA fluorescence in cells deleted for vma1 or vma1 and btn1.  There was no significant difference in fluorescence intensity as a measure of pH between cells severely compromised in vacuole acidification (vma1) and those lacking vma1 and btn1.

• Supplemental Figure 5 -

  Fig. S5. Trafficking of Ypt7p to the vacuole is independent of Btn1p. Localization of GFP-Ypt7 (green) in wild-type cells (left panels) or cells deleted for btn1 (right panels) and pre-labelled with FM4-64 (red,). GFP-Ypt7 traffics to the vacuole in btn1D cells. Bar, 4 mm.