Fig. 1. SH4-domain alignments and localization of Vac8 mutants. (A) Domain structure of Vac8 and Vac8 mutants used in this study. Alanine mutations within the N-terminal region are indicated in bold. The top panel shows the domain structure of Vac8 (SH4, SH4 domain; NR, asparagine rich domain). Constructs were named according to their non-mutated cysteines. Numbers indicates the amino acid positions within the SH4 domain. (B) Localization of GFP fusion proteins in yeast. BJ3505 vac8 cells were transformed with plasmids expressing the indicated GFP fusion proteins. Their localization was visualized by fluorescence microscopy. Bars, 5 µm. (C) Subcellular localization of Vac8 mutants. Cells expressing the respective mutant proteins were lysed essentially as for vacuole purification. Lysates were cleared by low-speed centrifugation (300 g, 4°C) and half was saved (T), the other was fractionated by centrifugation (10 minutes, 13,000 g, 4°C) into pellet (P) and supernatant (S). Pellets and TCA-precipitated supernatant were analyzed by SDS-PAGE and western blotting with anti-Vac8. Fractionation was confirmed by control antibodies (see Fig. 4D). (D) In vivo localization of Vac8-GFP mutants with single cysteines. Fusion proteins encoding Vac8-Cys4-GFP, Vac8-Cys5-GFP or Vac8-Cys7-GFP were expressed in BJ3505 vac8 cells, and analyzed by fluorescence microscopy. Bars, 5 µm. (E) Subcellular localization. Cells with the indicated Vac8 cysteine mutations were fractionated and analyzed as described in Fig. 1C. Western blots were probed with antibodies to Vac8.