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First published online 23 May 2006
doi: 10.1242/jcs.02972

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Palmitoylation determines the function of Vac8 at the yeast vacuole

K. Subramanian^1,2,*, L. E. P. Dietrich^1,*,, H. Hou^1,2, T. J. LaGrassa^1,, C. T. A. Meiringer^1,2 and C. Ungermann^1,2,¶

¹ Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
² University of Osnabrück, Department of Biology, Barbarastr. 13, 49069 Osnabrück, Germany

View larger version (76K): [in a new window]   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.

View larger version (46K): [in a new window]   Fig. 2. Analysis of membrane association and palmitoylation of Vac8 mutants. (A) Expression levels of endogenous Vac8 mutants. Proteins were extracted from BJ3505 strains expressing the respective mutants and analyzed by SDS-PAGE and western blotting with antibodies to Vac8 and Vti1. (B,C) Localization of Vac8 mutants to purified vacuoles. Vacuoles were isolated from strains carrying the indicated mutations in Vac8 as described in the Materials and Methods, then washed with 20 mM PIPES (pH 6.8), 150 mM KCl. Isolated vacuoles (30 µg) and total cell lysates were analyzed by SDS-PAGE and western blotting (B). (C) Effect of SH4 mutations in Vac8 on vacuole, binding and inheritance. For vacuole binding, Vac8 bands in B were quantified by laser densitometry (grey bars). Fusion (black bars) was determined by the standard fusion assay (see Materials and Methods). Where indicated, error bars are standard deviations (n=3). To determine vacuole inheritance (white bars), yeast cells of the respective mutants were analyzed by FM4-64 staining and fluorescence microscopy. For each strain at least 80 cells were counted. Inheritance of the wild-type control was on average 72%, which was set to 100% in the Figure. (D) Palmitoylation of Vac8 detected by the biotin-switch method (Hou et al., 2005). Yeast cells were lysed, and free cysteines were quenched by NEM. The extract was then subjected to the biotin-switch procedure using hydroxylamine (HA) and Biotin-BMCC as a crosslinker. A fraction of the lysate (6%) was removed and proteins were TCA precipitated. The remaining modified proteins were captured by Neutravidin-agarose pull-down assay, and analyzed by SDS-PAGE and western blotting with the indicated antibodies. The wild-type control is the same as in Hou et al. (Hou et al., 2005). (E) Yeast cells expressing the respective Vac8 mutant analyzed as in D for palmitoylation.

View larger version (66K): [in a new window]   Fig. 3. Targeting of GFP by Vac8 wild-type and mutant SH4 domains in yeast. GFP-fusion proteins encoding the first 18 amino acids of Vac8 (A) or the indicated mutations (B) were expressed in BJ3505 vac8 cells and analyzed by fluorescence microscopy. Bars, 5 µm. (C) Subcellular localization of Vac8(1-18)-GFP. The indicated cells were analyzed as in Fig. 1C. Proteins were analyzed by SDS-PAGE and western blotting with anti-GFP antibodies. (D) Palmitoylation of the SH4 constructs. The biotin-switch method was used as described in Fig. 2D and Hou et al. (Hou et al., 2005). Western blots were decorated as in C.

View larger version (37K): [in a new window]   Fig. 4. Vacuole targeting of Vac8 through the Src kinase SH4 domain. (A) Comparison of the SH4 sequence of Vac8 and the Src kinase. Alignment of the two SH4 domains is shown. (B) Localization of Src(1-16)-GFP by confocal fluorescence microscopy. The GFP-fusion protein was expressed from a plasmid in the vac8 background. Vacuoles were visualized with the lipophilic dye FM4-64. (C) Vacuole targeting of Vac8 through the Src kinase SH4 domain. Src-Vac8-GFP protein was expressed from a plasmid in vac8 cells and monitored by confocal microscopy in FM4-64 stained cells. Bars, 5 µm. (D) Subcellular localization of Src-Vac8. Fractionation was done as in Fig. 1C. Pellets and TCA-precipitated supernatant were analyzed by SDS-PAGE and western blotting with anti-Vac8, anti-Rpl13 and anti-Vti1 antibodies. Rpl13 is a ribosomal subunit (cytosolic marker), Vti1 is found on vacuoles (membrane marker). See Materials and Methods for details.

View larger version (43K): [in a new window]   Fig. 5. Vac8 function depends on palmitoylation. (A) Vacuole fusion. Vacuoles were isolated from the two vac8 tester strains carrying no Vac8 protein (vac8), Vac8 wild-type, the Cys- mutant (cys-) or the Src-Vac8 chimera, and incubated in a standard vacuole fusion reaction for 90 min at 26°C (see Materials and Methods), and then assayed for alkaline phosphatase activity. The average of four independent experiments is shown; error bars are standard deviations. (B) Vacuole inheritance. Analysis of yeast cells carrying the respective mutants was done as in Fig. 2C. (C) Morphology and inheritance of the mutant strains. Inheritance of FM4-64 labeled vacuoles was visualized by fluorescence microscopy. Bars, 5 µm. (D) AP-1 maturation assay. Cells from the indicated strains were fractionated into total (T) and a membrane containing pellet (P) fraction. Proteins were analyzed by SDS-PAGE and western blots were probed with antibodies to Ape1. pr, immature protein; m, mature protein. (E) Triton X-114 partitioning of wild-type Vac8 and Src-Vac8. Cells expressing the indicated Vac8 variant were fractionated into a P13 and S13 fraction, and subjected to Triton X-114 partitioning. Proteins in the aqueous (A) and detergent (D) phase were TCA precipitated and analyzed by SDS-PAGE and western blotting with antibodies to Vac8. (F) Membrane association of Vac8 and Src-Vac8. Vacuoles (30 µg) from strains expressing the respective Vac8 variants were subjected to addition of 100 mM Na2CO3 (carb.), 6 M urea, 1 M NaCl or 1% Triton X-100. Samples were vortexed, incubated on ice for 30 minutes, and then centrifuged (30 minutes, 100,000 g, 4°C) (Ungermann and Wickner, 1998). Pellet and TCA-precipitated supernatant were dissolved in SDS sample buffer, and analyzed by SDS-PAGE and western blotting with the indicated antibodies. The same wild-type control has also been used in Hou et al. (Hou et al., 2005). (G) Morphology and Inheritance of Src (1-16; S4C)-Vac8. Analysis was done as in Fig. 2C. Phase-contrast and FM4-64 images were superimposed using Photoshop 7.0. Bar, 5 µm. (H) Summary of data from this study. The asterisk on Cys4,5 indicates that similar results were obtained for all single Cys-Ala mutations; ND, not determined.