The Vac14p-Fig4p complex acts independently of Vac7p and couples PI3,5P2 synthesis and turnover

Abstract

Phosphoinositide-signaling lipids function in diverse cellular pathways. Dynamic changes in the levels of these signaling lipids regulate multiple processes. In particular, when Saccharomyces cerevisiae cells are exposed to hyperosmotic shock, PI3,5P2 (phosphatidylinositol [PI] 3,5-bisphosphate) levels transiently increase 20-fold. This causes the vacuole to undergo multiple acute changes. Control of PI3,5P2 levels occurs through regulation of both its synthesis and turnover. Synthesis is catalyzed by the PI3P 5-kinase Fab1p, and turnover is catalyzed by the PI3,5P2 5-phosphatase Fig4p. In this study, we show that two putative Fab1p activators, Vac7p and Vac14p, independently regulate Fab1p activity. Although Vac7p only regulates Fab1p, surprisingly, we find that Vac14 regulates both Fab1p and Fig4p. Moreover, Fig4p itself functions in both PI3,5P2 synthesis and turnover. In both the absence and presence of Vac7p, the Vac14p-Fig4p complex controls the hyperosmotic shock-induced increase in PI3,5P2 levels. These findings suggest that the dynamic changes in PI3,5P2 are controlled through a tight coupling of synthesis and turnover.

Figure 1.

Selected point mutants in the Fab1p kinase domain or a region just proximal to the kinase domain result in suppression of vacuole volume defects in vac7Δ and/or vac14Δ. (A) Schematic of Fab1p. FAB1 was subjected to random mutagenesis in the region encoding the kinase domain. Mutants were screened in vac7Δ or vac14Δ cells for their ability to restore normal growth at 37°C and normal vacuole volume. (B) Wild-type (LWY7235), fig4Δ (LWY6474), vac14Δ (LWY5177), and vac7Δ (LWY2054) cells were labeled with FM4-64 to visualize vacuole membranes. Schematics under the images depict vacuole volume and approximate number of lobes. Representative fields shown. (C) fab1-6 through fab1-13 mutants were isolated for their ability to suppress defects in vac14Δ cells. Images of the fab1-6 mutant. (D) fab1-14 through fab1-16 mutants were isolated for their ability to suppress defects in vac7Δ cells. Images of the fab1-14 mutant.

Figure 2.

The Fab1p mutants require Fig4p, Vac7p, and Vac14p for maximum production of PI3,5P₂. FAB1, fab1-6, or fab1-14 alleles were expressed in fab1Δ (LWY2055), fab1Δfig4Δ (LWY7002), fab1Δvac14Δ (LWY5956), and fab1Δvac7Δ (LWY2046). Strains were labeled with [³H]inositol for 12 h and were untreated (A) or treated (B) with 0.9 M NaCl for 10 min. Total cellular PI was deacylated and analyzed by HPLC. To normalize for the number of cells and [³H]inositol incorporation, each value is shown as the percentage of total extracted [³H]PI. Mean of three independent experiments. Error bars represent SD.

Figure 3.

In the absence of Vac7p, Vac14p, and Fig4p, the fab1-14 mutant shows no elevation of PI3,5P₂ levels or decrease in vacuole volume after hyperosmotic shock. PI3,5P₂ levels and vacuole morphology were determined for vac7Δfab1-14 (LWY7179) (A) and vac7Δvac14Δfig4Δfab1-14 (LWY7197) (B) cells before and after a 10-min incubation in 0.9 M NaCl. Each data point is a mean of three independent experiments. Representative fields shown.

Figure 4.

Maximum elevation of PI3,5P₂ levels requires Vac7p, Vac14p, and Fig4p. (A) Overexpression of VAC14 or VAC7 increases the levels of PI3,5P₂ under basal conditions. VAC7 alone increases the levels of PI3,5P₂ in response to hyperosmotic shock. In a vac7Δvac14Δfig4Δfab1-14 (LWY7197) strain, each individual activator was expressed from a multicopy plasmid. PI3,5P₂ levels were determined before and after a 10-min incubation in 0.9 M NaCl. Mean of three independent experiments. (B) Coexpression of FIG4 and VAC14 increases PI3,5P₂ levels in response to hyperosmotic shock. Simultaneous expression of all three putative Fab1p activators was performed in vac7Δfab1-14 (LWY7179). Error bars represent SD.

Figure 5.

fig4Δ and vac14Δ mutants are defective in hyperosmotic shock–induced turnover of PI3,5P₂. (A) vac14Δ and fig4Δ cells have a defect in PI3,5P₂ turnover after hyperosmotic shock. Data was compiled from Duex et al. (2006). (B) fig4Δ and vac14Δ cells with fab1-14 as the sole copy of FAB1 have higher levels of PI3,5P₂. Thus, defects in hyperosmotic shock–induced PI3,5P₂ turnover can be readily detected. fab1-14 was expressed in fab1Δ (LWY2055), fab1Δfig4Δ (LWY7002), fab1Δvac14Δ (LWY5956), and fab1Δvac7Δ (LWY2046) cells. Strains were treated with 0.9 M NaCl for 0, 10, or 30 min, and the levels of PI3,5P₂ were determined. Data are the means of three independent experiments. Error bars represent SD. (C) Fig4p levels are reduced in vac14Δ cells. fab1Δ (LWY2055), wild-type (LWY7235), fig4Δ (LWY6474), and vac14Δ (LWY5177) cells were lysed in the absence of detergent, and extracts were separated into membrane (P) and soluble (S) fractions by centrifugation at 13,000 g. The P13 fraction contains large organelles such as the vacuole, Golgi, ER, and nucleus. Western blot analysis was performed using rabbit anti-Fig4p. Equal protein was loaded in each set of P13 and S13 fractions. Data is representative of three independent experiments.

Figure 6.

Fig4p function requires Vac14p. (A–C) VAC7, VAC14, and FIG4 were overexpressed alone and in combination in vac7Δvac14Δfig4Δfab1-14 (LWY7197). All three genes were simultaneously overexpressed in vac7Δfab1-14 (LWY7179). PI3,5P₂ levels were determined before and after treatment with 0.9 M NaCl. Mean of three independent experiments. Error bars represent SD.

Figure 7.

Elevation and turnover of PI3,5P₂ is partially defective in the fig4^G519R mutant. (A) Vac14p localization is normal in the fig4 ^G519R mutant. vac14Δ (LWY5177), wild-type (LWY7235), fig4Δ (LWY6474), and fig4Δ cells expressing fig4 ^G519R were lysed; extracts were spun at 13,000 g to generate membrane (P) and soluble (S) fractions. Equal protein was loaded between each P13 and S13 fraction. Western blot analysis was performed using goat anti-Vac14p. Data are representative of three independent experiments. (B–D) The fig4 ^G519R mutant is partially defective in both elevation and turnover of PI3,5P₂. PI3,5P₂ levels after exposure to 0.9 M NaCl for the indicated amount of time in fig4Δ cells expressing FIG4, fig4 ^G519R, or vector alone. In C and D, FIG4 data are shown in gray. Mean of three independent experiments. Error bars represent SD.

Figure 8.

Vac7p is the major activator of Fab1p during hyperosmotic shock. Fig4p is the major enzyme required for hyperosmotic shock–induced turnover of PI3,5P₂. Vac7p and Vac14p independently activate Fab1p under basal conditions. After hyperosmotic shock, Vac7p further activates Fab1p. The Vac14p–Fig4p complex also activates Fab1p, but to a lesser extent than Vac7p. Maximum Fab1p activation requires all three Fab1p activators. Rapid turnover of PI3,5P₂ after hyperosmotic shock predominantly requires the Vac14p–Fig4p complex.