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Assembly of the PtdIns 4-kinase Stt4 complex at the plasma membrane requires Ypp1 and Efr3.

Baird D, Stefan C, Audhya A, Weys S, Emr SD - J. Cell Biol. (2008)

Bottom Line: We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches.Efr3 is essential for assembly of both Ypp1 and Stt4 at PIK patches.We conclude that Ypp1 and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based PtdIns4P signaling.

View Article: PubMed Central - PubMed

Affiliation: Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
The phosphoinositide phosphatidylinositol 4-phosphate (PtdIns4P) is an essential signaling lipid that regulates secretion and polarization of the actin cytoskeleton. In Saccharomyces cerevisiae, the PtdIns 4-kinase Stt4 catalyzes the synthesis of PtdIns4P at the plasma membrane (PM). In this paper, we identify and characterize two novel regulatory components of the Stt4 kinase complex, Ypp1 and Efr3. The essential gene YPP1 encodes a conserved protein that colocalizes with Stt4 at cortical punctate structures and regulates the stability of this lipid kinase. Accordingly, Ypp1 interacts with distinct regions on Stt4 that are necessary for the assembly and recruitment of multiple copies of the kinase into phosphoinositide kinase (PIK) patches. We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches. Efr3 is essential for assembly of both Ypp1 and Stt4 at PIK patches. We conclude that Ypp1 and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based PtdIns4P signaling.

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Efr3 recruits PIK patch components to the PM. (A) Growth assay of EFR3 wild type and efr3-1 temperature-sensitive yeast at 26 and 38°C. (B) Phosphoinositide levels of EFR3 and efr3-1 at 38°C. Data are presented as means and SD (error bars) of two independent experiments. (C) Cellular localization of the PtdIns4P reporter GFP-2xPHOsh2 in efr3-1 yeast at the permissive (top) and restrictive (bottom) temperatures. Bars, 4 μm. (D) The localization profile of teto-EFR3 GFP-Stt4 in the absence (top) or presence (bottom) of doxycycline and the corresponding DIC image. Arrows indicate PIK patch localization. Bars, 4 μm. (E) The localization profile of teto-YPP1 Efr3-GFP in the absence (top) or presence (bottom) of doxycycline and the corresponding DIC images. Arrows indicate PIK patch localization. Bars, 4 μm.
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fig8: Efr3 recruits PIK patch components to the PM. (A) Growth assay of EFR3 wild type and efr3-1 temperature-sensitive yeast at 26 and 38°C. (B) Phosphoinositide levels of EFR3 and efr3-1 at 38°C. Data are presented as means and SD (error bars) of two independent experiments. (C) Cellular localization of the PtdIns4P reporter GFP-2xPHOsh2 in efr3-1 yeast at the permissive (top) and restrictive (bottom) temperatures. Bars, 4 μm. (D) The localization profile of teto-EFR3 GFP-Stt4 in the absence (top) or presence (bottom) of doxycycline and the corresponding DIC image. Arrows indicate PIK patch localization. Bars, 4 μm. (E) The localization profile of teto-YPP1 Efr3-GFP in the absence (top) or presence (bottom) of doxycycline and the corresponding DIC images. Arrows indicate PIK patch localization. Bars, 4 μm.

Mentions: Next, we addressed whether Efr3 functions in PtdIns4P synthesis and Stt4 PIK patch recruitment to the PM. For this, we generated a temperature-conditional mutant allele (see Materials and methods), efr3-1, which was unable to grow at the restrictive temperature of 38°C (Fig. 8 A). PtdIns4P levels were then determined at the nonpermissive temperature. Temperature-sensitive efr3-1 cells displayed reduced PtdIns4P levels as compared with wild-type EFR3 cells (Fig. 8 B). The depressed PtdIns4P appears specific to the PM pool because the PtdIns4P reporter GFP-2xPHOsh2 persists at the Golgi but is depleted from the PM in efr3-1 cells (Fig. 8 C). To assay the impact of Efr3 on PIK patch distribution, we placed EFR3 under the control of a doxycycline-repressible promoter (teto-EFR3) to selectively deplete expression of Efr3 in yeast harboring a GFP-tagged PIK patch component, Stt4 or Ypp1. Treatment of the teto-EFR3 GFP-Stt4 strain with doxycycline for 12 h resulted in mislocalization of the lipid kinase to the diffuse cytoplasm (Fig. 8 D). In contrast to Stt4, Efr3-GFP localization was still observed at the PM after depletion of Ypp1 in teto-YPP1 cells treated with doxycycline (Fig. 8 E). However, the patch-like punctate distribution of Efr3-GFP at the PM was reduced (Fig. 8 E). In total, these results suggest that Efr3 mediates PIK patch localization to the PM but also imply that the Stt4–Ypp1 complex could control the clustering of proteins within the PIK patch.


Assembly of the PtdIns 4-kinase Stt4 complex at the plasma membrane requires Ypp1 and Efr3.

Baird D, Stefan C, Audhya A, Weys S, Emr SD - J. Cell Biol. (2008)

Efr3 recruits PIK patch components to the PM. (A) Growth assay of EFR3 wild type and efr3-1 temperature-sensitive yeast at 26 and 38°C. (B) Phosphoinositide levels of EFR3 and efr3-1 at 38°C. Data are presented as means and SD (error bars) of two independent experiments. (C) Cellular localization of the PtdIns4P reporter GFP-2xPHOsh2 in efr3-1 yeast at the permissive (top) and restrictive (bottom) temperatures. Bars, 4 μm. (D) The localization profile of teto-EFR3 GFP-Stt4 in the absence (top) or presence (bottom) of doxycycline and the corresponding DIC image. Arrows indicate PIK patch localization. Bars, 4 μm. (E) The localization profile of teto-YPP1 Efr3-GFP in the absence (top) or presence (bottom) of doxycycline and the corresponding DIC images. Arrows indicate PIK patch localization. Bars, 4 μm.
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fig8: Efr3 recruits PIK patch components to the PM. (A) Growth assay of EFR3 wild type and efr3-1 temperature-sensitive yeast at 26 and 38°C. (B) Phosphoinositide levels of EFR3 and efr3-1 at 38°C. Data are presented as means and SD (error bars) of two independent experiments. (C) Cellular localization of the PtdIns4P reporter GFP-2xPHOsh2 in efr3-1 yeast at the permissive (top) and restrictive (bottom) temperatures. Bars, 4 μm. (D) The localization profile of teto-EFR3 GFP-Stt4 in the absence (top) or presence (bottom) of doxycycline and the corresponding DIC image. Arrows indicate PIK patch localization. Bars, 4 μm. (E) The localization profile of teto-YPP1 Efr3-GFP in the absence (top) or presence (bottom) of doxycycline and the corresponding DIC images. Arrows indicate PIK patch localization. Bars, 4 μm.
Mentions: Next, we addressed whether Efr3 functions in PtdIns4P synthesis and Stt4 PIK patch recruitment to the PM. For this, we generated a temperature-conditional mutant allele (see Materials and methods), efr3-1, which was unable to grow at the restrictive temperature of 38°C (Fig. 8 A). PtdIns4P levels were then determined at the nonpermissive temperature. Temperature-sensitive efr3-1 cells displayed reduced PtdIns4P levels as compared with wild-type EFR3 cells (Fig. 8 B). The depressed PtdIns4P appears specific to the PM pool because the PtdIns4P reporter GFP-2xPHOsh2 persists at the Golgi but is depleted from the PM in efr3-1 cells (Fig. 8 C). To assay the impact of Efr3 on PIK patch distribution, we placed EFR3 under the control of a doxycycline-repressible promoter (teto-EFR3) to selectively deplete expression of Efr3 in yeast harboring a GFP-tagged PIK patch component, Stt4 or Ypp1. Treatment of the teto-EFR3 GFP-Stt4 strain with doxycycline for 12 h resulted in mislocalization of the lipid kinase to the diffuse cytoplasm (Fig. 8 D). In contrast to Stt4, Efr3-GFP localization was still observed at the PM after depletion of Ypp1 in teto-YPP1 cells treated with doxycycline (Fig. 8 E). However, the patch-like punctate distribution of Efr3-GFP at the PM was reduced (Fig. 8 E). In total, these results suggest that Efr3 mediates PIK patch localization to the PM but also imply that the Stt4–Ypp1 complex could control the clustering of proteins within the PIK patch.

Bottom Line: We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches.Efr3 is essential for assembly of both Ypp1 and Stt4 at PIK patches.We conclude that Ypp1 and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based PtdIns4P signaling.

View Article: PubMed Central - PubMed

Affiliation: Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
The phosphoinositide phosphatidylinositol 4-phosphate (PtdIns4P) is an essential signaling lipid that regulates secretion and polarization of the actin cytoskeleton. In Saccharomyces cerevisiae, the PtdIns 4-kinase Stt4 catalyzes the synthesis of PtdIns4P at the plasma membrane (PM). In this paper, we identify and characterize two novel regulatory components of the Stt4 kinase complex, Ypp1 and Efr3. The essential gene YPP1 encodes a conserved protein that colocalizes with Stt4 at cortical punctate structures and regulates the stability of this lipid kinase. Accordingly, Ypp1 interacts with distinct regions on Stt4 that are necessary for the assembly and recruitment of multiple copies of the kinase into phosphoinositide kinase (PIK) patches. We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches. Efr3 is essential for assembly of both Ypp1 and Stt4 at PIK patches. We conclude that Ypp1 and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based PtdIns4P signaling.

Show MeSH
Related in: MedlinePlus