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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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Stt4 and Ypp1 are codependent for proper localization and organization into PIK patches at the PM. (A) Relative abundance of GFP-Stt4 after doxycycline treatment in teto-YPP1 cells over the time course of 16 h. The relative abundance of G6PDH is shown as a control. (B) The localization profile of teto-YPP1 GFP-Stt4 in the absence (top) or presence (second panel) of doxycycline and the corresponding DIC image. The localization teto-STT4 GFP-Ypp1 in the absence (third panel) or presence (bottom) of doxycycline and the corresponding DIC images. Bars, 4 μm. (C) Localization of PM anchored Psr11-28-GFP-Ypp1 expressed under wild-type conditions (left) or in the sac1Δ stt4Δ background (right). The graph is the relative fluorescence intensity along the PM within the indicated spatial region. Arrows indicating the start and end of the fluorescent images correspond to the x axis of the graph.
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fig5: Stt4 and Ypp1 are codependent for proper localization and organization into PIK patches at the PM. (A) Relative abundance of GFP-Stt4 after doxycycline treatment in teto-YPP1 cells over the time course of 16 h. The relative abundance of G6PDH is shown as a control. (B) The localization profile of teto-YPP1 GFP-Stt4 in the absence (top) or presence (second panel) of doxycycline and the corresponding DIC image. The localization teto-STT4 GFP-Ypp1 in the absence (third panel) or presence (bottom) of doxycycline and the corresponding DIC images. Bars, 4 μm. (C) Localization of PM anchored Psr11-28-GFP-Ypp1 expressed under wild-type conditions (left) or in the sac1Δ stt4Δ background (right). The graph is the relative fluorescence intensity along the PM within the indicated spatial region. Arrows indicating the start and end of the fluorescent images correspond to the x axis of the graph.

Mentions: We next analyzed the organization of the PM Stt4 PIK patch. We asked if Ypp1 recruits Stt4 to the PM and/or structurally organizes Stt4 to regulate PtdIns4P metabolism. We detected very low amounts of GFP-Stt4 with either Western blot or fluorescence microscopy in the sac1Δ ypp1Δ strain compared with wild type or sac1Δ. Therefore, we hypothesized that Ypp1 may be required for the stability of Stt4. We determined that attenuation of Ypp1 levels in the teto-YPP1 GFP-STT4 strain caused a corresponding decrease of GFP-Stt4 (Fig. 5 A). Within 9 h, GFP-Stt4 decreased to <10% its initial levels and is barely visible by 16 h. Therefore, we found it very difficult to assess Stt4 distribution in the absence of Ypp1.


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)

Stt4 and Ypp1 are codependent for proper localization and organization into PIK patches at the PM. (A) Relative abundance of GFP-Stt4 after doxycycline treatment in teto-YPP1 cells over the time course of 16 h. The relative abundance of G6PDH is shown as a control. (B) The localization profile of teto-YPP1 GFP-Stt4 in the absence (top) or presence (second panel) of doxycycline and the corresponding DIC image. The localization teto-STT4 GFP-Ypp1 in the absence (third panel) or presence (bottom) of doxycycline and the corresponding DIC images. Bars, 4 μm. (C) Localization of PM anchored Psr11-28-GFP-Ypp1 expressed under wild-type conditions (left) or in the sac1Δ stt4Δ background (right). The graph is the relative fluorescence intensity along the PM within the indicated spatial region. Arrows indicating the start and end of the fluorescent images correspond to the x axis of the graph.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600738&req=5

fig5: Stt4 and Ypp1 are codependent for proper localization and organization into PIK patches at the PM. (A) Relative abundance of GFP-Stt4 after doxycycline treatment in teto-YPP1 cells over the time course of 16 h. The relative abundance of G6PDH is shown as a control. (B) The localization profile of teto-YPP1 GFP-Stt4 in the absence (top) or presence (second panel) of doxycycline and the corresponding DIC image. The localization teto-STT4 GFP-Ypp1 in the absence (third panel) or presence (bottom) of doxycycline and the corresponding DIC images. Bars, 4 μm. (C) Localization of PM anchored Psr11-28-GFP-Ypp1 expressed under wild-type conditions (left) or in the sac1Δ stt4Δ background (right). The graph is the relative fluorescence intensity along the PM within the indicated spatial region. Arrows indicating the start and end of the fluorescent images correspond to the x axis of the graph.
Mentions: We next analyzed the organization of the PM Stt4 PIK patch. We asked if Ypp1 recruits Stt4 to the PM and/or structurally organizes Stt4 to regulate PtdIns4P metabolism. We detected very low amounts of GFP-Stt4 with either Western blot or fluorescence microscopy in the sac1Δ ypp1Δ strain compared with wild type or sac1Δ. Therefore, we hypothesized that Ypp1 may be required for the stability of Stt4. We determined that attenuation of Ypp1 levels in the teto-YPP1 GFP-STT4 strain caused a corresponding decrease of GFP-Stt4 (Fig. 5 A). Within 9 h, GFP-Stt4 decreased to <10% its initial levels and is barely visible by 16 h. Therefore, we found it very difficult to assess Stt4 distribution in the absence of Ypp1.

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