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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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Deletion of SAC1 bypasses ypp1Δ or stt4Δ. (A) Relative growth profiles of wild type, sac1Δ, sac1Δ stt4Δ, sac1Δ ypp1Δ, stt4Δ, and ypp1Δ at 26°C. (B) Phosphoinositide levels of wild type, sac1Δ, sac1Δ stt4Δ, and sac1Δ ypp1Δ. Data are presented as means and SD (error bars) of three independent experiments. (C) Cellular localization of the PtdIns4P reporter GFP-2xPHOsh2 in wild type, pik1ts, and stt4ts (top) at the restrictive temperature. (bottom) The fluorescent localization of GFP-2xPHOsh2 in teto-YPP1 grown in the presence (bottom) or absence (top) of doxycycline for 20 h. Bars, 4 μm.
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fig4: Deletion of SAC1 bypasses ypp1Δ or stt4Δ. (A) Relative growth profiles of wild type, sac1Δ, sac1Δ stt4Δ, sac1Δ ypp1Δ, stt4Δ, and ypp1Δ at 26°C. (B) Phosphoinositide levels of wild type, sac1Δ, sac1Δ stt4Δ, and sac1Δ ypp1Δ. Data are presented as means and SD (error bars) of three independent experiments. (C) Cellular localization of the PtdIns4P reporter GFP-2xPHOsh2 in wild type, pik1ts, and stt4ts (top) at the restrictive temperature. (bottom) The fluorescent localization of GFP-2xPHOsh2 in teto-YPP1 grown in the presence (bottom) or absence (top) of doxycycline for 20 h. Bars, 4 μm.

Mentions: Our focus turned toward studying steady-state Stt4 activity in the absence of Ypp1. For this, we isolated a bypass suppressor of ypp1Δ, the PtdIns 4-phosphatase SAC1. We found that yeast lacking SAC could bypass deletion of YPP. Additionally, sac1Δ could bypass stt4Δ. The relative growth strength of these various strains demonstrate that the sac1Δ ypp1Δ yeast are slightly healthier than sac1Δ stt4Δ, implying that the PtdIns 4-kinase may retain some function in the absence of Ypp1 (Fig. 4 A). To further analyze the importance of this finding, we measured PIP levels of each strain.


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)

Deletion of SAC1 bypasses ypp1Δ or stt4Δ. (A) Relative growth profiles of wild type, sac1Δ, sac1Δ stt4Δ, sac1Δ ypp1Δ, stt4Δ, and ypp1Δ at 26°C. (B) Phosphoinositide levels of wild type, sac1Δ, sac1Δ stt4Δ, and sac1Δ ypp1Δ. Data are presented as means and SD (error bars) of three independent experiments. (C) Cellular localization of the PtdIns4P reporter GFP-2xPHOsh2 in wild type, pik1ts, and stt4ts (top) at the restrictive temperature. (bottom) The fluorescent localization of GFP-2xPHOsh2 in teto-YPP1 grown in the presence (bottom) or absence (top) of doxycycline for 20 h. Bars, 4 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Deletion of SAC1 bypasses ypp1Δ or stt4Δ. (A) Relative growth profiles of wild type, sac1Δ, sac1Δ stt4Δ, sac1Δ ypp1Δ, stt4Δ, and ypp1Δ at 26°C. (B) Phosphoinositide levels of wild type, sac1Δ, sac1Δ stt4Δ, and sac1Δ ypp1Δ. Data are presented as means and SD (error bars) of three independent experiments. (C) Cellular localization of the PtdIns4P reporter GFP-2xPHOsh2 in wild type, pik1ts, and stt4ts (top) at the restrictive temperature. (bottom) The fluorescent localization of GFP-2xPHOsh2 in teto-YPP1 grown in the presence (bottom) or absence (top) of doxycycline for 20 h. Bars, 4 μm.
Mentions: Our focus turned toward studying steady-state Stt4 activity in the absence of Ypp1. For this, we isolated a bypass suppressor of ypp1Δ, the PtdIns 4-phosphatase SAC1. We found that yeast lacking SAC could bypass deletion of YPP. Additionally, sac1Δ could bypass stt4Δ. The relative growth strength of these various strains demonstrate that the sac1Δ ypp1Δ yeast are slightly healthier than sac1Δ stt4Δ, implying that the PtdIns 4-kinase may retain some function in the absence of Ypp1 (Fig. 4 A). To further analyze the importance of this finding, we measured PIP levels of each strain.

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