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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 stabilizes Ypp1 molecules on a membrane-bound fraction of the cell. (A) Subcellular fractionation of cells expressing HA-Ypp1 (left) in a wild-type background and HA-Ypp1 distribution in the absence of SAC1 and STT4 (right). Protein component fractions in the pellet (P100; rcf of 100,000) or soluble (S100; soluble portion) fractions. Fractionation of G6PDH (soluble) and Pep13 (membrane-tethered Golgi component) are shown as controls. (B) FPLC elution profile of HA-Ypp1 isolated from the S100 portion of sac1Δ stt4Δ cells (top). Fractions were collected at the indicated elution volumes, separated by SDS-PAGE, and detected using an anti-HA antibody. The molecular mass corresponding to each elution is indicated below the blot. (C) Coimmunoprecipitation of HA-Ypp1 from yeast lysate that coexpressed GFP alone, GFP-Ypp1, or GFP-Stt4. (D) Coimmunoprecipitation of HA-Ypp1 with GFP-Ypp1 in the wild type, sac1Δ, and sac1Δ stt4Δ background. (E) FPLC sizing of E. coli–expressed and purified HIS6-Ypp1 using a Superdex 200 analytical column.
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fig6: Stt4 stabilizes Ypp1 molecules on a membrane-bound fraction of the cell. (A) Subcellular fractionation of cells expressing HA-Ypp1 (left) in a wild-type background and HA-Ypp1 distribution in the absence of SAC1 and STT4 (right). Protein component fractions in the pellet (P100; rcf of 100,000) or soluble (S100; soluble portion) fractions. Fractionation of G6PDH (soluble) and Pep13 (membrane-tethered Golgi component) are shown as controls. (B) FPLC elution profile of HA-Ypp1 isolated from the S100 portion of sac1Δ stt4Δ cells (top). Fractions were collected at the indicated elution volumes, separated by SDS-PAGE, and detected using an anti-HA antibody. The molecular mass corresponding to each elution is indicated below the blot. (C) Coimmunoprecipitation of HA-Ypp1 from yeast lysate that coexpressed GFP alone, GFP-Ypp1, or GFP-Stt4. (D) Coimmunoprecipitation of HA-Ypp1 with GFP-Ypp1 in the wild type, sac1Δ, and sac1Δ stt4Δ background. (E) FPLC sizing of E. coli–expressed and purified HIS6-Ypp1 using a Superdex 200 analytical column.

Mentions: We used differential centrifugation to fractionate the various subcellular components of yeast to compare relative PIK patch densities with known cellular components. HA-Ypp1 fractionates into the membrane-associated P100 fraction in a wild-type background (Fig. 6 A). However, deletion of STT4 in the sac1Δ background completely redistributes HA-Ypp1 from P100 fraction to the S100 fraction, indicating that its normal distribution to membrane-bound structures depends on Stt4 (Fig. 6 A, right). We collected this soluble fraction and determined the relative size of a Ypp1 protein complex by measuring its elution from a Superdex S300 column in the absence of Stt4. Soluble HA-Ypp1 (99 kD) from the stt4Δ sac1Δ strain was stable and eluted in several fractions with a peak corresponding to a molecular mass of 150 kD (Fig. 6 B). The fast protein liquid chromatography (FPLC) elution profile suggests that Ypp1 may dimerize or bind to other PIK patch-associated components.


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 stabilizes Ypp1 molecules on a membrane-bound fraction of the cell. (A) Subcellular fractionation of cells expressing HA-Ypp1 (left) in a wild-type background and HA-Ypp1 distribution in the absence of SAC1 and STT4 (right). Protein component fractions in the pellet (P100; rcf of 100,000) or soluble (S100; soluble portion) fractions. Fractionation of G6PDH (soluble) and Pep13 (membrane-tethered Golgi component) are shown as controls. (B) FPLC elution profile of HA-Ypp1 isolated from the S100 portion of sac1Δ stt4Δ cells (top). Fractions were collected at the indicated elution volumes, separated by SDS-PAGE, and detected using an anti-HA antibody. The molecular mass corresponding to each elution is indicated below the blot. (C) Coimmunoprecipitation of HA-Ypp1 from yeast lysate that coexpressed GFP alone, GFP-Ypp1, or GFP-Stt4. (D) Coimmunoprecipitation of HA-Ypp1 with GFP-Ypp1 in the wild type, sac1Δ, and sac1Δ stt4Δ background. (E) FPLC sizing of E. coli–expressed and purified HIS6-Ypp1 using a Superdex 200 analytical column.
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fig6: Stt4 stabilizes Ypp1 molecules on a membrane-bound fraction of the cell. (A) Subcellular fractionation of cells expressing HA-Ypp1 (left) in a wild-type background and HA-Ypp1 distribution in the absence of SAC1 and STT4 (right). Protein component fractions in the pellet (P100; rcf of 100,000) or soluble (S100; soluble portion) fractions. Fractionation of G6PDH (soluble) and Pep13 (membrane-tethered Golgi component) are shown as controls. (B) FPLC elution profile of HA-Ypp1 isolated from the S100 portion of sac1Δ stt4Δ cells (top). Fractions were collected at the indicated elution volumes, separated by SDS-PAGE, and detected using an anti-HA antibody. The molecular mass corresponding to each elution is indicated below the blot. (C) Coimmunoprecipitation of HA-Ypp1 from yeast lysate that coexpressed GFP alone, GFP-Ypp1, or GFP-Stt4. (D) Coimmunoprecipitation of HA-Ypp1 with GFP-Ypp1 in the wild type, sac1Δ, and sac1Δ stt4Δ background. (E) FPLC sizing of E. coli–expressed and purified HIS6-Ypp1 using a Superdex 200 analytical column.
Mentions: We used differential centrifugation to fractionate the various subcellular components of yeast to compare relative PIK patch densities with known cellular components. HA-Ypp1 fractionates into the membrane-associated P100 fraction in a wild-type background (Fig. 6 A). However, deletion of STT4 in the sac1Δ background completely redistributes HA-Ypp1 from P100 fraction to the S100 fraction, indicating that its normal distribution to membrane-bound structures depends on Stt4 (Fig. 6 A, right). We collected this soluble fraction and determined the relative size of a Ypp1 protein complex by measuring its elution from a Superdex S300 column in the absence of Stt4. Soluble HA-Ypp1 (99 kD) from the stt4Δ sac1Δ strain was stable and eluted in several fractions with a peak corresponding to a molecular mass of 150 kD (Fig. 6 B). The fast protein liquid chromatography (FPLC) elution profile suggests that Ypp1 may dimerize or bind to other PIK patch-associated components.

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