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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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Ypp1 colocalizes with Stt4 at static cortical structures at the PM. (A) Cells expressing Ypp1-GFP were grown at 26°C and examined by fluorescence microscopy. Cells shown are representative of over 200 cells observed. Arrows show regions of highly concentrated Ypp1-GFP. Bar, 4 μm. (B) Cells coexpressing GFP-Stt4 and Ypp1-mCherry were grown at 26°C and examined by confocal microscopy. As previously reported (Audhya and Emr, 2002), GFP-Stt4 localizes to cortical patches on the PM (top left) and with a similar distribution as Ypp1-mCherry (top right). An overlay of the two images (bottom left) shows overlap of the fluorescent patches. Arrows indicate regions of the most conspicuous colocalization. Bar, 4 μm. (C) Schematic representation of Stt4, Ygr198w (Ypp1), and the Ypp1 human homologue TTC7a (available from GenBank/EMBL/DDBJ under accession no. NM_020458). Stt4 is a 215-kD protein with a lipid kinase unique domain (LKU) and a PtdIns 4-kinase domain (PI4K) as its only recognizable motifs. Ypp1 is a 95-kD protein encompassing multiple TPR domains and with conserved homologues in higher species. The schematics are not to scale.
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fig1: Ypp1 colocalizes with Stt4 at static cortical structures at the PM. (A) Cells expressing Ypp1-GFP were grown at 26°C and examined by fluorescence microscopy. Cells shown are representative of over 200 cells observed. Arrows show regions of highly concentrated Ypp1-GFP. Bar, 4 μm. (B) Cells coexpressing GFP-Stt4 and Ypp1-mCherry were grown at 26°C and examined by confocal microscopy. As previously reported (Audhya and Emr, 2002), GFP-Stt4 localizes to cortical patches on the PM (top left) and with a similar distribution as Ypp1-mCherry (top right). An overlay of the two images (bottom left) shows overlap of the fluorescent patches. Arrows indicate regions of the most conspicuous colocalization. Bar, 4 μm. (C) Schematic representation of Stt4, Ygr198w (Ypp1), and the Ypp1 human homologue TTC7a (available from GenBank/EMBL/DDBJ under accession no. NM_020458). Stt4 is a 215-kD protein with a lipid kinase unique domain (LKU) and a PtdIns 4-kinase domain (PI4K) as its only recognizable motifs. Ypp1 is a 95-kD protein encompassing multiple TPR domains and with conserved homologues in higher species. The schematics are not to scale.

Mentions: To understand Stt4 PIK patch assembly and regulation, we sought novel Stt4-interacting partners. We examined several candidate PtdIns 4-kinase regulators by experimentally testing the various Stt4-interacting proteins cataloged in the Saccharomyces Genome Database and identified one uncharacterized essential protein: YGR198w (recently named Ypp1 [Flower et al., 2007]). This interaction was originally published by Hazbun et al. (2003), who isolated Ypp1 and corresponding binding partners through tandem affinity purification and subsequent mass spectrometry to identify Stt4 as the primary binding partner. To determine the localization of Ypp1 in live yeast cells, the sequence encoding GFP was integrated at the 3′ end of Ypp1's chromosomal locus to generate Ypp1-GFP (Fig. 1 A). Similar to GFP-Stt4 (Audhya and Emr, 2002), Ypp1-GFP localized to several punctate structures on the periphery of the cell with a majority of these patches concentrated in the mother cell. Furthermore, strong colocalization of Ypp1-mCherry with GFP-Stt4 on these punctate patches suggests they reside at the same cortical structure on the PM (Fig. 1 B).


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)

Ypp1 colocalizes with Stt4 at static cortical structures at the PM. (A) Cells expressing Ypp1-GFP were grown at 26°C and examined by fluorescence microscopy. Cells shown are representative of over 200 cells observed. Arrows show regions of highly concentrated Ypp1-GFP. Bar, 4 μm. (B) Cells coexpressing GFP-Stt4 and Ypp1-mCherry were grown at 26°C and examined by confocal microscopy. As previously reported (Audhya and Emr, 2002), GFP-Stt4 localizes to cortical patches on the PM (top left) and with a similar distribution as Ypp1-mCherry (top right). An overlay of the two images (bottom left) shows overlap of the fluorescent patches. Arrows indicate regions of the most conspicuous colocalization. Bar, 4 μm. (C) Schematic representation of Stt4, Ygr198w (Ypp1), and the Ypp1 human homologue TTC7a (available from GenBank/EMBL/DDBJ under accession no. NM_020458). Stt4 is a 215-kD protein with a lipid kinase unique domain (LKU) and a PtdIns 4-kinase domain (PI4K) as its only recognizable motifs. Ypp1 is a 95-kD protein encompassing multiple TPR domains and with conserved homologues in higher species. The schematics are not to scale.
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fig1: Ypp1 colocalizes with Stt4 at static cortical structures at the PM. (A) Cells expressing Ypp1-GFP were grown at 26°C and examined by fluorescence microscopy. Cells shown are representative of over 200 cells observed. Arrows show regions of highly concentrated Ypp1-GFP. Bar, 4 μm. (B) Cells coexpressing GFP-Stt4 and Ypp1-mCherry were grown at 26°C and examined by confocal microscopy. As previously reported (Audhya and Emr, 2002), GFP-Stt4 localizes to cortical patches on the PM (top left) and with a similar distribution as Ypp1-mCherry (top right). An overlay of the two images (bottom left) shows overlap of the fluorescent patches. Arrows indicate regions of the most conspicuous colocalization. Bar, 4 μm. (C) Schematic representation of Stt4, Ygr198w (Ypp1), and the Ypp1 human homologue TTC7a (available from GenBank/EMBL/DDBJ under accession no. NM_020458). Stt4 is a 215-kD protein with a lipid kinase unique domain (LKU) and a PtdIns 4-kinase domain (PI4K) as its only recognizable motifs. Ypp1 is a 95-kD protein encompassing multiple TPR domains and with conserved homologues in higher species. The schematics are not to scale.
Mentions: To understand Stt4 PIK patch assembly and regulation, we sought novel Stt4-interacting partners. We examined several candidate PtdIns 4-kinase regulators by experimentally testing the various Stt4-interacting proteins cataloged in the Saccharomyces Genome Database and identified one uncharacterized essential protein: YGR198w (recently named Ypp1 [Flower et al., 2007]). This interaction was originally published by Hazbun et al. (2003), who isolated Ypp1 and corresponding binding partners through tandem affinity purification and subsequent mass spectrometry to identify Stt4 as the primary binding partner. To determine the localization of Ypp1 in live yeast cells, the sequence encoding GFP was integrated at the 3′ end of Ypp1's chromosomal locus to generate Ypp1-GFP (Fig. 1 A). Similar to GFP-Stt4 (Audhya and Emr, 2002), Ypp1-GFP localized to several punctate structures on the periphery of the cell with a majority of these patches concentrated in the mother cell. Furthermore, strong colocalization of Ypp1-mCherry with GFP-Stt4 on these punctate patches suggests they reside at the same cortical structure on the PM (Fig. 1 B).

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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