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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 is a component of PIK patches at the PM. (A) Tetrad dissection of diploid yeast with a deleted chromosomal copy of efr3. (B) Full-length and truncation mutants of GST-tagged Efr3 were purified from E. coli lysates, immobilized on glutathione beads, and normalized to equal amounts. Beads containing the GST-Efr3 constructs were then incubated for 1 h with yeast lysate expressing Ypp1-GFP. Protein that bound the GST-Efr3 fragments was probed using an anti-GFP antibody. (C) Subcellular fractionation of Efr3-GFP from wild-type cell extracts. (D) Cells coexpressing Efr3-GFP and Ypp1-mCherry were grown at 26°C and examined by fluorescence microscopy. Efr3-GFP localizes to cortical patches on the PM (left) and with a similar distribution as Ypp1-mCherry (right). Arrows on the Efr3-GFP image indicate regions of colocalization. Bar, 4 μm.
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fig7: Efr3 is a component of PIK patches at the PM. (A) Tetrad dissection of diploid yeast with a deleted chromosomal copy of efr3. (B) Full-length and truncation mutants of GST-tagged Efr3 were purified from E. coli lysates, immobilized on glutathione beads, and normalized to equal amounts. Beads containing the GST-Efr3 constructs were then incubated for 1 h with yeast lysate expressing Ypp1-GFP. Protein that bound the GST-Efr3 fragments was probed using an anti-GFP antibody. (C) Subcellular fractionation of Efr3-GFP from wild-type cell extracts. (D) Cells coexpressing Efr3-GFP and Ypp1-mCherry were grown at 26°C and examined by fluorescence microscopy. Efr3-GFP localizes to cortical patches on the PM (left) and with a similar distribution as Ypp1-mCherry (right). Arrows on the Efr3-GFP image indicate regions of colocalization. Bar, 4 μm.

Mentions: Our results indicated that Ypp1 played an essential role in stabilizing Stt4 in the PM PIK patch. However, because neither Stt4 nor Ypp1 contain a recognizable membrane-targeting motif, we predicted that there were additional components critical for PIK patch membrane association. Accordingly, during the course of our Ypp1 characterization, a genome-wide protein interaction study uncovered Efr3 as another candidate component of the Stt4 PIK patch (Tarassov et al., 2008). Similar to STT4 and YPP1, we found that EFR3 is an essential gene (Fig. 7 A). Primary sequence analysis of Efr3 revealed potential homologues in higher eukaryotes, including humans. The best characterized of these is the Drosophila melanogaster RBO gene product, a multipass transmembrane protein essential for viability (Faulkner et al., 1998). Recent work has shown the RBO protein to be localized to presynaptic boutons of Drosophila neuromuscular junction synapses, where it has been suggested to have an essential role in synaptic vesicle exocytosis (Huang et al., 2004, 2006).


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 is a component of PIK patches at the PM. (A) Tetrad dissection of diploid yeast with a deleted chromosomal copy of efr3. (B) Full-length and truncation mutants of GST-tagged Efr3 were purified from E. coli lysates, immobilized on glutathione beads, and normalized to equal amounts. Beads containing the GST-Efr3 constructs were then incubated for 1 h with yeast lysate expressing Ypp1-GFP. Protein that bound the GST-Efr3 fragments was probed using an anti-GFP antibody. (C) Subcellular fractionation of Efr3-GFP from wild-type cell extracts. (D) Cells coexpressing Efr3-GFP and Ypp1-mCherry were grown at 26°C and examined by fluorescence microscopy. Efr3-GFP localizes to cortical patches on the PM (left) and with a similar distribution as Ypp1-mCherry (right). Arrows on the Efr3-GFP image indicate regions of colocalization. Bar, 4 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Efr3 is a component of PIK patches at the PM. (A) Tetrad dissection of diploid yeast with a deleted chromosomal copy of efr3. (B) Full-length and truncation mutants of GST-tagged Efr3 were purified from E. coli lysates, immobilized on glutathione beads, and normalized to equal amounts. Beads containing the GST-Efr3 constructs were then incubated for 1 h with yeast lysate expressing Ypp1-GFP. Protein that bound the GST-Efr3 fragments was probed using an anti-GFP antibody. (C) Subcellular fractionation of Efr3-GFP from wild-type cell extracts. (D) Cells coexpressing Efr3-GFP and Ypp1-mCherry were grown at 26°C and examined by fluorescence microscopy. Efr3-GFP localizes to cortical patches on the PM (left) and with a similar distribution as Ypp1-mCherry (right). Arrows on the Efr3-GFP image indicate regions of colocalization. Bar, 4 μm.
Mentions: Our results indicated that Ypp1 played an essential role in stabilizing Stt4 in the PM PIK patch. However, because neither Stt4 nor Ypp1 contain a recognizable membrane-targeting motif, we predicted that there were additional components critical for PIK patch membrane association. Accordingly, during the course of our Ypp1 characterization, a genome-wide protein interaction study uncovered Efr3 as another candidate component of the Stt4 PIK patch (Tarassov et al., 2008). Similar to STT4 and YPP1, we found that EFR3 is an essential gene (Fig. 7 A). Primary sequence analysis of Efr3 revealed potential homologues in higher eukaryotes, including humans. The best characterized of these is the Drosophila melanogaster RBO gene product, a multipass transmembrane protein essential for viability (Faulkner et al., 1998). Recent work has shown the RBO protein to be localized to presynaptic boutons of Drosophila neuromuscular junction synapses, where it has been suggested to have an essential role in synaptic vesicle exocytosis (Huang et al., 2004, 2006).

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