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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 functions in the PtdIns4P signaling pathway to positively regulate Stt4 kinase activity. (A) The relative growth capabilities of wild type, stt4-4, and ypp1-7 at 26 (top left) and 37°C (bottom left). The relative growth capability of ypp1-7 with overexpression of YPP1 from a 2μ plasmid, plasmid alone (pRS426), or overexpressed STT4 at 37°C (top right). Similar growth profile of stt4-4 at 37°C in the presence of vector alone or in the presence of overexpressed YPP1 (bottom right). (B) Whole cell lysate from teto-YPP1-HA cells grown in the presence or absence of doxycycline were analyzed by Western blot with an anti-HA antibody. The relative abundance of Ypp1-HA under the indicated conditions (top) and the relative abundance of a control protein, G6PDH, from the same lysate (bottom) are shown. (C) Phosphoinositide levels in teto-YPP1 cells with and without doxycycline and control R1158 in the presence of doxycycline. Data are presented as means and SD (error bars) of three independent experiments.
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fig3: Ypp1 functions in the PtdIns4P signaling pathway to positively regulate Stt4 kinase activity. (A) The relative growth capabilities of wild type, stt4-4, and ypp1-7 at 26 (top left) and 37°C (bottom left). The relative growth capability of ypp1-7 with overexpression of YPP1 from a 2μ plasmid, plasmid alone (pRS426), or overexpressed STT4 at 37°C (top right). Similar growth profile of stt4-4 at 37°C in the presence of vector alone or in the presence of overexpressed YPP1 (bottom right). (B) Whole cell lysate from teto-YPP1-HA cells grown in the presence or absence of doxycycline were analyzed by Western blot with an anti-HA antibody. The relative abundance of Ypp1-HA under the indicated conditions (top) and the relative abundance of a control protein, G6PDH, from the same lysate (bottom) are shown. (C) Phosphoinositide levels in teto-YPP1 cells with and without doxycycline and control R1158 in the presence of doxycycline. Data are presented as means and SD (error bars) of three independent experiments.

Mentions: Our current study utilizes the strongest of these alleles, ypp1-7, in combination with the stt4-4 mutant strain to detect synthetic growth defects. The ypp1-7 allele, which exhibited strong growth at 26°C, is dead at 37°C (Fig. 3 A). We dissected >20 tetrads generated from diploids with a deleted chromosomal copy of stt4 and ypp1 but carrying stt4-4 and ypp1-7 on plasmids. We failed to isolate a single mutant strain with the stt4-4 ypp1-7 combination. The synthetic lethality of the combined mutant alleles suggests the two gene products function in parallel pathways or the same pathway. Additionally, we found that overexpression of YPP1 from a 2μ plasmid could not rescue the growth defect of stt4-4 at 37°C (Fig. 3 A, bottom right); however, overexpression of the STT4 gene from a 2μ plasmid mildly rescues the growth defect of ypp1-7 at 37°C (Fig. 3 A, top right).


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 functions in the PtdIns4P signaling pathway to positively regulate Stt4 kinase activity. (A) The relative growth capabilities of wild type, stt4-4, and ypp1-7 at 26 (top left) and 37°C (bottom left). The relative growth capability of ypp1-7 with overexpression of YPP1 from a 2μ plasmid, plasmid alone (pRS426), or overexpressed STT4 at 37°C (top right). Similar growth profile of stt4-4 at 37°C in the presence of vector alone or in the presence of overexpressed YPP1 (bottom right). (B) Whole cell lysate from teto-YPP1-HA cells grown in the presence or absence of doxycycline were analyzed by Western blot with an anti-HA antibody. The relative abundance of Ypp1-HA under the indicated conditions (top) and the relative abundance of a control protein, G6PDH, from the same lysate (bottom) are shown. (C) Phosphoinositide levels in teto-YPP1 cells with and without doxycycline and control R1158 in the presence of doxycycline. Data are presented as means and SD (error bars) of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Ypp1 functions in the PtdIns4P signaling pathway to positively regulate Stt4 kinase activity. (A) The relative growth capabilities of wild type, stt4-4, and ypp1-7 at 26 (top left) and 37°C (bottom left). The relative growth capability of ypp1-7 with overexpression of YPP1 from a 2μ plasmid, plasmid alone (pRS426), or overexpressed STT4 at 37°C (top right). Similar growth profile of stt4-4 at 37°C in the presence of vector alone or in the presence of overexpressed YPP1 (bottom right). (B) Whole cell lysate from teto-YPP1-HA cells grown in the presence or absence of doxycycline were analyzed by Western blot with an anti-HA antibody. The relative abundance of Ypp1-HA under the indicated conditions (top) and the relative abundance of a control protein, G6PDH, from the same lysate (bottom) are shown. (C) Phosphoinositide levels in teto-YPP1 cells with and without doxycycline and control R1158 in the presence of doxycycline. Data are presented as means and SD (error bars) of three independent experiments.
Mentions: Our current study utilizes the strongest of these alleles, ypp1-7, in combination with the stt4-4 mutant strain to detect synthetic growth defects. The ypp1-7 allele, which exhibited strong growth at 26°C, is dead at 37°C (Fig. 3 A). We dissected >20 tetrads generated from diploids with a deleted chromosomal copy of stt4 and ypp1 but carrying stt4-4 and ypp1-7 on plasmids. We failed to isolate a single mutant strain with the stt4-4 ypp1-7 combination. The synthetic lethality of the combined mutant alleles suggests the two gene products function in parallel pathways or the same pathway. Additionally, we found that overexpression of YPP1 from a 2μ plasmid could not rescue the growth defect of stt4-4 at 37°C (Fig. 3 A, bottom right); however, overexpression of the STT4 gene from a 2μ plasmid mildly rescues the growth defect of ypp1-7 at 37°C (Fig. 3 A, top right).

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