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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 directly interacts with Ypp1 to form a stable complex. (A) Fluorescent image of GFP-Stt4 (left), Ypp1-GFP (right), and their corresponding kymographs (bottom). A representative section of the cell (the demarcated region in top panels) was examined for its relative movement at the cell membrane. A fluorescent time point of the demarcated region was taken every 5 s over the course of 3 min. The spatial variance of the selected fluorescent region with regard to time is represented in the bottom image. (B) FRAP of the PtdIns4P reporter GFP-2xPHOsh2 (top and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200804003/DC1) and GFP-Stt4 (bottom and Video 2). The white partitions demarcate the regions exposed to high intensity light, a FRAP region, and a control region exposed to normal excitation. The corresponding graph quantifies the fluorescent values with regard to time. (C) Cells expressing HA-Ypp1 and GFP alone or GFP-Stt4 were grown to mid-log phase, lysed, and incubated with an anti-GFP antibody. Coimmunoprecipitated proteins were detected with an anti-HA antibody. The relative abundance of coimmunoprecipitated HA-Ypp1 is shown in the top panel compared with a sample of the input from the whole cell lysate (WCL) in the bottom panel. Note that a small region of the immunoprecipitated GFP-Stt4 is cleaved at the C terminus. (D) Truncation mutants of GST-tagged Stt4 were purified from E. coli lysates, immobilized on glutathione beads, and normalized to equal amounts. Beads containing the GST-Stt4 truncations were then incubated for 1 h with lysate from E. coli overexpressing HIS6-Ypp1. Protein that bound the GST-Stt4 fragments was probed using an anti-HIS6 antibody. The relative amount of HIS6-Ypp1 that bound each respective GST-Stt4 fragment (top) and the relative amount of the Stt4 fragments that bound the glutathione beads (bottom) are shown. Notice that the primary Stt4 fragment (736–1346) is a C-terminal degradation product missing ∼200 amino acids. C-terminal degradation products are marked with an asterisk.
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fig2: Stt4 directly interacts with Ypp1 to form a stable complex. (A) Fluorescent image of GFP-Stt4 (left), Ypp1-GFP (right), and their corresponding kymographs (bottom). A representative section of the cell (the demarcated region in top panels) was examined for its relative movement at the cell membrane. A fluorescent time point of the demarcated region was taken every 5 s over the course of 3 min. The spatial variance of the selected fluorescent region with regard to time is represented in the bottom image. (B) FRAP of the PtdIns4P reporter GFP-2xPHOsh2 (top and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200804003/DC1) and GFP-Stt4 (bottom and Video 2). The white partitions demarcate the regions exposed to high intensity light, a FRAP region, and a control region exposed to normal excitation. The corresponding graph quantifies the fluorescent values with regard to time. (C) Cells expressing HA-Ypp1 and GFP alone or GFP-Stt4 were grown to mid-log phase, lysed, and incubated with an anti-GFP antibody. Coimmunoprecipitated proteins were detected with an anti-HA antibody. The relative abundance of coimmunoprecipitated HA-Ypp1 is shown in the top panel compared with a sample of the input from the whole cell lysate (WCL) in the bottom panel. Note that a small region of the immunoprecipitated GFP-Stt4 is cleaved at the C terminus. (D) Truncation mutants of GST-tagged Stt4 were purified from E. coli lysates, immobilized on glutathione beads, and normalized to equal amounts. Beads containing the GST-Stt4 truncations were then incubated for 1 h with lysate from E. coli overexpressing HIS6-Ypp1. Protein that bound the GST-Stt4 fragments was probed using an anti-HIS6 antibody. The relative amount of HIS6-Ypp1 that bound each respective GST-Stt4 fragment (top) and the relative amount of the Stt4 fragments that bound the glutathione beads (bottom) are shown. Notice that the primary Stt4 fragment (736–1346) is a C-terminal degradation product missing ∼200 amino acids. C-terminal degradation products are marked with an asterisk.

Mentions: Subsequently, we studied the dynamics of Stt4 and Ypp1 at PIK patches and compared it to the Stt4 product PtdIns4P. Fig. 2 A is a fluorescent image with the corresponding kymograph of GFP-Stt4 and Ypp1-GFP. Unlike cortical actin patches (Kaksonen et al., 2005), GFP-Stt4 PIK patches are stable over the course of 3 min. To test whether the Stt4 molecules residing in these patches were also static instead of dynamically exchanging, we performed FRAP on a region of the PM (Fig. 2 B and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200804003/DC1). 3 min after photobleaching a PIK patch–rich region of the PM, none of the fluorescence had recovered. This is in stark contrast to the Stt4 product PtdIns4P as observed with a PtdIns4P reporter, GFP-2xPHOsh2 (Yu et al., 2004). After photobleaching a PtdIns4P-rich region of the PM, the fluorescence of the GFP-2xPHOsh2 reporter recovers rapidly (Fig. 2 B and Video 3).


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 directly interacts with Ypp1 to form a stable complex. (A) Fluorescent image of GFP-Stt4 (left), Ypp1-GFP (right), and their corresponding kymographs (bottom). A representative section of the cell (the demarcated region in top panels) was examined for its relative movement at the cell membrane. A fluorescent time point of the demarcated region was taken every 5 s over the course of 3 min. The spatial variance of the selected fluorescent region with regard to time is represented in the bottom image. (B) FRAP of the PtdIns4P reporter GFP-2xPHOsh2 (top and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200804003/DC1) and GFP-Stt4 (bottom and Video 2). The white partitions demarcate the regions exposed to high intensity light, a FRAP region, and a control region exposed to normal excitation. The corresponding graph quantifies the fluorescent values with regard to time. (C) Cells expressing HA-Ypp1 and GFP alone or GFP-Stt4 were grown to mid-log phase, lysed, and incubated with an anti-GFP antibody. Coimmunoprecipitated proteins were detected with an anti-HA antibody. The relative abundance of coimmunoprecipitated HA-Ypp1 is shown in the top panel compared with a sample of the input from the whole cell lysate (WCL) in the bottom panel. Note that a small region of the immunoprecipitated GFP-Stt4 is cleaved at the C terminus. (D) Truncation mutants of GST-tagged Stt4 were purified from E. coli lysates, immobilized on glutathione beads, and normalized to equal amounts. Beads containing the GST-Stt4 truncations were then incubated for 1 h with lysate from E. coli overexpressing HIS6-Ypp1. Protein that bound the GST-Stt4 fragments was probed using an anti-HIS6 antibody. The relative amount of HIS6-Ypp1 that bound each respective GST-Stt4 fragment (top) and the relative amount of the Stt4 fragments that bound the glutathione beads (bottom) are shown. Notice that the primary Stt4 fragment (736–1346) is a C-terminal degradation product missing ∼200 amino acids. C-terminal degradation products are marked with an asterisk.
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Related In: Results  -  Collection

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fig2: Stt4 directly interacts with Ypp1 to form a stable complex. (A) Fluorescent image of GFP-Stt4 (left), Ypp1-GFP (right), and their corresponding kymographs (bottom). A representative section of the cell (the demarcated region in top panels) was examined for its relative movement at the cell membrane. A fluorescent time point of the demarcated region was taken every 5 s over the course of 3 min. The spatial variance of the selected fluorescent region with regard to time is represented in the bottom image. (B) FRAP of the PtdIns4P reporter GFP-2xPHOsh2 (top and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200804003/DC1) and GFP-Stt4 (bottom and Video 2). The white partitions demarcate the regions exposed to high intensity light, a FRAP region, and a control region exposed to normal excitation. The corresponding graph quantifies the fluorescent values with regard to time. (C) Cells expressing HA-Ypp1 and GFP alone or GFP-Stt4 were grown to mid-log phase, lysed, and incubated with an anti-GFP antibody. Coimmunoprecipitated proteins were detected with an anti-HA antibody. The relative abundance of coimmunoprecipitated HA-Ypp1 is shown in the top panel compared with a sample of the input from the whole cell lysate (WCL) in the bottom panel. Note that a small region of the immunoprecipitated GFP-Stt4 is cleaved at the C terminus. (D) Truncation mutants of GST-tagged Stt4 were purified from E. coli lysates, immobilized on glutathione beads, and normalized to equal amounts. Beads containing the GST-Stt4 truncations were then incubated for 1 h with lysate from E. coli overexpressing HIS6-Ypp1. Protein that bound the GST-Stt4 fragments was probed using an anti-HIS6 antibody. The relative amount of HIS6-Ypp1 that bound each respective GST-Stt4 fragment (top) and the relative amount of the Stt4 fragments that bound the glutathione beads (bottom) are shown. Notice that the primary Stt4 fragment (736–1346) is a C-terminal degradation product missing ∼200 amino acids. C-terminal degradation products are marked with an asterisk.
Mentions: Subsequently, we studied the dynamics of Stt4 and Ypp1 at PIK patches and compared it to the Stt4 product PtdIns4P. Fig. 2 A is a fluorescent image with the corresponding kymograph of GFP-Stt4 and Ypp1-GFP. Unlike cortical actin patches (Kaksonen et al., 2005), GFP-Stt4 PIK patches are stable over the course of 3 min. To test whether the Stt4 molecules residing in these patches were also static instead of dynamically exchanging, we performed FRAP on a region of the PM (Fig. 2 B and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200804003/DC1). 3 min after photobleaching a PIK patch–rich region of the PM, none of the fluorescence had recovered. This is in stark contrast to the Stt4 product PtdIns4P as observed with a PtdIns4P reporter, GFP-2xPHOsh2 (Yu et al., 2004). After photobleaching a PtdIns4P-rich region of the PM, the fluorescence of the GFP-2xPHOsh2 reporter recovers rapidly (Fig. 2 B and Video 3).

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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