The dual PH domain protein Opy1 functions as a sensor and modulator of PtdIns(4,5)P 2 synthesis
2012; Springer Nature; Volume: 31; Issue: 13 Linguagem: Inglês
10.1038/emboj.2012.127
ISSN1460-2075
AutoresYading Ling, Christopher J. Stefan, Jason A. MacGurn, Anjon Audhya, Scott D. Emr,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoArticle4 May 2012free access Source Data The dual PH domain protein Opy1 functions as a sensor and modulator of PtdIns(4,5)P2 synthesis Yading Ling Yading Ling Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Christopher J Stefan Christopher J Stefan Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Jason A MacGurn Jason A MacGurn Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Anjon Audhya Anjon Audhya Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA Search for more papers by this author Scott D Emr Corresponding Author Scott D Emr Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Yading Ling Yading Ling Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Christopher J Stefan Christopher J Stefan Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Jason A MacGurn Jason A MacGurn Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Anjon Audhya Anjon Audhya Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA Search for more papers by this author Scott D Emr Corresponding Author Scott D Emr Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Author Information Yading Ling1,‡, Christopher J Stefan1,‡, Jason A MacGurn1, Anjon Audhya2 and Scott D Emr 1 1Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA 2Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI, USA ‡These authors contributed equally to this work *Corresponding author. Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, 441 Weill Hall, Ithaca, NY 14853, USA. Tel.: +1 607 255 0816; Fax: +1 607 255 5961; E-mail: [email protected] The EMBO Journal (2012)31:2882-2894https://doi.org/10.1038/emboj.2012.127 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Phosphatidylinositol-4,5-bisphosphate, PtdIns(4,5)P2, is an essential signalling lipid that regulates key processes such as endocytosis, exocytosis, actin cytoskeletal organization and calcium signalling. Maintaining proper levels of PtdIns(4,5)P2 at the plasma membrane (PM) is crucial for cell survival and growth. We show that the conserved PtdIns(4)P 5-kinase, Mss4, forms dynamic, oligomeric structures at the PM that we term PIK patches. The dynamic assembly and disassembly of Mss4 PIK patches may provide a mechanism to precisely modulate Mss4 kinase activity, as needed, for localized regulation of PtdIns(4,5)P2 synthesis. Furthermore, we identify a tandem PH domain-containing protein, Opy1, as a novel Mss4-interacting protein that partially colocalizes with PIK patches. Based upon genetic, cell biological, and biochemical data, we propose that Opy1 functions as a coincidence detector of the Mss4 PtdIns(4)P 5-kinase and PtdIns(4,5)P2 and serves as a negative regulator of PtdIns(4,5)P2 synthesis at the PM. Our results also suggest that additional conserved tandem PH domain-containing proteins may play important roles in regulating phosphoinositide signalling. Introduction Phosphorylated derivatives of phosphatidylinositol, collectively known as phosphoinositide (PIP) lipids, regulate cell polarity, growth and development, and membrane trafficking (Martin, 1998; Behnia and Munro, 2005; Di Paolo and De Camilli, 2006; Vicinanza et al, 2008). A wide range of protein effectors are directly regulated by PIP lipids through conserved PIP-binding modules that specify effector recruitment and activation at distinct organelle membranes (Lemmon, 2008). However, how levels of distinct PIP isoforms are accurately maintained by the opposing actions of PIP kinases and PIP phosphatases is not fully understood. One of the major PIP speies, PtdIns(4,5)P2, is enriched in the inner leaflet of the plasma membrane (PM). PtdIns(4,5)P2 was initially shown to function as a precursor of the second messengers diacylglycerol (DAG) and 1,4,5-inositol trisphosphate (IP3) generated by phospholipase C (Hokin, 1985). More recent studies have highlighted direct roles for PtdIns(4,5)P2 in the regulation of the endocytic machinery (Haucke, 2005), exocytosis (Liu et al, 2007; James et al, 2008), the actin cytoskeleton (Yin and Janmey, 2003) and septin assembly during cell division (Logan and Mandato, 2006; Bertin et al, 2010). In the yeast Saccharomyces cerevisiae, PtdIns(4,5)P2 is synthesized by the conserved PtdIns(4)P 5-kinase Mss4. Mss4 localizes to the PM and generates essential pools of PtdIns(4,5)P2 (Desrivieres et al, 1998; Homma et al, 1998; Audhya and Emr, 2002). Mss4 also undergoes nuclear-cytoplasmic shuttling and nuclear sequestration may control Mss4 function (Audhya and Emr, 2003). However, little is known about how regulation of Mss4 at the PM is achieved. We have found that Mss4 assembles into oligomeric protein complexes at the PM that we term PIK (PIP kinase) patches. Mss4 PIK patches are dynamic and are distinct from other previously described cortical structures, such as actin patches (Pruyne and Bretscher, 2000a, 2000b) and eisosomes (Walther et al, 2006). Assembly of Mss4 PIK patches requires the C-terminus of Mss4 and its substrate PtdIns(4)P. In addition, by genetic and quantitative proteomic approaches, we identify the dual PH domain-containing protein Opy1 as a novel regulator of Mss4. We propose that assembly of Mss4 PIK patches permits coordinate regulation of multiple Mss4 kinase molecules by accessory factors, including the conserved Opy1 protein. Results Mss4 forms dynamic protein complexes, PIK patches, at the PM Mss4 is a cytoplasmic protein that associates with the inner face of the PM (Audhya and Emr, 2003). To understand how Mss4 is regulated, we employed cells solely expressing a functional Mss4–GFP fusion. Mss4–GFP formed cortical, punctate structures at the PM: PIK (phosphoinositide kinase) patches (Figure 1A, observed at both mid and top sections of cells; Supplementary Movie S1) similar to previously reported patterns for Mss4 localization (Audhya and Emr, 2002; Sun et al, 2007; Smaczynska-de et al, 2008). Reconstructions (2D projections of Z series) of cells expressing Mss4–GFP indicated that there are ∼30–50 Mss4 PIK patches in each individual yeast cell. However, the oligomeric status or the dynamics of Mss4 PIK patches at the PM has not been addressed. To determine whether multiple copies of Mss4 assemble at PIK patches, we tested if an Mss4–13xmyc fusion isolated with an Mss4–3xHA fusion in coimmunoprecipitation experiments. Mss4–13xmyc was present in anti-HA immunoprecipitates from cell lysates coexpressing Mss4–3xHA, but not from control cell lysates lacking Mss4–3xHA (Figure 1B), suggesting that Mss4 oligomerizes. To estimate the copy numbers of Mss4 molecules present in PIK patches, we expressed Mss4–GFP in cells coexpressing a Cse4–3xGFP fusion (both are integrated; Supplementary Figure S1A). Cse4–3xGFP forms a complex containing 96 GFP molecules in the nucleus of yeast cells (Markus et al, 2009). By comparing the GFP signal intensities of Mss4 PIK patches and Cse4–3xGFP in the nucleus, we found a distribution of ∼5–30 copies of Mss4–GFP in each PIK patch (Figure 1C). However, most PIK patches contained ∼10–20 Mss4–GFP molecules (Figure 1C). Thus, multiple copies of the Mss4 lipid kinase assemble at PIK patches. Figure 1.Mss4 forms oligomeric, dynamic cortical structures at the PM. (A) Mss4–GFP localization in mid and top sections of yeast cells. Cells shown are representative of over 100 cells observed. Lines indicate individual Mss4 PIK patches at the PM. The inset (boxed area) shows a region magnified two-fold. Levels of the colour images overlayed on DIC images were adjusted with Adobe Photoshop. Scale bar, 5 μm. (B) Mss4–13myc was coimmunoprecipitated with Mss4–3HA. Lysates from cells expressing Mss4–3HA, Mss4–13myc or both were incubated with crosslinker and immunoprecipitated with anti-HA beads, and analysed by immunoblotting to detect Mss4–Mss4 interaction. (C) Quantification of numbers of Mss4 molecules in PIK patches (n=32). Cells coexpressing integrated Mss4–GFP and Cse4–3xGFP were analysed by fluorescence microscopy. Numbers of Mss4 were calculated based on fluorescence signal intensity of Mss4 PIK patches and Cse4–3xGFP in the nucleus as an internal standard (corresponding to 96 GFP molecules; see Supplementary Figure S1A; Supplementary data set 1). Using this approach, the average number of Mss4 molecules per PIK patch was 15±6 (s.d.). (D) Mss4 PIK patches are dynamic structures. Cells expressing Mss4–GFP were examined by time-lapse fluorescence microscopy. Images of Mss4–GFP at the cell surface were captured every 5 s. At time t 0=0 s, Mss4–GFP is shown in green, at t 1=30 s Mss4–GFP is shown in red. Scale bar, 5 μm. (E) Three representative examples of Mss4 PIK patch lifetimes at the cell surface, images were captured every 5 s. Figure source data can be found with the Supplementary data. Source Data for Figure 1 [embj2012127-sup-0009.pdf] Download figure Download PowerPoint To address the dynamics of Mss4 PIK patches, we performed time-lapse imaging experiments following Mss4–GFP at the cell surface by focusing on the top of cells. Strikingly, Mss4 PIK patches were highly dynamic and short-lived structures (Supplementary Movie S2). More than 75% of Mss4 PIK patches appear to change localization within 30 s (Figure 1D), and the lifetime of Mss4 PIK patches range from 10 to 40 s (Figure 1E; Supplementary Figure S3A). This dramatic rearrangement in distribution likely occurs by the dynamic assembly and disassembly of Mss4 PIK patches as well as lateral movements along the surface of the PM, as Mss4 PIK patches did not move into the interior of the cell by following Mss4–GFP in mid sections of cells (Supplementary Movie S3). Mss4 PIK patches were distinct from cortical actin patches (Pruyne and Bretscher, 2000a, 2000b) and did not require actin polymerization for assembly (Supplementary Figure S1B). Likewise, Mss4 PIK patches were distinct from eisosomes and independent of the eisosome component Pil1 (Supplementary Figure S1C and D). These results suggested that Mss4 assembles into unique dynamic structures at the PM. We thus sought to further understand how Mss4 organization and function are regulated. The Mss4 kinase domain and PtdIns(4)P are required for PIK patch assembly Mss4 consists of an uncharacterized N-terminal domain, a central nuclear localization signal (NLS), and a conserved C-terminal PIP 5-kinase domain (Figure 2A). To map regions in Mss4 necessary for PIK patch assembly, we generated a series of truncated Mss4 mutants tagged with GFP. The large N-terminal region of Mss4 (residues 2–346) and the NLS in Mss4 (residues 347–364) were dispensable for PM localization (Figure 2A). In contrast, a mutant form lacking the last 54 amino acids of Mss4 (residues 726–779), did not localize to the PM and instead accumulated in the cytoplasm and nucleus (Figure 2A). We then tested if the truncated Mss4–GFP fusions were functional using a plasmid shuffle growth assay. For this, we transformed an mss4Δ strain carrying an URA3-marked wild-type MSS4 plasmid or with plasmids encoding truncated forms of Mss4–GFP. As expected, cells expressing Mss4Δ726–779–GFP alone failed to grow on 5-FOA media (due to loss of the URA3-marked wild-type MSS4 plasmid). However, neither the N-terminal region nor the NLS were required for Mss4 function, as cells expressing mutant forms of Mss4 lacking these regions were able to grow on 5-FOA plates (Figure 2B). Figure 2.The Mss4 kinase domain and PtdIns(4)P are required for PIK patch assembly. (A) Localization of wild-type and truncated Mss4–GFP proteins. From top to bottom: full-length Mss4–GFP, N-terminally truncated Mss4–GFP (lacking residues 2–346), NLS-deleted Mss4–GFP (lacking residues 347–364), and C-terminally truncated Mss4–GFP (lacking residues 726–779). Lines indicate the nucleus of cells. Scale bar, 5 μm. Cyto, cytoplasm, N, nucleus. (B) Complementation assays of the truncated Mss4 mutants. The mss4Δ cells carrying a centromeric URA3-marked MSS4 plasmid was cotransformed with plasmids expressing various mutant Mss4–GFP forms as indicated. Cells were spotted onto –Ura –Trp plates to retain both plasmids or plates containing 5-FOA to select for loss of the URA3-marked MSS4 plasmid; only cells harbouring functional MSS4–GFP plasmids grew on 5-FOA plates. (C) Mss4–GFP localization in stt4tspik1ts double-mutant cells at 26 and 38°C. Cells were grown at 26°C to mid-log phase and shifted to 38°C for 60 min prior to observation by fluorescence microscopy. Scale bar, 5 μm. (D) Cellular PtdIns(4,5)P2 levels measured by 3H-inositiol labelling and HPLC analysis of mss4ts cells expressing empty vector, full-length Mss4–GFP, N-terminally truncated Mss4–GFP (lacking residues 2–346), or C-terminally truncated Mss4–GFP (lacking residues 726–779). The lipid labelling was performed at 37°C, a non-permissive temperature for the mss4ts cells. Results from a representative experiment are shown. Additional data are provided in Supplementary Table S1. (E) Expression of N-terminally truncated Mss4 impairs the growth of sjl1Δ sjl2tssjl3Δ inp54Δ cells deficient in PIP 5-phosphatase activity. PtdIns(4,5)P2 metabolism is regulated by the Mss4 PIP 5-kinase and a set of conserved 5-phosphatases, the synaptojanin-like (Sjl) proteins and Inp54. Full-length Mss4–GFP or N-terminally truncated Mss4Δ2–346–GFP was expressed in sjl1Δ sjl2tssjl3Δ inp54Δ cells as indicated. Serial dilutions of yeast cells were grown on –Ura plates to retain the MSS4–GFP plasmids at either 26 or 34°C for 4 days. Download figure Download PowerPoint The truncation resulting in Mss4 mislocalization occurs in a conserved region of the C-terminus, termed the activation loop (Supplementary Figure S2A; Kunz et al, 2000). The activation loops of mammalian PtdIns(4)P 5-kinases control substrate recognition and subcellular targeting (Kunz et al, 2000). Substitution of two highly conserved lysine residues to negatively charged aspartate residues in the activation loop resulted in Mss4 mislocalization (Supplementary Figure S2B). In contrast, substitutions with arginine, retaining the positive charge, did not affect Mss4 PM localization (Supplementary Figure S2B). Thus, binding to the anionic substrate PtdIns(4)P may be important for Mss4 localization. Stt4 and Pik1 are the two major PtdIns 4-kinases responsible for PtdIns(4)P synthesis in yeast (Audhya et al, 2000). To deplete PtdIns(4)P, we used a temperature conditional stt4tspik1ts double-mutant strain. Mss4–GFP localized to the cytoplasm in stt4tspik1ts double-mutant cells at the restrictive temperature (Figure 2C). However, Mss4–GFP localized to the PM in stt4ts and pik1ts single-mutant cells at the non-permissive temperature (Supplementary Figure S2C), suggesting that both PtdIns 4-kinases contribute to Mss4 PM targeting. Mss4 localization was not dependent on PtdIns(4,5)P2 levels, as a kinase inactive form of Mss4–GFP localized to the PM when PtdIns(4,5)P2 is depleted in mss4ts cells (Supplementary Figure S2D; Stefan et al, 2002). Consistent with a role for PtdIns(4)P in targeting Mss4 to the PM, the purified Mss4 kinase domain, His6–SUMO–Mss4454–779, bound to PtdIns(4)P, as well as PtdIns(3)P and weakly to PtdIns(4,5)P2, in lipid overlay experiments (Supplementary Figure S2E). Thus, the C-terminus of Mss4 and PtdIns(4)P target Mss4 to the PM. The N-terminal region of Mss4 functions as a negative regulatory domain Our initial results implicated the conserved C-terminus of Mss4 in PIK patch assembly. However, the role of the Mss4 N-terminal region remained unclear. We further examined Mss4 regulation by performing 3H-inositol labelling experiments to measure cellular PtdIns(4,5)P2 levels in mss4ts cells coexpressing various forms of Mss4–GFP. As expected, the C-terminal truncated Mss4Δ726–779–GFP was defective in PtdIns(4,5)P2 synthesis (Figure 2D; Supplementary Table S1). However, deletion of the N-terminus increased Mss4 activity, as PtdIns(4,5)P2 levels were ∼2-fold higher in mss4ts cells expressing Mss4Δ2–346–GFP compared with cells expressing full-length Mss4–GFP (Figure 2D; Supplementary Table S1). Both Mss4Δ726–779–GFP and Mss4Δ2–346–GFP were expressed at levels similar to full-length Mss4–GFP, indicating that the altered activities of the mutant proteins were not due to changes in protein stability (Supplementary Figure S2F). In addition, PIK patches containing Mss4–GFP and Mss4Δ2–346–GFP displayed similar dynamics and GFP fluorescence intensity distributions (Supplementary Figure S3A and B; Supplementary Movie S4). Thus, the increase in PtdIns(4,5)P2 levels in cells expressing Mss4Δ2–346–GFP was not due to increases in Mss4 assembly or PIK patch lifetime. Elevated PtdIns(4,5)P2 levels are toxic in cells with impaired PtdIns(4,5)P2 phosphatase activity (Stefan et al, 2002). Expression of the N-terminal truncated form but not full-length Mss4 impaired the growth of cells deficient in PIP 5-phosphatase function (sjl1Δ sjl2tssjl3Δ inp54Δ cells) at a semi-permissive temperature (Figure 2E), further suggesting that deletion of the N-terminal region results in increased Mss4 PIP kinase activity. The PH domain-containing protein Opy1 binds Mss4 The mechanisms of Mss4 localization and regulation at the PM are poorly characterized. We reasoned that the localization and regulation of Mss4 are mediated in part by associated factors. To identify candidate proteins that interact with Mss4, we undertook a quantitative proteomics approach (Figure 3A). Cells were grown in media containing either light (for the control strain) or heavy (for cells expressing Mss4–3xFlag) isotope amino acids. We then performed crosslinking immunopurification (IP) experiments and processed the protein samples for quantitative mass spectrometry analysis. This approach detects the enrichment of proteins that specifically interact with Mss4, and allows for the identification of weak/transient interactions. One protein, Opy1, was highly enriched with purified Mss4–3xFlag (Figure 3B). To confirm the Mss4–Opy1 interaction, we repeated the crosslinking coIP experiment and monitored the results by immunoblotting. As expected, Opy1–3xHA was present in immunoprecipitates from cells expressing Mss4–3xFlag, but not control cells lacking Mss4–3xFlag (Figure 3C). Figure 3.Identification of Mss4-interacting proteins by chemical crosslinking experiments and quantitative mass spectrometry. (A) Outline of the quantitative SILAC–MS approach. See the Results and Materials and methods for additional details. (B) Expression ratio (Xpress ratio=Mss4–3xFlag IP/ control IP) for proteins identified by the SILAC–MS experiments. An Xpress ratio of >10 was used as a set point to define specific Mss4-interacting proteins. Opy1 was enriched >50-fold in the Mss4–3xFlag IP compared with control IP. The inset shows the number of Mss4 and Opy1 peptides identified in the heavy (containing Mss4–3xFlag) and light samples. (C) Opy1–3HA crosslinks and coimmunoprecipitates with Mss4–Flag. Lysates from cells expressing Opy1–3HA or Mss4–Flag and Opy1–3HA were incubated with crosslinker and incubated with anti-Flag beads. Immunoprecipitates were analysed by immunoblotting to detect Mss4–Opy1 interactions. (D) Opy1 localizes to cortical structures and the cytoplasm. Wild-type cells expressing Opy1–GFP were grown to mid-log and examined by fluorescence microscopy. Cells shown are representative of over 100 cells observed. Scale bar, 4 μm. Diagram of the Opy1 protein is shown under the fluorescence images. PH, pleckstrin homology domain. Figure source data can be found with the Supplementary data. Source Data for Figure 3 [embj2012127-sup-0010.pdf] Download figure Download PowerPoint Opy1 consists of two PH domains and the C-terminal PH domain has been proposed to bind PtdIns(4,5)P2 (Figure 3D; Szentpetery et al, 2009). By examining full-length Opy1–GFP in vivo, we observed Opy1 in the cytoplasm and at cortical punctate structures (Figure 3D). Interestingly, the cortical Opy1 structures partially colocalized with Mss4 PIK patches at the PM (Supplementary Figure S3D), Taken together, these results suggested that Opy1 interacts with Mss4 at PIK patches. Opy1 is a novel regulator of PtdIns(4,5)P2 synthesis To study the function of Opy1 in vivo, we first deleted the OPY1 gene. Mss4 assembled into PIK patches at the PM in cells lacking OPY1 with wild-type dynamics and Mss4–GFP fluorescence intensities, and thus Opy1 was not essential for Mss4 PIK patch formation (Supplementary Figure S3A and C; Supplementary Movie S5). To test if Opy1 regulates PtdIns(4,5)P2 metabolism, we performed 3H-inositol labelling experiments to measure cellular PIP levels. We detected a two-fold increase in PtdIns(4,5)P2 levels in opy1Δ cells compared with wild-type cells (Figure 4A; Supplementary Table S1). In addition, overexpression of Opy1 in wild-type cells resulted in a 40% decrease in PtdIns(4,5)P2 levels (Figure 4A; Supplementary Table S1). This was not due to decreases in Mss4 PIK patch lifetime (Supplementary Figure S3A; Supplementary Movie S6) or assembly (as measured by relative Mss4–GFP fluorescence intensity; Supplementary Figure S3C). Moreover, overexpression of Opy1 impaired the growth of mss4ts cells at a semi-permissive temperature (34°C; Figure 4B), further suggesting that Opy1 regulates PtdIns(4,5)P2 metabolism, either by inhibiting its synthesis or by promoting its turnover. Figure 4.Opy1 inhibits PtdIns(4,5)P2 synthesis at the PM. (A) Cellular PtdIns(4,5)P2 levels were determined in wild-type, opy1Δ cells, or wild-type cells overexpressing OPY1 by 3H-inositiol labelling and HPLC analysis. Three independent labelling experiments were performed at 26°C. Data represent the mean of three independent experiments (±s.d.). (B) Overexpression of Opy1 impairs the growth of mss4ts cells with impaired PIP 5-kinase activity at a semi-permissive temperature (34oC). Serial dilutions of mss4ts cells were grown on –Ura plates to retain the high-copy OPY1 plasmid at either 26 or 34°C for 3 days. (C) Cellular PtdIns(4,5)P2 levels as measured by 3H-inositiol labelling and HPLC analysis in sjl1Δ sjl2tssjl3Δ cells carrying empty vector, SJL2, or OPY1 plasmids. Two independent labelling experiments were performed at 38°C. Results from a representative experiment are shown. Additional data are provided in Supplementary Table S1. (D) Overexpression of full-length Opy1, but not the PH1 or PH2 domains from Opy1 alone, rescues the growth defect of sjl1Δ sjl2tssjl3Δ cells at 38°C. Serial dilutions of sjl1Δ sjl2tssjl3Δ cells carrying empty vector, SJL2, full-length OPY1 or truncated opy1 (PH1 domain or PH2 domain) plasmids as indicated were spotted onto –Ura plates to retain the plasmids and grown at either 26 or 38°C for 4 days. Download figure Download PowerPoint In an independent genetic screen to identify regulators of PtdIns(4,5)P2 signalling, we isolated OPY1 as a high copy suppressor of synaptojanin mutant cells deficient in PIP 5-phosphatase activity (sjl1Δ sjl2tssjl3Δ cells; Figure 4D). Consistent with this, OPY1 overexpression resulted in decreased PtdIns(4,5)P2 levels in sjl1Δ sjl2tssjl3Δ cells at the restrictive temperature (Figure 4C; Supplementary Table S1). Thus, Opy1 modulates PtdIns(4,5)P2 metabolism independently of the synaptojanin-like PIP phosphatases. Consistent with this, loss of Opy1 did not alter the cortical localization of Sjl2 (Supplementary Figure S4A). Furthermore, triple deletion of SJL1, SJL2, and OPY1 resulted in an additive growth defect, as sjl1Δ sjl2Δ opy1Δ cells failed to grow upon loss of a URA3-marked SJL2 plasmid on 5-FOA media (Supplementary Figure S4B), suggesting that Opy1 and the synaptojanins regulate PtdIns(4,5)P2 by distinct mechanisms. In control experiments, OPY1 overexpression did not rescue the growth defects of other PIP phosphatase mutant cells that accumulate toxic levels of PtdIns(4)P and PtdIns(3)P, sac1tssjl2Δ sjl3Δ cells and ymr1tssjl2Δ sjl3Δ cells, respectively (Supplementary Figure S4C; Foti et al, 2001; Parrish et al, 2004). We observed that PtdIns(4)P levels were slightly elevated (1.5-fold) in cells lacking Opy1 (opy1Δ cells; Supplementary Table S1). It was therefore possible that Opy1 could activate a PIP 4-phosphatase such as the Sac1 enzyme (Foti et al, 2001; Stefan et al, 2011), and thus loss of Opy1 could lead to elevated levels of both PtdIns(4)P and PtdIns(4,5)P2. However, PtdIns(4,5)P2 levels were increased (1.8-fold) in sac1Δ opy1Δ double-mutant cells, as compared with sac1Δ single-mutant cells (Supplementary Figure S4D; Supplementary Table S1). Thus, coupled with our co-IP and colocalization results, we propose that Opy1 may inhibit Mss4 activity at PIK patches rather than activate a PIP phosphatase, such as the synaptojanins or Sac1. Opy1 is a coincidence detector of PtdIns(4,5)P2 and Mss4 As Opy1 consists of two PH domains, we next addressed how Opy1 localizes to PIK patches at the PM. The N-terminal PH domain of Opy1 fused to GFP (GFP–PH1) did not localize to the PM in vivo (Figure 5A; Yu et al, 2004). In contrast, the C-terminal PH domain fused to GFP (GFP–PH2) was sufficient for PM targeting (Figure 5A; Yu et al, 2004). Similar to a previous study, the GFP–PH2 fusion still localized to the PM in mss4ts cells at the non-permissive temperature (Yu et al, 2004; our unpublished observations). However, we found that GFP–PH2 became mis-localized from the PM to the cytoplasm in pik1tsstt4ts double-mutant cells upon an extended shift to 37°C (Figure 5A). A previous study reported that GFP–PH2 localized to the PM in pik1tsstt4ts mutant cells (Yu et al, 2004). However, GFP–PH2 was overexpressed from a high copy plasmid in this study. In addition, Yu et al found that overexpression of the PH2 domain caused growth defects in pik1tsstt4ts cells, suggesting that the PH2 domain may compete for some factor that becomes limiting in these cells. As pik1tsstt4ts double-mutant cells have significantly reduced levels of PtdIns(4)P, PtdIns(4,5)P2, and Mss4 PIK patches at the PM (Audhya et al, 2000; Figure 2C in this study), we further addressed the lipid and protein binding activities for full-length Opy1 and the Opy1 PH domains. Figure 5.The Opy1 PH2 domain is sufficient for PM targeting in vivo and PtIns(4,5)P2-binding in vitro. (A) Top panels: Localization of GFP-tagged Opy1 PH1 or PH2 domains in wild-type cells. Wild-type cells expressing GFP–PH1 or GFP–PH2 were grown to mid-log and examined by fluorescence microscopy. Bottom panels: Localization of GFP-tagged Opy1 PH2 domain in cells with impaired PtdIns 4-kinase activity. Double-mutant pik1tsstt4ts cells expressing GFP–PH2 were grown to mid-log at the permissive temperature (26°C) and examined by fluorescence microscopy at 26°C and following a 1-h incubation at the restrictive temperature (37°C). Scale bar, 3 μm. Cyto, cytoplasm. (B) Full-length Opy1 and the Opy1 PH2 domain bind PtdIns(4,5)P2-containing liposomes in vitro. Recombinant GST, GST–PH1, GST–PH2, and GST–Opy1 fusion proteins were expressed, purified from bacteria, and incubated with PC:PtdIns(4,5)P2-containing liposomes (0.3 mM total lipid, 3% mol PtdIns(4,5)P2). Liposomes were separated from unbound protein by floatation on Nycodenz equilibrium gradients (see Materials and methods). GST fusion proteins in unbound (Un) and liposome-bound (B) fractions were detected by immunoblotting with GST antisera. (C) Opy1 PM targeting is dependent on PtdIns(4,5)P2 synthesis. Opy1–GFP localization in wild-type and mss4ts cells incubated at 37°C. Cells were grown at 26°C to mid-log phase, shifted to 37°C for 60 min, and examined by fluorescence microscopy. Scale bar, 5 μm. Cyto, cytoplasm. Figure source data can be found with the Supplementary data. Source Data for Figure 5 [embj2012127-sup-0011.pdf] Download figure Download PowerPoint First, we examined whether Opy1 bound PtdIns(4,5)P2-containing liposomes in vitro. For these experiments, we used GST, GST–PH1, GST–PH2, and GST–Opy1 fusion proteins purified from bacteria. GST–Opy1 (52% of the total protein) efficiently bound and floated with PC:PtdIns(4,5)P2-containing liposomes (3% mol PtdIns4,5P2) on an equilibrium density gradient (Figure 5B). Likewise, GST–PH2 (41% of the total protein) was present with liposomes following equilibrium density fractionation, suggesting that
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