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• W2085524881 abstract "Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis of an Frq1-binding fragment, Pik1-(10–192), showed that residues within 80–192 are necessary and sufficient for Frq1 association in vitro. A synthetic peptide (residues 151–199) competed for binding of [35S]Pik1-(10–192) to bead-immobilized Frq1, whereas shorter peptides (164–199 and 174–199) did not. Correspondingly, a deletion mutant, Pik1(Δ152–191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature. Site-directed mutagenesis of Pik1-(10–192) suggested that recognition determinants lie over an extended region. Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110–192), or the 151–199 peptide to Frq1 shows high affinity (K d ∼100 nm) and is largely entropic, consistent with hydrophobic interaction. Stoichiometry of Pik1-(110–192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography. Cys-15 in Frq1 is notS-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Δ cells. This study defines the segment of Pik1 required for high affinity binding of Frq1. Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis of an Frq1-binding fragment, Pik1-(10–192), showed that residues within 80–192 are necessary and sufficient for Frq1 association in vitro. A synthetic peptide (residues 151–199) competed for binding of [35S]Pik1-(10–192) to bead-immobilized Frq1, whereas shorter peptides (164–199 and 174–199) did not. Correspondingly, a deletion mutant, Pik1(Δ152–191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature. Site-directed mutagenesis of Pik1-(10–192) suggested that recognition determinants lie over an extended region. Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110–192), or the 151–199 peptide to Frq1 shows high affinity (K d ∼100 nm) and is largely entropic, consistent with hydrophobic interaction. Stoichiometry of Pik1-(110–192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography. Cys-15 in Frq1 is notS-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Δ cells. This study defines the segment of Pik1 required for high affinity binding of Frq1. Recognition that phosphoinositides and inositol phosphates are key regulators of many processes in eukaryotic cells has brought increased attention to the enzymes that regulate the synthesis and turnover of these molecules (reviewed in Refs. 1Tolias K.F. Cantley L.C. Chem. Phys. Lipids. 1999; 98: 69-77Crossref PubMed Scopus (71) Google Scholar, 2York J.D. Guo S. Odom A.R. Spiegelberg B.D. Stolz L.E. Adv. Enzyme Regul. 2001; 41: 57-71Crossref PubMed Scopus (80) Google Scholar, 3Toker A. Cell. Mol. Life Sci. 2002; 59: 761-779Crossref PubMed Scopus (181) Google Scholar). Of particular interest are the enzymes responsible for producing the various polyphosphoinositides situated on the cytosolic face of cellular membranes, which initiate several different signaling pathways by serving as highly specific recognition determinants for the selective recruitment of proteins to membranes (reviewed in Refs. 4Lemmon M.A. Ferguson K.M. Biochem. Soc. Trans. 2001; 29: 377-384Crossref PubMed Scopus (86) Google Scholar, 5Hurley J.H. Meyer T. Curr. Opin. Cell Biol. 2001; 13: 146-152Crossref PubMed Scopus (218) Google Scholar, 6Simonsen A. Stenmark H. Nat. Cell Biol. 2001; 3: E179-E182Crossref PubMed Scopus (64) Google Scholar, 7De Camilli P. Chen H. Hyman J. Panepucci E. Bateman A. Brunger A.T. FEBS Lett. 2002; 20: 11-18Crossref Scopus (123) Google Scholar) and as the precursors for several intracellular second messengers (reviewed in Refs. 8Zhang X. Majerus P.W. Semin. Cell Dev. Biol. 1998; 9: 153-160Crossref PubMed Scopus (60) Google Scholar, 9Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4451) Google Scholar, 10Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (835) Google Scholar). The first committed step in the synthesis of the polyphosphoinositide, phosphatidylinositol 4,5-bisphosphate, is considered to be ATP-dependent phosphorylation of the hydrophilicmyo-inositol head group of phosphatidylinositol (PtdIns) 1The abbreviations used are: PtdIns, phosphatidylinositol; PtdIns 4-kinase, phosphatidylinositol 4-kinase; PI4Kβ, phosphatidylinositol 4-kinase beta isoform; PtdIns(4)P, phosphatidylinositol-4-phosphate; PI(4)P 5-kinase, phosphatidylinositol-4-phosphate 5-kinase; CEN , centromeric; Gal, galactose; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; Glc, glucose; HSQC, heteronuclear single-quantum coherence spectroscopy; K av, average retention coefficient; LKU, lipid kinase unique domain; mAb, monoclonal antibody; NCS, neuronal calcium sensor; NOE, nuclear Overhauser effect; NTA, nitrilotriacetate; Raf, raffinose; SC, synthetic complete medium; Suc, sucrose; ts, temperature-sensitive; Fmoc, N-(9-fluorenyl)methoxycarbonyl at thed-4 position by PtdIns 4-kinase (ATP:1-phosphatidyl-1D-myo-inositol 4-phosphotransferase, EC2.7.1.67) (reviewed in Refs. 11Balla T. Biochim. Biophys. Acta. 1998; 1436: 69-85Crossref PubMed Scopus (85) Google Scholar, 12Gehrmann T. Heilmeyer L.M.J. Eur. J. Biochem. 1998; 253: 357-370Crossref PubMed Scopus (109) Google Scholar, 13Meyers R.E. Cantley L.C. Methods Mol. Biol. 1998; 105: 99-108PubMed Google Scholar) . The resulting product, PtdIns(4)P, can be phosphorylated on the d-5 position by PtdIns(4)P 5-kinase to generate PtdIns(4,5)P2, PtdIns(4,5)P2 can be phosphorylated on the d-3 position by yet other lipid kinases, and the phosphoinositides so generated can be converted to other species by specific phosphatases and phospholipases (reviewed in Refs. 14Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 15Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar, 16Majerus P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 17Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1220) Google Scholar). The first PtdIns 4-kinase to be purified to homogeneity from any organism (18Flanagan C.A. Thorner J. J. Biol. Chem. 1992; 267: 24117-24125Abstract Full Text PDF PubMed Google Scholar), and to have the corresponding gene cloned (19Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (171) Google Scholar, 20Garcia-Bustos G.F. Marini F. Stevenson I. Frei C. Hall M.N. EMBO J. 1994; 13: 2352-2361Crossref PubMed Scopus (102) Google Scholar), was Pik1 from the yeast Saccharomyces cerevisiae. Thereafter, a second isoform, Stt4, which is the product of a discrete gene, was described (21Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1172Abstract Full Text PDF PubMed Google Scholar). Absence of either Pik1 or Stt4 is lethal, and overproduction of each protein cannot compensate for absence of the other, indicating that these enzymes participate in distinct cellular processes and generate discrete pools of PtdIns(4)P that are essential for yeast cell viability. Indeed, subsequent work has shown that, together, Pik1 and Stt4 account for all of the PtdIns(4)P generated in the yeast cell (22Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar) and that Pik1 is required for vesicular trafficking in the late secretory pathway (23Hama H. Schnieders E.A. Thorner J. Takemoto J.Y. DeWald D.B. J. Biol. Chem. 1999; 274: 34294-34300Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 24Walch-Solimena C. Novick P. Nat. Cell Biol. 1999; 1: 523-525Crossref PubMed Scopus (269) Google Scholar) and perhaps for cytokinesis (20Garcia-Bustos G.F. Marini F. Stevenson I. Frei C. Hall M.N. EMBO J. 1994; 13: 2352-2361Crossref PubMed Scopus (102) Google Scholar), whereas Stt4 plays roles in cell wall integrity, maintenance of vacuole morphology, and aminophospholipid transport from the endoplasmic reticulum to the Golgi (25Yoshida S. Ohya Y. Nakano A. Anraku Y. Mol. Gen. Genet. 1994; 242: 631-640Crossref PubMed Scopus (77) Google Scholar, 26Trotter P.J. Wu W.I. Pedretti J. Yates R. Voelker D.R. J. Biol. Chem. 1998; 273: 13189-13196Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 27Audhya A. Emr S.D. Dev. Cell. 2002; 2: 593-605Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). The presence of Pik1- and Stt4-like isoforms is also conserved in metazoans (11Balla T. Biochim. Biophys. Acta. 1998; 1436: 69-85Crossref PubMed Scopus (85) Google Scholar, 12Gehrmann T. Heilmeyer L.M.J. Eur. J. Biochem. 1998; 253: 357-370Crossref PubMed Scopus (109) Google Scholar, 28Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar). We have shown previously that Frq1, a small calcium-binding protein, co-purifies with Pik1 and is required for optimal activity of the enzyme (29Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar). Frq1 is the yeast ortholog of a protein called frequenin, first described in Drosophila (30Pongs O. Lindemeier J. Zhu X.R. Theil T. Engelkamp D. Krah-Jentgens I. Lambrecht H.G. Koch K.W. Schwemer J. Rivosecchi R. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (282) Google Scholar), but referred to as neuronal-calcium-sensor-1 (NCS-1) in mammalian cells. Members of a large subfamily of small, EF-hand-containing, calcium-binding proteins that includes frequenin (31Braunewell K.H. Gundelfinger E.D. Cell Tissue Res. 1999; 295: 1-12Crossref PubMed Scopus (236) Google Scholar, 32An W.F. Bowlby M.R. Betty M. Cao J. Ling H.P. Mendoza G. Hinson J.W. Mattsson K.I. Strassle B.W. Trimmer J.S. Rhodes K.J. Nature. 2000; 403: 553-556Crossref PubMed Scopus (840) Google Scholar, 33Burgoyne R.D. Weiss J.L. Biochem. J. 2001; 353: 1-12Crossref PubMed Scopus (379) Google Scholar, 34Haeseleer F. Imanishi Y. Sokal I. Filipek S. Palczewski K. Biochem. Biophys. Res. Commun. 2002; 290: 615-623Crossref PubMed Scopus (139) Google Scholar) are characterized by a consensus signal for N-terminal myristoylation and four Ca2+-binding sites (of which the first and, in some cases, the fourth or another contain substitutions that make them non-functional). We have shown previously that Frq1 binds three Ca2+ (35Ames J.B. Hendricks K.B. Strahl T. Huttner I.G. Hamasaki N. Thorner J. Biochemistry. 2000; 39: 12149-12161Crossref PubMed Scopus (107) Google Scholar). Available evidence indicates that frequenin/NCS-1 may also modulate PtdIns 4-kinase activity in animal cells (36Bourne Y. Dannenberg J. Pollmann V. Marchot P. Pongs O. J. Biol. Chem. 2001; 276: 11949-11955Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 37Zhao X. Varnai P. Tuymetova G. Balla A. Toth Z.E. Oker-Blom C. Roder J. Jeromin A. Balla T. J. Biol. Chem. 2001; 276: 40183-40189Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Frq1, which is itself essential for the viability of yeast cells (29Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar), associates with membranes in a manner that depends on both theN-myristoyl group and conformational changes induced upon Ca2+ binding (35Ames J.B. Hendricks K.B. Strahl T. Huttner I.G. Hamasaki N. Thorner J. Biochemistry. 2000; 39: 12149-12161Crossref PubMed Scopus (107) Google Scholar). Thus, in addition to its stimulation of enzymic activity, Frq1 may contribute to the optimal function of Pik1 by assisting with its membrane recruitment, because Pik1 itself lacks any obvious membrane-targeting motifs. Indeed prior work indicated thatN-myristoylation of Frq1 is important, but not essential, for its function (29Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar). In some Ca2+-binding regulatory proteins, in addition to the N-terminal myristoyl group, palmitoylation of a cysteine residue near the N terminus is also required for efficient membrane association (38Godsel L.M. Engman D.M. EMBO J. 1999; 18: 2057-2065Crossref PubMed Scopus (124) Google Scholar, 39Takimoto K. Yangi E.K. Conforti L. J. Biol. Chem. 2002; 277: 26904-26911Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Frq1 has only two Cys residues, one is near its N terminus and the other buried in the interior (35Ames J.B. Hendricks K.B. Strahl T. Huttner I.G. Hamasaki N. Thorner J. Biochemistry. 2000; 39: 12149-12161Crossref PubMed Scopus (107) Google Scholar). In this study, as a prelude to structural analysis to determine at atomic resolution how Frq1 recognizes Pik1, we have applied several independent methods to determine the affinity and stoichiometry of the Frq1-Pik1 interaction, used different approaches to delineate the sequences in Pik1 responsible for high affinity binding of Frq1 and utilized both biochemical and genetic techniques to explore the role, if any, of S-palmitoylation in the function of Frq1. Yeast strains used in this study are listed below in Table I. Standard rich (YP) and synthetic complete (SC) media (40Sherman F. Fink G.R. Hicks J.A. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar) were supplemented with carbon sources (either 2% Glc, or 2% Raf/0.2% Suc, or 2% Gal/0.2% Suc, as indicated) and with appropriate nutrients for the selection and maintenance of plasmids. Yeast was cultivated at 30 °C, unless otherwise noted. Conventional methods for DNA-mediated transformation and other genetic manipulations of yeast cells were used (41Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 182-187Crossref PubMed Scopus (673) Google Scholar).Table IS. cerevisiae strainsStrainGenotypeSourceYPH499MAT a ade-101oc his3-Δ200 leu2-Δ1 lys2–801am trp1-Δ1 ura3–52(58Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar)YPH501MAT a/MATα (otherwise isogenic to YPH499)(58Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar)YKBH1YPH501frq1Δ∷HIS3/FRQ1(29Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar)YKBH4YPH499frq1–1ts∷HIS3(29Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar)YES10YPH501pik1-Δ1∷LEU2/PIK1(45Schnieders E.A. Biochemical and Genetic Analysis of a Phosphatidylinositol 4-Kinase (the PIK1 Gene Product) in the Yeast Saccharomyces cerevisiae.Ph.D. thesis. University of California at Berkeley, Berkeley, CA1996Google Scholar)BJ2168MAT a leu2 trp1 ura3–52 prb1–1122 pep4–3 prc1–407 gal2(59Jones E.W. Methods Enzymol. 1991; 194: 428-435Crossref PubMed Scopus (367) Google Scholar) Open table in a new tab Plasmids were constructed using standard methods for the manipulation of recombinant DNA (42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar).Escherichia coli strain DH5α (43Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8190) Google Scholar) was used for routine manipulation and propagation of plasmids. Unless otherwise indicated, all PCR reactions were performed using Pfu DNA polymerase (Stratagene, La Jolla, CA). All recombinant plasmids were verified by dideoxy chain termination sequencing. Fragments of the N terminus of Pik1 were tagged with a C-terminal His6 tract and expressed in E. coli, as follows. pET23d–PIK1(10–192) was constructed by inserting via blunt-end ligation the HindIII fragment ofPIK1 into pET23d (Novagen, Madison, WI) that had been cleaved with NcoI and EcoRI. pET23d–PIK1(10–192, Δ31–79) was produced by cleaving pET23d–PIK1(10–192) with EcoRI andNcoI and religating the plasmid after filling in the recessed 3′-ends using Klenow fragment of E. coli DNA polymerase I. pET23d–PIK1(10–163) and pET23d–PIK1(10–125) were generated by PCR amplification of pET23d–PIK1(10–192) using as primers an oligonucleotide spanning the NcoI site of PIK1 and oligonucleotides introducing a NotI site 3′ to the sequences encoding, respectively, either codon 163 or codon 125 of thePIK1 open-reading-frame. The resulting PCR fragments were ligated into pET23d–PIK1(10–192) that had been cleaved with NcoI and NotI. AnXbaI-XhoI fragment of pET23d–PIK1(10–192) was inserted into Litmus28 (New England BioLabs, Beverly, MA) cleaved with the same enzymes, to generate the cloning intermediate, Litmus28–PIK1(10–192). Litmus28–pik1(10–192; P181A,V183A) and Litmus28–pik1(10–192; L175A,P181A,V183) were generated by exchanging the TfiI fragment in Litmus28–PIK1(10–192) with TfiI-digested PCR products carrying the appropriate substitutions, which were introduced by site-directed mutagenesis using a commercial kit (QuikChange, Stratagene, La Jolla, CA), according to the manufacturer's instructions. The XbaI-XhoI fragments from Litmus28–pik1(10–192; P181A,V183A) and Litmus28–pik1(10–192; L175A,P181A,V183) were then transferred back into pET23d that had been cleaved with XbaI and XhoI, to generate pET23d–pik1(10–192; P181A,V183A) and pET23d–pik1(10–192; L175A,P181A,V183). A PCR product carrying the appropriate substitutions and a XhoI site 3′ to sequences encoding residue 192 of Pik1, all introduced by site-directed mutagenesis, was cleaved with EcoRI and XhoI and inserted into the corresponding sites of pET23d–PIK1(10–192), yielding pET23d–pik1(10–192; R188A,R189A). pET23d–pik1(10–192; E154A,N155A,V156A,P158A) was constructed using a three-way ligation strategy, as follows. pET23d–PIK1(10–192) was cleaved with EcoRI andPstI and ligated with a PCR product (carrying the appropriate substitutions to produce E154A, N155A, and V156A) that had been cleaved with EcoRI and PvuII and a second PCR product (carrying the appropriate substitutions to produce N155A, V156A, and P158A) cleaved with PvuII andPstI. pET23d-FRQ1 has been described previously (29Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar, 35Ames J.B. Hendricks K.B. Strahl T. Huttner I.G. Hamasaki N. Thorner J. Biochemistry. 2000; 39: 12149-12161Crossref PubMed Scopus (107) Google Scholar). Multicopy (2-μm, DNA-based) URA3-marked plasmids, YEp352-FRQ1, YEp352-GAL-FRQ1, and YEp352-FRQ1(G2A) have been described previously (29Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar). YEp352-GAL-FRQ1-(His) 6 was constructed by excising the corresponding fragment from pET23d-FRQ1-(His) 6(29Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar) and inserting it into YEp352-GAL (44Benton B.M. Eng W.-K. Dunn J.J. Studier F.W. Sternglanz R. Fisher P.A. Mol. Cell. Biol. 1990; 10: 353-360Crossref PubMed Scopus (56) Google Scholar). YEp352-FRQ1(G2A,C15A) was generated by replacing anHindIII-BglII fragment of YEp352FRQ1(G2A) with a PCR product, carrying the appropriate mutations to encode C15A, that was digested with the same enzymes. The low copy (CEN-based) URA3-marked plasmid, pRS316-FRQ1(G2A,C15A), was generated by inserting an EcoRI fragment from YEp352-FRQ1(G2A,C15A) into pRS316-FRQ1 (29Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar). Construction of the TRP1-markedCEN plasmids, RS314-PIK1, pRS314-GAL1,10, and pRS314-GAL-mycPIK is described elsewhere (45Schnieders E.A. Biochemical and Genetic Analysis of a Phosphatidylinositol 4-Kinase (the PIK1 Gene Product) in the Yeast Saccharomyces cerevisiae.Ph.D. thesis. University of California at Berkeley, Berkeley, CA1996Google Scholar). The cloning intermediate, Litmus28-PIK1, was produced by inserting aBamHI-SacI fragment containing the entire coding sequence of PIK1, excised from pRS314-PIK1, into Litmus28. Litmus28PIK1(Δ152–191) was generated by insertion of a PCR-derived HindIII fragment, encoding residues 10–151 of Pik1, into Litmus28-PIK1, that had been cleaved with HindIII. pRS314-PIK1(Δ152–191) was produced by subcloning the EcoRI fragment of Litmus28-PIK1(Δ152–191) into pRS314-PIK1. Inserting the BamHI-SacI fragment of pRS314-PIK1(Δ152–191) into pRS314-GAL1,10 generated pRS314-GAL-PIK1(Δ152–191). pRS314-GAL-mycPIK1(Δ152–191) was constructed by replacing the NcoI-SacI fragment of pRS314-GAL-mycPIK1 with the corresponding fragment from pRS314-GAL-PIK1(Δ152–191). Vectors for in vitroproduction of mRNA were constructed, as follows. A fragment containing the FRQ1 coding sequence, generated by PCR and containing an NcoI site overlapping the translation initiation codon and a BamHI site introduced 3′ to the stop codon, was inserted into pBAT4 (46Peranen J. Rikkonen M. Hyvonen M. Kaariainenen L. Anal. Biochem. 1996; 236: 371-373Crossref PubMed Scopus (223) Google Scholar), which had been cleaved withNcoI and BamHI, yielding pBAT4–FRQ1. A PCR fragment was amplified from pET23d–PIK1(10–192)using appropriate primers to introduce a SmaI site 5′ to the coding sequence and a HindIII site 3′ to codon 192 of thePIK1 open reading frame, cleaved with SmaI andHindIII, and ligated into pBAT4 that had been cleaved with the same enzymes, generating pBAT4–PIK1(10–192).35S-Labeled proteins were produced by coupled in vitro transcription and translation in the presence of [35S]Met and [35S]Cys (PerkinElmer Life Sciences, Boston, MA) using the TnTTM coupled reticulocyte lysate system (Promega, Madison, WI), according to the manufacturer's instructions. Translation mixtures were clarified by centrifugation (10 min, 4 °C) at maximum speed in a microcentrifuge. If not used immediately, the resulting supernatant fractions were mixed with an equal volume of glycerol and stored at −20 °C. The Pik1 and Frq1 constructs containing a C-terminal His6 tag were expressed in E. coli strain BL21(DE3) (Novagen, Madison, WI) and purified using Ni2+-saturated NTA-agarose (Qiagen, Valencia, CA) under denaturing conditions according to the manufacturer's specifications. To confirm their identity and purity, proteins recovered by binding to Ni2+-saturated NTA-agarose were resolved by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue and by immunoblotting. Protein concentration was determined by the dye-binding method of Bradford (47Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216412) Google Scholar) using a commercial kit (Bio-Rad, Inc., Hercules, CA) with bovine serum albumin as the standard. Protease-deficient strain, BJ2168 (Table I), transformed with YEp352-GAL-FRQ1-(His) 6, was grown in SC-Raf lacking uracil at 30 °C to mid-exponential phase and induced by addition of Gal (2% final concentration). After incubation for 6 h at 30 °C, cells were collected by centrifugation and resuspended in an equal volume of distilled water. The cell suspension was frozen by dripping into liquid nitrogen, and the resulting pellets were crushed with a pestle in a precooled mortar. The resulting frozen cell powder was dissolved in lysis buffer (5 mm imidazole, 145 mm NaCl, 50 mm Na-PO4 (pH 7.5); 20 ml/liter yeast culture) containing a mixture of protease inhibitors (CompleteTM, Promega, Madison, WI). The crude lysate was clarified by centrifugation at maximum rpm in a microcentrifuge at 4 °C for 15 min and then at 72,000 × g for 90 min in a L8–80M ultracentrifuge (Beckman-Coulter Inc., Fullerton, CA). The resulting supernatant fraction was then applied to a Ni2+-saturated NTA-agarose column (1.5-ml bed volume) that had been pre-equilibrated with three volumes of lysis buffer. After washing with 10 bed volumes of lysis buffer and 6 volumes of wash buffer (20 mm imidazole, 145 mm NaCl, 50 mm Na-PO4 (pH 7.5)), the bound Frq1-His6 was eluted with 3 volumes of elution buffer (120 mm imidazole, 145 mm NaCl, 50 mmNa-PO4 (pH 7.5)). The eluate was concentrated by ultrafiltration through an anisotropic membrane (3-kDa cut-off, Centricon YM-3, Amicon, Beverly, MA) until a final concentration of 0.5 μg/ml was reached. To confirm identity and purity, the resulting fraction was resolved by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue and by immunoblotting. Prior to use, Ni2+-saturated NTA-agarose beads used in protein binding and peptide competition experiments were pre-blocked by incubation for 30 min in 8 volumes of buffer A (10 mm imidazole, 100 mm NaCl, 1 μm CaCl2, 1 mm dithiothreitol, 50 mm Tris-HCl (pH 7.4)) containing 5 mg/ml ovalbumin at room temperature. All in vitro binding assays were carried out at 4 °C. Radiolabeled Frq1, prepared by coupled in vitro transcription and translation, was mixed with an equal volume of a slurry of pre-blocked Ni2+-saturated NTA-agarose beads in buffer A and incubated on a roller drum for 30 min. The beads and any nonspecifically bound radioactivity were removed by brief sedimentation in a microcentrifuge, and the resulting pre-cleared supernatant fraction was collected. Aliquots (400 μl) of the pre-cleared fraction were mixed either with 40 μl of pre-blocked Ni2+-saturated NTA-agarose beads, as a control for background binding, or with Ni2+-saturated NTA-agarose beads on which had been immobilized Pik1-(10–192)-(His)6 or its deletion derivatives (30 μg of protein/40 μl of beads) and incubated for 1 h on a rollerdrum. The beads were collected by centrifugation for 15 s in a microcentrifuge and washed three times with buffer A. Bound proteins were eluted from the beads in 50 μl of buffer A containing 300 mm imidazole, and samples of the resulting eluate were resolved by SDS-PAGE on a 12% gel and visualized by autoradiography. Synthetic peptides corresponding to Pik1-(174–199), Pik1-(164–199), and Pik1-(151–199) were prepared by standard solid phase peptide synthesis (using Fmoc chemistry) on an automated synthesizer (Model ABI 431A, PerkinElmer Life Sciences-Applied Biosystems, Foster City, CA), purified by high-performance liquid chromatography, and confirmed by electrospray ionization mass spectrometry. A slurry (30 μl), of either pre-blocked Ni2+-saturated NTA-agarose beads or the same beads pre-coated to saturation with purified Frq1-His6, was mixed with 500 μl of 2×-concentrated buffer A, 290 μl of H2O, and 100 μl of either an aqueous solution of the indicated peptide in 10% acetonitrile or H2O containing 10% acetonitrile as a control. After preincubation of the samples on a roller drum for 30 min, 80 μl of35S-labeled Pik1-(10–192), produced by coupled in vitro transcription and translation, was added to each tube, and the mixture was incubated for a further 1.5 h. The beads were collected by sedimentation in a microcentrifuge and washed twice with buffer A. Bead-bound radioactivity was solubilized by boiling in SDS-PAGE sample buffer (48Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar), and the resulting eluate was subjected to SDS-PAGE. The species corresponding to [35S]Pik1-(10–192) was quantitated using a PhosphorImagerTM (Amersham Biosciences, Sunnyvale, CA). At each peptide concentration, the amount of radioactivity bound nonspecifically to empty beads was subtracted from the amount of radioactivity bound to the beads coated with Frq1-His6. 15N-Labeled and unlabeled samples of unmyristoylated Frq1 were prepared as described previously (35Ames J.B. Hendricks K.B. Strahl T. Huttner I.G. Hamasaki N. Thorner J. Biochemistry. 2000; 39: 12149-12161Crossref PubMed Scopus (107) Google Scholar). Chemical synthesis of the 49-residue synthetic peptide corresponding to residues 151–199 of Pik1 was described in the preceding section. Using PCR with appropriate primers, an 83-residue segment from the N terminus of Pik1 consisting of residues 110–192, Pik1-(110–192), was tagged at its C terminus with an His6tract, inserted as an NcoI-XhoI fragment into the corresponding sites in the vector, pET23d, and expressed in E. coli strain BL21(DE3), as described above; in this construct, the Pik1-derived sequence is preceded by a 2-residue leader (Met-Ala-). Pik1-(110–192)-(His)6 was produced primarily in inclusion bodies, which were solubilized using 6 m guanidine hydrochloride (49Wingfield P.T. Palmer I. Liang S.M. Coligan J.E. Current Protocols in Protein Science. 1. John Wiley & Sons, Inc., New York, NY1995: 6.5.1-6.5.27Google Scholar) and purified using Ni2+-saturated NTA-agarose chromatography, essentially as described above. Samples for NMR analysis were prepared by dissolving 15N-labeled Frq1 (0.4 mm) with various amounts (0, 1, or 2 molar equiv" @default.
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• W2085524881 date "2003-02-01" @default.
• W2085524881 modified "2023-10-09" @default.
• W2085524881 title "Molecular Interactions of Yeast Frequenin (Frq1) with the Phosphatidylinositol 4-Kinase Isoform, Pik1" @default.
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• W2085524881 doi "https://doi.org/10.1074/jbc.m207920200" @default.
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