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Immunocytochemical Localization and Crystal Structure of Human Frequenin (Neuronal Calcium Sensor 1)

2001; Elsevier BV; Volume: 276; Issue: 15 Linguagem: Inglês

10.1074/jbc.m009373200

1083-351X

Yves Bourne, Jens Dannenberg, Verena Pollmann, P. Marchot, Olaf Pongs,

Neuroscience and Neuropharmacology Research

Frequenin, a member of a large family of myristoyl-switch calcium-binding proteins, functions as a calcium-ion sensor to modulate synaptic activity and secretion. We show that human frequenin colocalizes with ARF1 GTPase in COS-7 cells and occurs in similar cellular compartments as the phosphatidylinositol-4-OH kinase PI4Kβ, the mammalian homolog of the yeast kinase PIK1. In addition, the crystal structure of unmyristoylated, calcium-bound human frequenin has been determined and refined to 1.9 Å resolution. The overall fold of frequenin resembles those of neurocalcin and the photoreceptor, recoverin, of the same family, with two pairs of calcium-binding EF hands and three bound calcium ions. Despite the similarities, however, frequenin displays significant structural differences. A large conformational shift of the C-terminal region creates a wide hydrophobic crevice at the surface of frequenin. This crevice, which is unique to frequenin and distinct from the myristoyl-binding box of recoverin, may accommodate a yet unknown protein ligand. Frequenin, a member of a large family of myristoyl-switch calcium-binding proteins, functions as a calcium-ion sensor to modulate synaptic activity and secretion. We show that human frequenin colocalizes with ARF1 GTPase in COS-7 cells and occurs in similar cellular compartments as the phosphatidylinositol-4-OH kinase PI4Kβ, the mammalian homolog of the yeast kinase PIK1. In addition, the crystal structure of unmyristoylated, calcium-bound human frequenin has been determined and refined to 1.9 Å resolution. The overall fold of frequenin resembles those of neurocalcin and the photoreceptor, recoverin, of the same family, with two pairs of calcium-binding EF hands and three bound calcium ions. Despite the similarities, however, frequenin displays significant structural differences. A large conformational shift of the C-terminal region creates a wide hydrophobic crevice at the surface of frequenin. This crevice, which is unique to frequenin and distinct from the myristoyl-binding box of recoverin, may accommodate a yet unknown protein ligand. Frequenin (Frq), 1Frq, frequenin; NCS-1, neuronal sensor 1; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; TGN, trans-Golgi-network; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HEK, human embryonic kidney cells; r.m.s.d., root mean square deviation; PIK1, yeast phosphatidylinositol-4-OH kinase; PI4Kβ, mammalian homolog of phosphatidylinositol-4-OH kinase.1Frq, frequenin; NCS-1, neuronal sensor 1; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; TGN, trans-Golgi-network; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HEK, human embryonic kidney cells; r.m.s.d., root mean square deviation; PIK1, yeast phosphatidylinositol-4-OH kinase; PI4Kβ, mammalian homolog of phosphatidylinositol-4-OH kinase. or neuronal calcium-sensor 1, is a member of a family of related calcium-myristoyl-switch proteins that have been proposed to function as calcium-ion sensors. Members of this family include recoverin, GCAP, neurocalcin, visinin, and others (1Polans A. Baehr W. Palczewski K. Trends Neurosci. 1996; 19: 547-554Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Recoverin and GCAP have been implicated in the control of recovery and adaptation in visual signal transduction. In vertebrate rod outer segments, GCAP apparently inhibits guanylate cyclase when the cytosolic concentration of Ca2+ is high in the dark, whereas recoverin may inhibit rhodopsin kinase (2Dizhoor A.M. Lowe D.G. Olshevskaya E.V. Laura R.P. Hurley J.B. Neuron. 1994; 12: 1345-1352Abstract Full Text PDF PubMed Scopus (272) Google Scholar, 3Chen C.K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Lowering the cytosolic Ca2+ during light illumination attenuates the inhibitory activities of GCAP and recoverin, leading to an activation of these enzymes. Frq, on the other hand, has attracted much attention because it may function as a calcium-ion sensor to modulate synaptic activity and secretion (4Pongs O. Lindemeir J. Zhu X.R. Engelkamp D. Krah-Jentsens I. Lambrecht H.G. Koch K.W. Schwemer J. Rivosecchi R. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (282) Google Scholar, 5McFerran B. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 6Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar, 7Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar). Drosophila Frq has been implicated in the facilitation of neurotransmitter release at neuromuscular junctions of third instar larvae (Drosophila melanogaster). Drosophilamutants that overexpress Frq show a facilitated neurotransmitter release that dramatically depend on the frequency of stimulation (4Pongs O. Lindemeir J. Zhu X.R. Engelkamp D. Krah-Jentsens I. Lambrecht H.G. Koch K.W. Schwemer J. Rivosecchi R. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (282) Google Scholar). Similarly, overexpression of a rat homolog of Frq in PC12 cells evokes an increased release of growth hormone in response to agonists like ATP (5McFerran B. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). These results are consistent with the idea that Frq activity and regulated secretion are coupled. More recently, it has been shown that the yeast homolog of Frq functions as a Ca2+-sensing subunit of the yeast phosphatidylinositol (PtdIns)-4-OH kinase, PIK1, a key enzyme in the phosphoinositide signaling system (6Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar). In Saccharomyces cerevisiae, PIK1 participates in the mating pheromone-signal transduction cascade and regulates secretion at the Golgi. PIK1 is essential in yeast cells for normal secretion, Golgi and vacuole membrane dynamics, and endocytosis (7Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar). Yeast PIK1tsmutants exhibit severe protein trafficking defects and accumulate morphologically aberrant Golgi membranes (8Walch-Solimena C. Novick P. Nat. Cell Biol. 1999; 1: 523-525Crossref PubMed Scopus (269) Google Scholar). The aberrant Golgi morphology is strikingly similar to that found in yeast cells lacking a functional ARF1 GTPase (7Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar). ARF1 has been implicated in multiple membrane trafficking events including the recruitment of the mammalian PIK1 homolog, PI4Kβ, to the Golgi membrane (9Godi A. Pertile P. Meyers R. Marra P. Di, T.ullio G. Iurisci C. Luini A. Corda D. De, Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (450) Google Scholar). Here we show that human Frq (HuFrq) colocalizes with ARF1 in COS-7 cells and occurs in similar cellular localizations as PI4Kβ. In addition, in a further step toward understanding the cellular function of Frq, we report the crystal structure of unmyristoylated Ca2+-bound human Frq (HuFrq) refined to 1.9 Å resolution. This structure confirms that frequenins belong to the large family of myristoyl-switch Ca2+-binding proteins and reveals the architecture of the Ca2+-binding sites. Most importantly, comparative analysis of the HuFrq structure with those of neurocalcin and recoverin highlights a unique wide crevice and a solvent-exposed carboxyl terminus that could be responsible for ligand recognition and account for the broad substrate specificity among members of the family. Human poly(A)RNA was isolated from HEK293 cells using the Fast Track II Kit (Invitrogen), and cDNA was synthesized with Superscript II Reverse Transcriptase (Life Technologies, Inc.). The HuFrq-encoding cDNA was amplified in a polymerase chain reaction (PCR) usingPfuTurbo-Polymerase (Life Technologies, Inc.) with the first strand cDNA as template and the primers 5′-ATACCATGGGGAAATCCAACAG-3′ (sense) and 5′-CTATACCAGCCCGTCGTAGAGG-3′ (antisense). Primer sequences were derived from the HuFrqnucleotide sequence (GenBankTM/EBI accession no. AF186409). The restriction sites NcoI and NdeI were used for subcloning into expression vector pET-16b (Promega) to generate expression plasmid pET-HuFrq. All nucleotide sequences were verified by automated sequencing. The expression plasmid pET-HuFrq was transformed into Escherichia coli strain BL21(DE3) (Novagen). Transformed cells were grown in Luria-Bertani (LB) medium containing ampicillin (100 μg/ml) at 37 °C. HuFrq expression was induced overnight at anA 600 of 0.8 with 0.5 mmisopropyl-1-thio-β-d-galactopyranoside and reached average yields of 20 mg/liter. Cells were harvested by centrifugation, resuspended in 20 ml of lysis buffer (50 mm HEPES, pH 7.4; 100 mm KCl; 1 mm EGTA; 1 mmdithiothreitol; 1 mm MgCl2) per 1 liter medium, and lysed in a French pressure cell. Protamine sulfate was added to a final concentration of 0.1% for 10 min and then the lysate was cleared by centrifugation (40,000 × g, 30 min, 4 °C). The supernatant was filtered (0.45 μm), adjusted to 1 mmCaCl2, and applied to a 15-ml phenyl-Sepharose CL4-B column (Amersham Pharmacia Biotech). The column was washed with buffer A (20 mm Tris/HCl, pH 7.9; 1 mm MgCl2; 1 mm dithiothreitol) containing 1 mmCaCl2 until A 280 was below 0.01, and then the protein was eluted with buffer A containing 2 mmEGTA. The eluate was applied to a 3-ml HiTrapQ column (Amersham Pharmacia Biotech). The column was washed with buffer A containing 60 mm NaCl and 1 mm CaCl2, and eluted with 120 mm NaCl in buffer A. The HuFrq-containing fractions were extensively dialyzed against water and concentrated to 10 mg/ml using microconcentrators (Pall Filtron). MALDI-TOF analysis of the purified HuFrq used the linear mode and a 337-nm nitrogen laser (Voyager-DE™RP BioSpectrometer work station, Perseptive Biosystems). Point mutations intoFrq cDNA were introduced by PCR using the following mutation primers: 5′-GGCAGGATCGTGTTCTCCGAATTC-3′ and 5′-TCGGAGAA CACGATCCTGCCAT-3′ for E81V (EF2); 5′-ACATCGCCAG AAACGAGATGCTG-3′ and 5′-CTGGTTTCTGC CGATGTAGCCGTC-3′ for T117A (EF3); 5′-GGGAAGCTAGCTCTTCAGGAGTTC-3′ and 5′-CCTGAAGAGCTAGCTTCCCATCA-3′ for T165A (EF4). PCR fragments were cloned into pET16b and sequenced before use. Protein samples were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The 45Ca2+ blotting was performed as previously described (10Maruyama K. Mikawa T. Ebashi S. J. Biochem. ( Tokyo ). 1984; 95: 511-519Crossref PubMed Scopus (628) Google Scholar); autoradiographic exposure time was 2 days. The polyclonal anti-HuFrq antibodies were raised in rabbit against purified HuFrq and purified by affinity chromatography on HuFrq-Sepharose 4B according to the manufacturer's instructions (Amersham Pharmacia Biotech); specificity analyses performed by enzyme-linked immunosorbent assay showed full recognition of the immunizing HuFrq but no recognition of either recoverin or neurocalcin (11Lindemeier J. Flup and flics. Zwei neue Frequenin-verwaudte, an der Signal transduktion beteiligte Ca2+-Bindende Proteine Thesis. Freie Universität, Berlin, Germany1995Google Scholar). COS-7 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% (v/v) fetal calf serum (Life Technologies). They were grown to 60% confluency on poly-l-lysine-coated glass-coverslips. The cells were fixed with 4% (v/v) paraformaldehyde in phosphate-buffered saline for 15 min at room temperature, washed twice with phosphate-buffered saline and blocked with 1% (v/v) goat serum and 1% (w/v) bovine serum albumin in phosphate-buffered saline. Permeabilization used a blocking solution containing 0.3% (v/v) Triton X-100. Successive incubations with primary and secondary antibodies were carried out for 1 h at room temperature. Cells were washed in phosphate-buffered saline, and coverslips were mounted with Fluoromount GC (Southern Technologies). The cells were visualized and confocal images acquired using a confocal laser scanning microscope (Leica TCS NT). Primary antibodies were detected by species-specific cyanine dye-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories): Cy3-conjugated antibodies were used for visualization of anti-HuFrq and anti-PI4Kβ (Upstate Biotechnology) staining, whereas Cy2- and Cy5-conjugated antibodies were used for anti-γ-adaptin (Sigma) and anti-ARF1 (Santa Cruz Biotechnology) staining, respectively. Crystals were obtained at 4 °C using the vapor diffusion technique. Typically, 5 μl of the HuFrq solution were mixed with 5 μl of a reservoir solution made of 0.1 m sodium cacodylate, pH 6.5, 0.2 m NaAc, 30% polyethylene glycol M r 8,000 (Crystal Screen I, solution 28, Hampton Research). Under rare circumstances, three different crystal forms grew from this solution: needle-like (form A), thin plates (form B), and thick plates (form C). Form A crystals belong to the hexagonal space group P61/5 with unit cell dimensions: a = b = 82.2 Å and c = 56 Å and contains one HuFrq molecule per asymmetric unit. Both form B and C crystals belong to the monoclinic space group P21 with unit cell dimensions:a = 53.8 Å, b = 55.5 Å,c = 77.7 Å, β = 107.6°, and a= 29.6 Å, b = 105.2 Å, c = 55.2 Å, β = 106.6°, respectively, and contain two HuFrq molecules per asymmetric unit. Crystals selected for data collection were briefly soaked into the reservoir solution supplemented with 10% (v/v) ethylene glycol, flash-cooled at 100 K in the nitrogen gas stream and stored in liquid nitrogen. No single crystal could be selected for form C. Data for forms A and B were collected on beamline ID14-EH2 of ESRF (Grenoble, France). Oscillation images were integrated with DENZO (12Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38527) Google Scholar) and scaled and merged with SCALA (13CCP4 Acta Crystallogr. Sect. D. 1994; 50: 760Crossref PubMed Scopus (19748) Google Scholar). Amplitude factors were generated with TRUNCATE (13CCP4 Acta Crystallogr. Sect. D. 1994; 50: 760Crossref PubMed Scopus (19748) Google Scholar). Form A crystals were found to be twinned and the collected data could not be used. Initial phases for form B crystals were obtained by molecular replacement using the structure of neurocalcin (14Vijay-Kumar S. Kumar V.D. Nat. Struct. Biol. 1999; 6: 80-88Crossref PubMed Scopus (105) Google Scholar) (PDB code 1BJF) as a search model with the AMoRe package (15Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5028) Google Scholar), giving a correlation coefficient of 36% and anR-factor value of 48% in the 15 to 4 Å resolution range. Rigid-body refinement, performed on each molecule with CNS (16Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) using data between 20 and 3 Å, gave an R-factor of 49%. For 2% of the reflections against which the model was not refined,R-free was 48%. The model was refined to 1.9 Å resolution using CNS, including bulk solvent and anisotropic B-factor corrections; the resulting 2Fo-Fc and Fo-Fc electron density maps were used to correct the model with the graphics program TURBO-FRODO (17Roussel A. Cambillau C. Silicon Graphics Geometry Partners Directory. Silicon Graphics Corp, Mountain View, CA1991: 81Google Scholar). Solvent molecules automatically added using CNS were carefully examined on the graphics display. The final model comprises residues Asn5–Val190 and Asn5–Gly188, respectively, for the two molecules in the asymmetric unit. High temperature factors and weak electron density are associated with residues 1–7, 49–60, and 133–138. The average r.m.s.d. between the two HuFrq molecules is 0.6 Å for 182 Cα atoms with the largest deviation (1.4 Å) for residue Gln54. The stereochemistry of the model was analyzed with PROCHECK (18Laskowski R. MacArthur M. Moss D. Thornton J. J. Appl. Crystallogr. 1993; 26: 91-97Crossref Google Scholar); no residues were found in the disallowed regions of the Ramachandran plot. Data collection and refinement statistics are summarized in Table I. The coordinates and structure factors of HuFrq have been deposited with the Protein Data Bank (1G8I). Fig. 1 B was generated by ALSCRIPT (19Barton G.J. Prot. Eng. 1993; 6: 37-40Crossref PubMed Scopus (1110) Google Scholar) and Figs. Figure 4, Figure 5 with SPOCK (20Christopher J.A. The Center for Macromolecular Design. Texas A&M University, College Station, TX1998Google Scholar) and Raster3D (21Merritt E.A. Murphy M.E.P. J. Appl. Crystallogr. 1994; 50: 869-873Crossref Scopus (2857) Google Scholar).Table IData collection and refinementCrystal formBResolution (Å)1.9No. observations281,683No. unique35,881R sym1-aRsym = ‖I − 〈I〉‖〈I〉, where I is intensity and 〈I〉 is the average I for all observations of equivalent reflections. Values in brackets are for the outer resolution shell.(%)6.0 (35)I/ς(I)8.9 (2.0)Redundancy3.3Completeness (%)99 (98.9)Resolution (Å)20–1.9R-factor1-bR-factor = ‖Fobs‖ − ‖Fcalc‖/‖Fobs‖. R-free same asR-factor for 2% of the data omitted from the refinement. −R free (%)22–25.6No. of reflections (no ς cutoff)35,849Rms deviationsbond length (Å)0.016bond angles (°)1.7dihedral angles (°)21.3improper angles (°)1.2Mean B factors (Å2)main/side chain26/30solvent/Ca2+34/22Rms deviations on B factors (Å2)main chain1.6side chain2.31-a Rsym = ‖I − 〈I〉‖〈I〉, where I is intensity and 〈I〉 is the average I for all observations of equivalent reflections. Values in brackets are for the outer resolution shell.1-b R-factor = ‖Fobs‖ − ‖Fcalc‖/‖Fobs‖. R-free same asR-factor for 2% of the data omitted from the refinement. Open table in a new tab Figure 4Quality of the map and overall fold of HuFrq. A, stereo view of the 1.9 Å resolution omit 2Fo-Fc averaged electron density map, contoured at 1ς, showing part of helix J. The coordinates of this region were omitted and the protein coordinates refined by simulated annealing before the phase calculation. B, stereo ribbon diagram of HuFrq with bound Na+ and Ca2+ ions. Secondary structure elements are indicated as in Fig. 1. Labels Ca2,Ca3, and Ca4 refer to the Ca2+ ions bound to EF hands EF2, EF3, and EF4, respectively. Functionally important side-chain residues in the vicinity of helix J are shown asblue/orange bonds with red oxygen andblue nitrogen atoms. Hydrogen bonds are shown asdotted lines. C, stereo overlay of HuFrq (yellow/cyan) and neurocalcin (orange/green) oriented as in B. Helix J is highlighted in cyanfor HuFrq and green for neurocalcin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Structural differences between HuFrq and homologous proteins. Molecular surface of HuFrq (A), viewed down the large hydrophobic crevice (orange) and oriented as in Fig. 2., neurocalcin (B) and recoverin (C) (same orientation). Secondary structure elements are labeled. The C-terminal helices, helix J in HuFrq (cyan) and in neurocalcin (green) and helices J and K in recoverin (green) are displayed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A search in the HTGS data bank with the HuFrq cDNA sequence revealed that theHuFrq gene is located on chromosome 9. A comparison of the publicly available chromosome 9 DNA sequence with the frequenin cDNA sequence showed that the exon-intron organization betweenDrosophila (4Pongs O. Lindemeir J. Zhu X.R. Engelkamp D. Krah-Jentsens I. Lambrecht H.G. Koch K.W. Schwemer J. Rivosecchi R. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (282) Google Scholar) and HuFrq genes has been conserved. Both open reading frames are interrupted by the same exon-intron borders and each are composed of same 8 exons (Fig.1 A). About 50 kilobases upstream of the first Frq exon are located several STS markers,e.g. DGS1924, A001W37, STSG22304, placing the Frqgene at 9q34.11. To our knowledge, a human disease has not been associated yet with this locus. Previously, mammalian homologs ofDrosophila Frq have been cloned (22Hauenschild A. Frequenin: Untersuchungen zur Struktur and Funktion eines neuronalen Proteins. Auflage, Wissenschaft and Technik-Verlag, Berlin1997Google Scholar, 23Olafsson P. Soares H.D. Herzog K.H. Wang T. Morgan J.I. Lu B. Brain Res. Mol. Brain Res. 1997; 44: 73-81Crossref PubMed Scopus (50) Google Scholar). We have used this information to clone HuFrq from human first strand cDNA (Fig.1 A). The predicted HuFrq sequence contains 190 amino acids (Fig. 1 B) with a theoretical monoisotopic mass of 21,865.91 and exhibits four EF hand motifs that represent potential Ca2+-binding domains. In HuFrq, as in other members of the family, the first of the four EF hand motifs is not likely to be a functional Ca2+-binding site as it lacks two Ca2+-coordinating amino acids. The HuFrq N terminus contains the consensus sequence MGXXX(S/T)K for myristoylation (24Towler D.A. Gordon J.I. Adams S.P. Glaser L. Annu. Rev. Biochem. 1988; 57: 69-99Crossref PubMed Google Scholar); hence it could be myristoylated like the rat homolog (5McFerran B. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The HuFrq sequence is 100% homologous to those of rat (5McFerran B. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and mouse (23Olafsson P. Soares H.D. Herzog K.H. Wang T. Morgan J.I. Lu B. Brain Res. Mol. Brain Res. 1997; 44: 73-81Crossref PubMed Scopus (50) Google Scholar) and it differs by a single amino acid from that ofXenopus (25Olafsson P. Wang T. Lu B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8001-8005Crossref PubMed Scopus (87) Google Scholar). Remarkably, the yeast (6Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar) and HuFrq protein sequences also show a high degree of conversation: 143 of 190 amino acids (75%) are either identical or correspond to conservative replacements (Fig. 1 B). In yeast, Frq has been shown to stimulate the activity of the PtdIns-4-OH kinase PIK1 (6Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (219) Google Scholar), an enzyme that is essential for normal secretion, Golgi and vacuole membrane dynamics, and endocytosis (7Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar).Xenopus Frq rescues a yeast Frq deletion mutant, indicating that Frq from higher eukaryotes is able to fulfill similar functions like yeast Frq (25Olafsson P. Wang T. Lu B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8001-8005Crossref PubMed Scopus (87) Google Scholar). Consistent with the functional role of Frq in yeast are the phenotypes that have been described forDrosophila mutants (4Pongs O. Lindemeir J. Zhu X.R. Engelkamp D. Krah-Jentsens I. Lambrecht H.G. Koch K.W. Schwemer J. Rivosecchi R. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (282) Google Scholar) and for mammalian cells overexpressing Frq (5McFerran B. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In both cases, it appears that evoked secretion is stimulated by Frq (26Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar). The activity of PI4Kβ, the likely mammalian homolog of yeast PIK1 (26Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar), is recruited by the small GTPase ARF1 to the Golgi and contributes to the regulation of Golgi membrane dynamics and Golgi-dependent vesicle formation (9Godi A. Pertile P. Meyers R. Marra P. Di, T.ullio G. Iurisci C. Luini A. Corda D. De, Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (450) Google Scholar). Accordingly, in immunocytochemical experiments we have compared the immunostaining patterns obtained with anti-PI4Kβ, anti-ARF1, and anti-HuFrq antibodies, respectively; overlapping immunostaining reactions were observed (Fig. 2). For comparison, we also included in our investigations experiments with anti-γ-adaptin antibodies, a typical trans-Golgi network (TGN) marker. Paraformaldehyde-fixed COS-7 cells were first incubated with primary antibodies, e.g. polyclonal anti-HuFrq rabbit antibodies, monoclonal anti-γ-adaptin mouse antibodies, polyclonal anti-PI4Kβ rabbit antibodies, and polyclonal anti-ARF1 goat antibodies, respectively. Then, we used secondary Cy2-, Cy3-, or Cy5-labeled antibodies for immunocytofluorescent staining and localization of γ-adaptin, HuFrq and PI4Kβ, and ARF1, respectively, using confocal microscopy (Fig. 2). The anti-HuFrq antibodies revealed a pattern with a crescent of staining on one side of the nucleus and some punctuate staining within the cytoplasm (Fig. 2 A). Staining could be eliminated by preincubation of the primary antibody with the immunizing HuFrq protein (not shown), indicating that the observed immunofluorescence is generated by HuFrq-specific antibodies. A similar staining pattern was obtained with γ-adaptin (Fig. 2 B), which in double-labeling experiments colocalized with the HuFrq-immunostaining pattern (Fig. 2 C). Previously, γ-adaptin has been shown to be localized in the TGN and the late endosomes (27Robinson M.S. Kreis T.E. Cell. 1994; 69: 129-138Abstract Full Text PDF Scopus (286) Google Scholar). The double-immunostaining patterns also indicate a colocalization for γ-adaptin and PI4Kβ (Fig. 2, D–F) in agreement with a recent report (28Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Finally, we coimmunostained the COS-7 cells with anti-HuFrq and anti-ARF1 antibodies (Fig. 2,G–I). Again, we observed a crescent of staining on one side of the nucleus (presumably the TNG) and some punctuate immunostain extending to the plasma membrane, which colocalized with the HuFrq immunostain (Fig. 2 I). The results indicate similar subcellular distributions for HuFrq, ARF1, and PI4Kβ. The colocalization is consistent with the proposal that frequenin proteins modulate PtdIns-4-OH kinase activity both in yeast and in mammalian cells and thus may have similar regulatory functions in secretion and Golgi membrane dynamics. We mutated EF hands EF2, EF3, and EF4 of HuFrq together or in pairwise combinations utilizing in vitro mutagenesis. In all four cases, we mutated the amino acid residues at the -X position as previously described forDrosophila Frq (Fig. 1 B) (4Pongs O. Lindemeir J. Zhu X.R. Engelkamp D. Krah-Jentsens I. Lambrecht H.G. Koch K.W. Schwemer J. Rivosecchi R. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (282) Google Scholar). Accordingly, we generated four HuFrq mutants: E81V/T117A/T165A (Frq2,3,4), E81V/T117A (Frq2,3), E81V/T165A (Frq2,4), and T117A/T165A (Frq3,4). Bacterial lysates containing approximately equal amounts of each HuFrq mutant were blotted onto nitrocellulose. The blot was incubated with45Ca2+ to investigate the Ca2+-binding capacity of the HuFrq mutants in comparison to wild-type HuFrq (Fig. 3). The results showed that wild-type HuFrq yielded the highest45Ca2+ signal. We noted for the recombinant HuFrq mutants with pairwise mutations attenuated45Ca2+-signals of comparably reduced intensity. The pairwise HuFrq mutants each contained a single intact EF hand (EF2 in Frq3,4, EF3 in Frq2,4, and EF4 in Frq2,3), yet they bound Ca2+ with high affinity; this suggests that EF hands EF2, EF3, and EF4 not only are functional in HuFrq but also are independent from each other. Previously, it was shown that single mutations in yeast Frq1 EF hands did not display a temperature-sensitive phenotype like the quadruple mutant in the frq1-Its allele, consistent with our observations. By contrast, the triple mutant Frq2,3,4 did not bind 45Ca2+ to a significant extent; hence in HuFrq, EF hand 1 does not constitute a high affinity Ca2+-binding site in HuFrq, as predicted earlier from sequence analysis. The crystal structure of HuFrq was solved by the molecular replacement method using neurocalcin (14Vijay-Kumar S. Kumar V.D. Nat. Struct. Biol. 1999; 6: 80-88Crossref PubMed Scopus (105) Google Scholar) as a search model and was refined to 1.9 Å resolution. The structure consists of residues Asn5–Val190 with good stereochemistry; clear electron density maps could be observed for all structural elements (Fig. 4 A). As predicted, HuFrq shares the typical α-helical fold found in homologous proteins, with overall dimensions of 35 × 60 × 40 Å. HuFrq contains 10 helices labeled A to J (Fig. 4 B). Consistent with the recently proposed NMR-derived model of yeast Frq (29Ames J.B. Hendricks K.B. Strahl T. Huttner I.G. Hamasaki N. Thorner J. Biochemist