The Yeast Inositol Polyphosphate 5-Phosphatase Inp54p Localizes to the Endoplasmic Reticulum via a C-terminal Hydrophobic Anchoring Tail
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m010471200
ISSN1083-351X
AutoresFenny Wiradjaja, Lisa M. Ooms, James C. Whisstock, Brad McColl, Leon Helfenbaum, Joseph Sambrook, Mary‐Jane Gething, Christina A. Mitchell,
Tópico(s)Fungal and yeast genetics research
ResumoThe budding yeast Saccharomyces cerevisiae has four inositol polyphosphate 5-phosphatase (5-phosphatase) genes, INP51, INP52, INP53, andINP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate. INP54 encodes a protein of 44 kDa which consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich region found in the other three yeast 5-phosphatases. We report that Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail. The hydrophobic tail comprises the last 13 amino acids of the protein and is sufficient to target green fluorescent protein to the endoplasmic reticulum. Protease protection assays demonstrated that the N terminus of Inp54p is oriented toward the cytoplasm of the cell, with the C terminus of the protein also exposed to the cytosol. Null mutation ofINP54 resulted in a 2-fold increase in secretion of a reporter protein, compared with wild-type yeast or cells deleted for any of the SacI domain-containing 5-phosphatases. We propose that Inp54p plays a role in regulating secretion, possibly by modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the cytoplasmic surface of the endoplasmic reticulum membrane. The budding yeast Saccharomyces cerevisiae has four inositol polyphosphate 5-phosphatase (5-phosphatase) genes, INP51, INP52, INP53, andINP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate. INP54 encodes a protein of 44 kDa which consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich region found in the other three yeast 5-phosphatases. We report that Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail. The hydrophobic tail comprises the last 13 amino acids of the protein and is sufficient to target green fluorescent protein to the endoplasmic reticulum. Protease protection assays demonstrated that the N terminus of Inp54p is oriented toward the cytoplasm of the cell, with the C terminus of the protein also exposed to the cytosol. Null mutation ofINP54 resulted in a 2-fold increase in secretion of a reporter protein, compared with wild-type yeast or cells deleted for any of the SacI domain-containing 5-phosphatases. We propose that Inp54p plays a role in regulating secretion, possibly by modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the cytoplasmic surface of the endoplasmic reticulum membrane. 5)P2, phosphatidylinositol (4,5)-bisphosphate inositol polyphosphate 5-phosphatase phosphatidylinositol (3Hinchliffe K.A. Ciruela A. Irvine R.F. Biochim. Biophys. Acta. 1998; 1436: 87-104Crossref PubMed Scopus (103) Google Scholar)-phosphate phosphatidylinositol (4Martin T.F. Annu. Rev. Cell Dev. Biol. 1998; 14: 231-264Crossref PubMed Scopus (453) Google Scholar)-phosphate 5)P2, phosphatidylinositol (3,5)-bisphosphate bovine pancreatic trypsin inhibitor binding protein leucine-rich domain base pair(s) open reading frame polymerase chain reaction polyacrylamide gel electrophoresis green fluorescent protein endoplasmic reticulum hemagglutinin Phosphoinositides are ubiquitous membrane components of various intracellular compartments, which regulate many diverse cellular functions including membrane trafficking events, secretion, actin cytoskeletal organization, cellular proliferation, and inhibition of apoptosis (reviewed in Refs. 1De, Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (664) Google Scholar, 2Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (248) Google Scholar, 3Hinchliffe K.A. Ciruela A. Irvine R.F. Biochim. Biophys. Acta. 1998; 1436: 87-104Crossref PubMed Scopus (103) Google Scholar, 4Martin T.F. Annu. Rev. Cell Dev. Biol. 1998; 14: 231-264Crossref PubMed Scopus (453) Google Scholar). Many of these functions are mediated by binding and recruiting signaling proteins which contain specific phosphoinositide-binding domains such as SH2 domains, pleckstrin homology domains, FYVE domains, C2 domains or polybasic domains, thereby localizing these effector proteins to specific membranes (reviewed in Refs. 3Hinchliffe K.A. Ciruela A. Irvine R.F. Biochim. Biophys. Acta. 1998; 1436: 87-104Crossref PubMed Scopus (103) Google Scholar and 4Martin T.F. Annu. Rev. Cell Dev. Biol. 1998; 14: 231-264Crossref PubMed Scopus (453) Google Scholar). Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2)1serves as a precursor to second messenger molecules such as inositol (1,4,5)-trisphosphate and phosphatidylinositol (3,4,5)-trisphosphate, but also independent of further modification regulates the actin cytoskeleton and membrane trafficking (1De, Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (664) Google Scholar, 5Odorizzi G. Babst M. Emr S.D. Trends Biochem. Sci. 2000; 25: 229-235Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). PtdIns(4,5)P2binds to actin-binding proteins such as profilin and gelsolin (6Janmey P.A. Annu. Rev. Physiol. 1994; 56: 169-191Crossref PubMed Scopus (476) Google Scholar) and displaces capping proteins from actin filaments, allowing polymerization and formation of actin stress fibers (7Gilmore A.P. Burridge K. Nature. 1996; 381: 531-535Crossref PubMed Scopus (464) Google Scholar, 8De Corte V. Gettemans J. Vandekerckhove J. FEBS Lett. 1997; 401: 191-196Crossref PubMed Scopus (74) Google Scholar, 9Ren X.D. Schwartz M.A. Curr. Opin. Genet. Dev. 1998; 8: 63-67Crossref PubMed Scopus (78) Google Scholar). PtdIns(4,5)P2 also plays a role in regulating vesicle budding and in the recruitment and activation of proteins involved in the coating of vesicles (2Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (248) Google Scholar). Cellular levels of PtdIns(4,5)P2 are regulated by a series of lipid phosphorylation and dephosphorylation reactions mediated by specific lipid kinases and phosphatases. Inositol polyphosphate 5-phosphatases (5-phosphatases) regulate cellular PtdIns(4,5)P2 levels by hydrolyzing the 5-position phosphate from the inositol ring forming phosphatidylinositol 4-phosphate (PtdIns(4)P) (10Majerus P.W. Genes Dev. 1996; 10: 1051-1053Crossref PubMed Scopus (50) Google Scholar, 11Mitchell C.A. Brown S. Campbell J.K. Munday A.D. Speed C.J. Biochem. Soc. Trans. 1996; 24: 994-1000Crossref PubMed Scopus (54) Google Scholar). The budding yeastSaccharomyces cerevisiae has four 5-phosphatase genes,INP51, INP52, INP53, and INP54. Inp51p, Inp52p, and Inp53p each comprise an N-terminal SacI domain, a central 5-phosphatase domain, and a C-terminal proline-rich region (12Srinivasan S. Seaman M. Nemoto Y. Daniell L. Suchy S.F. Emr S. De Camilli P. Nussbaum R. Eur. J. Cell Biol. 1997; 74: 350-360PubMed Google Scholar, 13Stolz L.E. Huynh C.V. Thorner J. York J.D. Genetics. 1998; 148: 1715-1729Crossref PubMed Google Scholar). These enzymes share significant sequence homology with the mammalian homologue synaptojanin, which regulates the recycling of synaptic vesicles in nerve terminals (14McPherson P.S. Garcia E.P. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (497) Google Scholar). Synaptojanin, Inp52p, and Inp53p contain two catalytic domains, a central 5-phosphatase domain and an N-terminal SacI domain which hydrolyzes PtdIns(3,5)P2, PtdIns(4)P, and PtdIns(3)P forming PtdIns (15Guo S. Stolz L.E. Lemrow S.M. York J.D. J. Biol. Chem. 1999; 274: 12990-12995Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Null mutation of any two SacI domain-containing 5-phosphatases results in plasma membrane invaginations and thickened cell walls, defects in polarization of the actin cytoskeleton, and impaired endocytosis (12Srinivasan S. Seaman M. Nemoto Y. Daniell L. Suchy S.F. Emr S. De Camilli P. Nussbaum R. Eur. J. Cell Biol. 1997; 74: 350-360PubMed Google Scholar, 13Stolz L.E. Huynh C.V. Thorner J. York J.D. Genetics. 1998; 148: 1715-1729Crossref PubMed Google Scholar). However, double SacI domain-containing 5-phosphatase null mutants display normal secretion of invertase suggesting that Inp51p, Inp52p, and Inp53p do not play a role in regulating secretion (16Singer-Kruger B. Nemoto Y. Daniell L. Ferro-Novick S. De Camilli P. J. Cell Sci. 1998; 111: 3347-3356PubMed Google Scholar). A triple SacI domain-containing 5-phosphatase null mutant is nonviable suggesting Inp54p cannot function to rescue the loss of these three 5-phosphatases. Inp54p is a PtdIns(4,5)P2 5-phosphatase (17Raucher D. Stauffer T. Chen W. Shen K. Guo S. York J.D. Sheetz M.P. Meyer T. Cell. 2000; 100: 221-228Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar), as are all the yeast 5-phosphatases. Therefore it is not surprising that single null mutation of INP54 is not lethal (18Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. et al.Science. 1999; 285: 901-906Crossref PubMed Scopus (3257) Google Scholar). However, further characterization of the phenotype of this mutant has not been reported. In this study we demonstrate Inp54p is a C-terminal tail-anchored protein that localizes to the cytosolic face of the endoplasmic reticulum. This localization is mediated by a short 13-amino acid leucine-rich region at the extreme C terminus of the protein. Null mutation of INP54, but not any of the SacI domain-containing 5-phosphatases, results in increased levels of secretion from the endoplasmic reticulum. We propose the enzyme regulates PtdIns(4,5)P2 levels on the cytoplasmic surface of the endoplasmic reticulum and thereby regulates secretion. Restriction and DNA modifying enzymes were supplied by New England Biolabs, Fermentas, or Promega. Biomol GreenTM Reagent for phosphate detection was obtained from Biomol. Oligonucleotides were obtained from Bresatec, Australia, and the Department of Microbiology, Monash University, Australia. The Big Dye Terminator Cycle Sequencing kit was from PE Applied Systems (Foster City, CA). All other reagents were from Sigma unless otherwise stated. The plasmid pPS1303 for GFP expression was a kind gift from Professor Pamela Silver, Dana Farber Cancer Institute, Harvard University, the pRS416 vector from Dr. Mark Prescott, Monash University, Australia, the plasmid pJJ250 was a gift from Dr. Doris Germain, Peter MacCallum Cancer Institute, Australia. Bovine pancreatic trypsin inhibitor (BPTI) expression plasmid pEB316U was a kind gift from Professor Dane Wittrup, MIT. Yeast strains used in the study are listed in TableI. Yeast strains were cultured at 30 °C in standard YPD media or complete minimal media lacking specific amino acids to maintain selection of markers where appropriate.Table IYeast strains used in this studyStrainGenotypeSourceW303MATa/MATα ade2–1/ade2–1 trp-1–1/trp1-1, leu2–3,112/leu2–3,112— 1-aThese yeast strains were the kind gift of Dr. D. Germain.his3–11, 15/his3–11, 15, ura3–1/ura3–1W303αMATα ade2–1 trp1–1 leu2–3,112 his3–11,15 ura3–1— 1-aThese yeast strains were the kind gift of Dr. D. Germain.SEY6210MATα ura3–52 leu2–3, 112 his3-Δ200 trp-Δ901 lys2–801 suc2-Δ9Ref. 70Wilsbach K. Payne G.S. EMBO J. 1993; 12: 3049-3059Crossref PubMed Scopus (114) Google Scholarinp54MATα ade2–1 trp1–1 leu2–3, 112 his3–11,15 ura3–1, inp54∷LEU3This studyinp51MATα ade2–1 trp1–1 leu2–3,112 his3–11,15 ura3–1, inp51∷URA3Ref. 21Ooms L.M. McColl B.K. Wiradjaja F. Wijayaratnam A.P.W. Gleeson P. Gething M.-J. Sambrook J. Mitchell C.A. Mol. Cell. Biol. 2000; 20: 9376-9390Crossref PubMed Scopus (32) Google Scholarinp52MATα ade2–1 trp1–1 leu2–3,112 his3–11,15 ura3–1, inp52∷HIS3Ref. 21Ooms L.M. McColl B.K. Wiradjaja F. Wijayaratnam A.P.W. Gleeson P. Gething M.-J. Sambrook J. Mitchell C.A. Mol. Cell. Biol. 2000; 20: 9376-9390Crossref PubMed Scopus (32) Google Scholarinp53MATα ade2–1 trp1–1 leu2–3, 112 his3–11,15 ura3–1, inp53∷TRP1Ref. 21Ooms L.M. McColl B.K. Wiradjaja F. Wijayaratnam A.P.W. Gleeson P. Gething M.-J. Sambrook J. Mitchell C.A. Mol. Cell. Biol. 2000; 20: 9376-9390Crossref PubMed Scopus (32) Google Scholar1-a These yeast strains were the kind gift of Dr. D. Germain. Open table in a new tab The genomic sequence containing the INP54 ORF (open reading frame), 1600 bp upstream of the start codon and 700 bp downstream of the stop codon was amplified by PCR (incorporating a XhoI site at the 5′ end and a NotI site at the 3′ end) and cloned into theXhoI/NotI site of Bluescript. The construct was digested with PstI/SpeI to release a 1.7-kilobase pair fragment containing the full sequence of INP54, which was replaced with a LEU2 expression cassette obtained by digesting the plasmid pJJ250 with XbaI/PstI (19Jones J.S. Prakash L. Yeast. 1990; 6: 363-366Crossref PubMed Scopus (342) Google Scholar). The LEU2 gene flanked by the sequence upstream and downstream of INP54 ORF was recovered from the Bluescript vector by XhoI/NotI digestion. The DNA fragment was then transformed into W303 diploid cells by electroporation as described previously (20Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1991Google Scholar). Transformants were selected by their ability to grow on complete minimal media lacking leucine, and were subsequently screened for homologous integration of the LEU2marker by PCR. Null mutations of the SacI domain-containing 5-phosphatases has been described previously (21Ooms L.M. McColl B.K. Wiradjaja F. Wijayaratnam A.P.W. Gleeson P. Gething M.-J. Sambrook J. Mitchell C.A. Mol. Cell. Biol. 2000; 20: 9376-9390Crossref PubMed Scopus (32) Google Scholar). INP54was amplified from SEY6210 genomic DNA by PCR using synthetic oligonucleotide primers as described in Table II (GenBank accession numberZ74807). The primers amplified the complete coding region of the 5-phosphatase from nucleotides 1 to 1156 and incorporated anEcoRI site at the 5′ end and a BamHI site at the 3′ end. The PCR product was ligated into the pCRBlunt vector (Invitrogen), released by EcoRI restriction digest, and subcloned into the EcoRI site of the pTrcHisB vector to give the construct pTrcHisB-INP54. The identity of the PCR product was confirmed as INP54 by dideoxy sequence analysis.INP54331 was amplified and cloned as above with the 3′ oligonucleotide incorporating a stop codon plus anEcoRI site after nucleotide 1093 (Table II).Table IIList of constructs and oligonucleotide primers used in this studyConstructFeaturesPrimersRef.pPS1303GFP— 2-aThis plasmid was a kind donation from Prof. Pamela Silver.pTrcHisB6 × His tagInvitrogenINP54 384-pTrcHisBFull-length His-Inp54p5-gcgaatt catgaacaaa acgaattgThis study5-ccggatcca gatagtgg tacaactgINP54 331-pTrcHisBHis-Inp54p3315-gcgaatt catgaacaaa acgaattgThis study5-ccgaatt cttacggca ctggcgtccct gtINP54 384-pPS1303Full-length Inp54p-GFP5-gcggatcca aacaatga acaaaacga attggThis study5-cgggatcccca ggatt tttaatagt aaINP54 371-pPS1303Inp54p371-GFP5-gcggatcca aacaatga acaaaacga attggThis study5-cgggatccct atccaat aatgtacat tcttINP54 353-pPS1303Inp54p353-GFP5-gcggatcca aacaatga acaaaacga attggThis study5-cgggatcccgtcaccaa ttgtccaa tcagINP54 331-pPS1303Inp54p331-GFP5-gcggatcca aacaatga acaaaacga attggThis study5-cgggatccccg gcactg gcgtccctgtHA-INP54-pPS1303H 2-bpPS1303 vector with the GFP ORF deleted, bold letters represent the HA sequence.Full-length HA-Inp54p5-gcggatcca aacaatg tatccttatga cgtgcctg actatgccaacaaaacg aattggaa ggtaThis study5-gcggatcctta c aggatttttaatagta aINP54-HA-pPS1303H 2-bpPS1303 vector with the GFP ORF deleted, bold letters represent the HA sequence.Full-length Inp54p-HA5-gcggatcca aacaatga acaaaacga attggThis study5-gcggatccttaggcatagtcaggcac gtcataaggatacaggatttttaatagtaaLRD13-pPS1303LRD13-GFP5-cgggatcca aacaatgt taggttct ttactattaThis study5-cgggatcct tagcag ccggatcctt tgtaLRD12-pPS1303LRD12-GFP5-cgggatcca aacaatgt taggttct ttactattaThis study5-cgggatcct tagcag ccggatcctt tgtaLRD11-pPS1303LRD11-GFP5-cgggatcca aacaatgt ctttactat tatatttaThis study5-cgggatcct tagcag ccggatcctt tgtaLRD10-pPS1303LRD10-GFP5-cgggatcca aacaatgt tactattat atttactat taThis study5-cgggatcct tagcag ccggatcctt tgtaINP54-pRS416-GFPInp54p-GFP under its native promoter5-cgggatcccca ggatt tttaatagt aaThis study5-cgggatcct attgacg gaaccaag ggpEB316UBPTI expression plasmid with uracil marker— 2-cThis plasmid was a gift from Prof. Dane Wittrup.YEplac181-GalBPTIBPTI expression plasmid with leucine marker5-ggaaacag ctatgaccatgThis study5-gtaaaa cgacggccag tg2-a This plasmid was a kind donation from Prof. Pamela Silver.2-b pPS1303 vector with the GFP ORF deleted, bold letters represent the HA sequence.2-c This plasmid was a gift from Prof. Dane Wittrup. Open table in a new tab Two × 100-ml cultures of pTrcHisB-INP54 were grown at 37 °C to anA 600 of 0.5–0.6 prior to induction with 0.1 mm isopropylthio-β-d-galactoside for 2 h at 23 °C. Following induction, cells were pelleted and soluble proteins were extracted in 1/10 volume of buffer B (20 mmTris, pH 8, 250 mm NaCl) supplemented with 12 mm β-mercaptoethanol, 1 mmphenylmethylsulfonyl fluoride, 2 μg/ml aprotonin, 2 μg/ml leupeptin, 1 mm benzamidine, and 1% Triton X-100 at 4 °C overnight with gentle agitation. Triton extracts were centrifuged at 15,000 × g for 15 min then the 20-ml supernatant was incubated with 2.5 ml of Talon resin (CLONTECH) with gentle agitation in a 50-ml tube for 4 h at 4 °C. Following incubation, the resin was poured into a column and washed with 20 column volumes of buffer B. Bound proteins were eluted with 4 column volumes of buffer B at pH 6.5 supplemented with 100 mm imidazole and 700-μl fractions were collected. 50 μl of the starting material, flow-through, and the eluted fractions were analyzed by 12% SDS-PAGE (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (215638) Google Scholar), and either visualized by Coomassie Brilliant Blue staining or transferred to polyvinylidene fluoride membranes according to standard protocols (23Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (47090) Google Scholar) and immunoblotted using monoclonal antibodies to the (His)6-tag (Silenus). Immunoblots were developed using enhanced chemiluminescence (ECL) reagent (PerkinElmer Life Sciences) according to the manufacturer's instructions. The protein concentration in Coomassie-stained gels was quantitated using densitometry by comparison with a standard amount of protein loaded on the gel. PtdIns(4,5)P2 substrate was a mixture of 33.3 μm PtdIns(4,5)P2 and 3 μg of phosphatidylserine, dried under nitrogen, resuspended in 50 μl of lipid resuspension buffer (20 mm Hepes, pH 7.5, 1 mm MgCl2, 1 mm EGTA), and sonicated for 5 min. The recombinant Inp54p was incubated with the substrate in the presence of kinase buffer (20 mm Hepes, pH 7.4, 1 mm EGTA, 5 mm MgCl2) in a 100-μl total reaction volume. Assays were performed at 37 °C for 30 min using two linear protein concentrations in duplicate. The reaction was terminated by incubating with 1 ml of Biomol GreenTM reagent at room temperature for 30 min. The absorbance at 620 nm was measured and the amount of phosphate released calculated by comparison with known standards supplied in the Biomol kit. AllINP54 constructs were amplified by PCR using appropriate primers listed in Table II, from SEY6210 genomic DNA. The primers incorporated a BamHI site for cloning INP54 into the pPS1303 vector, in-frame with the C-terminal GFP. The PCR product was ligated into pCRBlunt, released by BamHI digestion, and ligated to the compatible BglII site in pPS1303. GFP-tagged LRD13, 12, 11, and 10 were amplified from pPS1303-INP54using oligonucleotides listed in Table II and a 3′ primer specific for the 3′ end of the GFP ORF. The resulting LRD-GFP products were cloned into pCRBlunt, excised using BamHI and cloned into theBglII site of pPS1303H. pPS1303H lacked the GFP ORF and was constructed by excising GFP from pPS1303 via HindIII digest followed by religation of the vector. The INP54-pPS1303 constructs were transformed into an inp54 null mutant strain using the S. cerevisiae EasyComp Transformation kit (Invitrogen) and the transformants were selected on complete minimal media agar plates lacking uracil. The identity of all constructs were confirmed using dideoxy sequencing analysis. Full-length INP54 was amplified together with 968 bp upstream of the open reading frame using oligonucleotides listed in Table II. The PCR product was cloned into theBamHI site of pRS416-GFP (21Ooms L.M. McColl B.K. Wiradjaja F. Wijayaratnam A.P.W. Gleeson P. Gething M.-J. Sambrook J. Mitchell C.A. Mol. Cell. Biol. 2000; 20: 9376-9390Crossref PubMed Scopus (32) Google Scholar) in-frame with the C-terminal GFP. The construct was then transformed into an inp54 null mutant strain using the S. cerevisiae EasyComp Transformation kit (Invitrogen) and the resulting transformants selected on complete minimal media lacking uracil. Single colonies were grown to mid-log phase in minimal media lacking uracil at 30 °C, fixed, and stained with an anti-GFP antibody (CLONTECH) to amplify the signal, and analyzed using confocal microscopy as described above. The HA-tagged Inp54p was cloned into the pPS1303H expression vector lacking the GFP coding region. The primers used to amplify INP54incorporated a HA tag at either the 5′ or 3′ end, with aBamHI site (see Table II), this facilitated cloning into theBglII site of pPS1303H vector. The resulting constructs were transformed into inp54 null mutants as described above. Yeast cells were grown overnight in complete minimal media + 2% glucose, diluted 1/200 in 2% raffinose, and induced the next day in 2% galactose for 4 h. Cells were fixed according to Franzusoff et al. (24Franzusoff A. Redding K. Crosby J. Fuller R.S. Schekman R. J. Cell Biol. 1991; 112: 27-37Crossref PubMed Scopus (203) Google Scholar) and stained with either an anti-HA antibody (diluted 1/1000) to visualize the HA-tagged proteins or an anti-BiP antibody (diluted 1/25) to co-localize the GFP-tagged proteins with the ER. A polyclonal antibody to BiP (binding protein) was raised in rabbits against the S. cerevisiae endoplasmic reticulum protein Kar2p (BiP) by standard protocols using mature Kar2p recombinant protein as an antigen. The primary antibodies were counterstained with anti-rabbit tetramethylrhodamine B isothiocyanate for BiP (1/200), or anti-mouse tetramethylrhodamine B isothiocyanate (1/200) for HA. For nuclear staining, spheroplasts were pretreated with RNase (200 μg/ml) at 37 °C for 1 h, then stained with propidium iodide (2 μg/ml) for 20 min. Cells were attached onto the microscope slides with poly-l-lysine (Sigma), coverslips were mounted on the slides using SlowFade (Molecular Probes). The cells were then analyzed using a Leica TCS-NT confocal microscope with an ArKr triple line laser, with green fluorescence collected in channel 1 (488 nm excitation, 530 ± 30 nm emission) and red fluorescence in channel 2 (568 nm excitation, LPS90 nm). This extraction method is a slight modification of that as described in Seedorf and Silver (25Seedorf M. Silver P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8590-8595Crossref PubMed Scopus (117) Google Scholar). Yeast cells expressing Inp54p tagged with GFP or HA were harvested and resuspended in PBSM buffer (phosphate-buffered saline, 5 mm MgCl2), 0.5 mm phenylmethylsulfonyl fluoride, and 3 μg/ml each of leupeptin, aprotonin, pepstatin, and chymostatin. Glass bead lysis was performed by vortexing and incubating on ice at 30-s intervals for 12 cycles, respectively. The lysate was centrifuged for 10 min at 4 °C, the supernatant represented cytosolic fraction, the pellet was resuspended in PBSM buffer with 1% Triton. After overnight Triton extraction at 4 °C, the lysate was spun for 10 min to separate the Triton-soluble and Triton-insoluble fractions. These were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes according to standard protocols (23Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (47090) Google Scholar), and immunoblotted using an antibody specific for GFP (P. Silver) or HA (Covance). To determine the 5-phosphatase activity of Inp54p tagged with GFP or HA, the fusion protein was immunoprecipitated from the Triton-soluble fraction using 50 μl (50% v/v) of protein A-Sepharose, 0.8 μg of anti-GFP antibody (Roche Molecular Biochemicals) or 6 μg of anti-HA antibody (Covance), and 7 μg of rabbit anti-mouse immunoglobulin (DAKO) which served as a linker. Immunoprecipitation was performed at 4 °C overnight with gentle agitation. The protein A-Sepharose pellet was washed 6 times with ice-cold Tris saline (20 mm Tris, pH 7.2, 150 mm NaCl), and the pellet used in PtdIns(4,5)P2 5-phosphatase enzyme assays as described previously. Microsomes were extracted from yeast by differential centrifugation fractionation according to Parlati et al. (26Parlati F. Dominguez M. Bergeron J.J. Thomas D.Y. J. Biol. Chem. 1995; 270: 244-253Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and Paddon et al. (27Paddon C. Loayza D. Vangelista L. Solari R. Michaelis S. Mol. Microbiol. 1996; 19: 1007-1017Crossref PubMed Scopus (21) Google Scholar). The medium-speed microsomal pellet, which was found to be enriched in ER markers, was treated with proteinase K according to the methods described by Bascom et al. (28Bascom R.A. Srinivasan S. Nussbaum R.L. J. Biol. Chem. 1999; 274: 2953-2962Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The control fractions (untreated) and proteinase K-treated fractions were separated by 10% SDS-PAGE, and immunoblotted with either an anti-HA antibody or anti-GFP antibody. The same fractions were immunoblotted with anti-BiP antibody as a control to ensure that the microsomal membranes were intact. W303α, inp51, inp52, andinp53 null mutant strains were transformed with YEplac181-GalBPTI plasmid containing a leucine nutritional marker. This was constructed by amplifying the Gal promoter and BPTI coding regions from pEB316U, using oligonucleotides listed in Table II and cloning the PCR product into the EcoRI/HindIII site of YEplac181 vector (29Gietz R.D. Sugino A. Gene ( Amst .). 1988; 74: 527-534Crossref PubMed Scopus (2593) Google Scholar). W303α and the inp54 mutant strain were transformed with a BPTI expression plasmid, pEB316U, containing a uracil nutritional marker. Yeast cells expressing BPTI were grown at 30 °C overnight in complete minimal media lacking either leucine or uracil, supplemented with 2% glucose. The next day, cultures were diluted 1/200 in raffinose containing media, grown overnight until they reached 107 cells per ml, and induced with galactose for the specified time frames. The amount of BPTI secreted in the culture media was quantified according to the method described by Parekhet al. (30Parekh R. Forrester K. Wittrup D. Protein Expression Purif. 1995; 6: 537-545Crossref PubMed Scopus (72) Google Scholar). A rescue experiment was done to investigate whether Δinp54 mutant could remove a sufficient amount of BPTI from the intracellular space and maintain viability when grown continuously on galactose-supplemented media. Since accumulation of high levels of BPTI is toxic, continuous induction would be lethal to the cells. Raffinose cultures of yeast cells were spotted in 10-fold serial dilutions in 5-μl aliquots onto complete minimal agar plates supplemented with either 2% glucose or galactose. Inp54p is one of four inositol polyphosphate 5-phosphatases (5-phosphatases) found in the yeast S. cerevisiae. Unlike the other previously characterized yeast 5-phosphatases, Inp54p predicts for a smaller molecular mass, 44 kDa, comprising a central 300-amino acid 5-phosphatase domain and no other defined signaling motif. However, a hydropathic profile of Inp54p (scale = Kyte-Doolittle × −1) indicates that the region spanning amino acids 372–384 is strongly hydrophobic, and is highly rich in leucine residues (Fig.1 A). To identify other 5-phosphatases containing a similar C-terminal hydrophobic tail we performed tBLASTn and PSI-BLAST (31Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61086) Google Scholar) searches of the nonredundant protein and nucleotide data bases at the NCBI. In all searches the filter excluding low compositional complexity regions was turned off. We were unable to identify any 5-phosphatase in any other organism that contained a similar hydrophobic anchor sequence in its C terminus. However, we noted that the C terminus of Inp54p strongly resembled that found in many C-terminal tail-anchored proteins (Fig. 1 B). Tail-anchored proteins are a class of integral membrane proteins which lack any N-terminal targeting sequence and insert into membranes via a single C-terminal hydrophobic sequence (32Kutay U. Hartmann E. Rapoport T.A. Trends Cell Biol. 1993; 3: 72-75Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 33Kuroda R. Kinoshita J. Honsho M. Mitoma J. Ito A. J. Biochem. ( Tokyo ). 1996; 120: 828-833Crossref PubMed Scopus (31) Google Scholar, 34Yang M. Ellenberg J. Bonifacino J.S. Weissman A.M. J. Biol. Chem. 1997; 272: 1970-1975Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 35da Fonseca F.G. Wolffe E.J. Weisberg A. Mo
Referência(s)