The Human Phosphatidylinositol Phosphatase SAC1 Interacts with the Coatomer I Complex
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m307983200
ISSN1083-351X
AutoresHolger M. Rohde, Fei Ying Cheong, Gerlinde Konrad, Karin Paiha, Peter Mayinger, Guido Boehmelt,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe Saccharomyces cerevisiae SAC1 gene encodes an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus. Yeast SAC1 mutants display a wide array of phenotypes including inositol auxotrophy, cold sensitivity, secretory defects, disturbed ATP transport into the ER, or suppression of actin gene mutations. At present, it is not clear how these phenotypes relate to the finding that SAC1 displays polyphosphoinositide phosphatase activity. Moreover, it is still an open question whether SAC1 functions similarly in mammalian cells, since some phenotypes are yeast-specific. Potential protein interaction partners and, connected to that, possible regulatory circuits have not been described. Therefore, we have cloned human SAC1 (hSAC1), show that it behaves similar to ySac1p in terms of substrate specificity, demonstrate that the endogenous protein localizes to the ER and Golgi, and identify for the first time members of the coatomer I (COPI) complex as interaction partners of hSAC1. Mutation of a putative COPI interaction motif (KXKXX) at its C terminus abolishes interaction with COPI and causes accumulation of hSAC1 in the Golgi. In addition, we generated a catalytically inactive mutant, demonstrate that its lipid binding capacity is unaltered, and show that it accumulates in the Golgi, incapable of interacting with the COPI complex despite the presence of the KXKXX motif. These results open the possibility that the enzymatic function of hSAC1 provides a switch for accessibility of the COPI interaction motif. The Saccharomyces cerevisiae SAC1 gene encodes an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus. Yeast SAC1 mutants display a wide array of phenotypes including inositol auxotrophy, cold sensitivity, secretory defects, disturbed ATP transport into the ER, or suppression of actin gene mutations. At present, it is not clear how these phenotypes relate to the finding that SAC1 displays polyphosphoinositide phosphatase activity. Moreover, it is still an open question whether SAC1 functions similarly in mammalian cells, since some phenotypes are yeast-specific. Potential protein interaction partners and, connected to that, possible regulatory circuits have not been described. Therefore, we have cloned human SAC1 (hSAC1), show that it behaves similar to ySac1p in terms of substrate specificity, demonstrate that the endogenous protein localizes to the ER and Golgi, and identify for the first time members of the coatomer I (COPI) complex as interaction partners of hSAC1. Mutation of a putative COPI interaction motif (KXKXX) at its C terminus abolishes interaction with COPI and causes accumulation of hSAC1 in the Golgi. In addition, we generated a catalytically inactive mutant, demonstrate that its lipid binding capacity is unaltered, and show that it accumulates in the Golgi, incapable of interacting with the COPI complex despite the presence of the KXKXX motif. These results open the possibility that the enzymatic function of hSAC1 provides a switch for accessibility of the COPI interaction motif. Phosphatidylinositol (PtdIns) 1The abbreviations used are: PtdInsphosphatidylinositolMTM1myotubularin 1ESTexpressed sequence tagCOPcoatomer proteinTMtransmembraneORFopen reading frameMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightGSTglutathione S-transferasePBSphosphate-buffered salineaaamino acid(s)GFPgreen fluorescent proteinERendoplasmic reticulumBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolhSAC1human SAC1ySAC1yeast SAC1rSAC1rat SAC1.1The abbreviations used are: PtdInsphosphatidylinositolMTM1myotubularin 1ESTexpressed sequence tagCOPcoatomer proteinTMtransmembraneORFopen reading frameMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightGSTglutathione S-transferasePBSphosphate-buffered salineaaamino acid(s)GFPgreen fluorescent proteinERendoplasmic reticulumBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolhSAC1human SAC1ySAC1yeast SAC1rSAC1rat SAC1. phosphates act as signaling components in various intracellular membranes and influence membrane trafficking, cytoskeletal organization, motility, and cellular survival, depending on their subcellular localization and the availability of specialized PtdIns phosphate-binding proteins, lipases, PtdIns kinases, and PtdIns phosphatases (for reviews, see Refs. 1Simonsen A. Wurmser A.E. Emr S.D. Stenmark H. Curr. Opin. Cell Biol. 2001; 13: 485-492Crossref PubMed Scopus (406) Google Scholar and 2Martin T.F. Annu. Rev. Cell Dev. Biol. 1998; 14: 231-264Crossref PubMed Scopus (449) Google Scholar). Several PtdIns phosphatases have recently gained much attention, because their loss of function is associated with disease. For example, mutations in the OCRL1 gene, which encodes a phosphatidylinositol-(4,5)-bisphosphate 5-phosphatase of the trans-Golgi network, are responsible for the oculocerebrorenal syndrome of Lowe (3Dressman M.A. Olivos-Glander I.M. Nussbaum R.L. Suchy S.F. J. Histochem. Cytochem. 2000; 48: 179-190Crossref PubMed Scopus (92) Google Scholar). The tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten) (for a review, see Ref. 4Maehama T. Dixon J.E. Trends Cell Biol. 1999; 9: 125-128Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar) functions as a plasma membrane-associated PtdIns-3-phosphatase that negatively regulates the PtdIns-3-kinase/AKT pathway (5Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2100) Google Scholar). Furthermore, myotubularin 1 (MTM1) belongs to a family of PtdIns-3-phosphatases and was originally identified by positional cloning of the gene responsible for X-linked myotubular myopathy (6Laporte J. Blondeau F. Buj-Bello A. Mandel J.L. Trends Genet. 2001; 17: 221-228Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 7Schaletzky J. Dove S.K. Short B. Lorenzo O. Clague M.J. Barr F.A. Curr. Biol. 2003; 13: 504-509Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Interestingly, PTEN and the MTM1 family harbor a core CX5R(T/S) catalytic motif, which was first identified in protein phosphatases (8Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 9Maehama T. Taylor G.S. Dixon J.E. Annu. Rev. Biochem. 2001; 70: 247-279Crossref PubMed Scopus (406) Google Scholar). phosphatidylinositol myotubularin 1 expressed sequence tag coatomer protein transmembrane open reading frame matrix-assisted laser desorption/ionization time-of-flight glutathione S-transferase phosphate-buffered saline amino acid(s) green fluorescent protein endoplasmic reticulum 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol human SAC1 yeast SAC1 rat SAC1. phosphatidylinositol myotubularin 1 expressed sequence tag coatomer protein transmembrane open reading frame matrix-assisted laser desorption/ionization time-of-flight glutathione S-transferase phosphate-buffered saline amino acid(s) green fluorescent protein endoplasmic reticulum 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol human SAC1 yeast SAC1 rat SAC1. This motif is also embedded in the SAC domain of PtdIns phosphatases like yeast Sac1p (suppressor of actin mutations), Fig4p, Inp51p/Sjl1p, Inp52p/Sjl2p, and Inp53p/Sjl3p (10Hughes W.E. Cooke F.T. Parker P.J. Biochem. J. 2000; 350: 337-352Crossref PubMed Scopus (109) Google Scholar). Interestingly, ySac1p and its rat homolog, rSAC1 (11Nemoto Y. Kearns B.G. Wenk M.R. Chen H. Mori K. Alb Jr., J.G. De Camilli P. Bankaitis V.A. J. Biol. Chem. 2000; 275: 34293-34305Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), exert a wider substrate specificity than PTEN or MTM1, since they were demonstrated to convert PtdIns(4)P, PtdIns(3)P, and PtdIns(3,5)P2 to PtdIns (11Nemoto Y. Kearns B.G. Wenk M.R. Chen H. Mori K. Alb Jr., J.G. De Camilli P. Bankaitis V.A. J. Biol. Chem. 2000; 275: 34293-34305Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 12Guo S. Stolz L.E. Lemrow S.M. York J.D. J. Biol. Chem. 1999; 274: 12990-12995Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 13Schorr M. Then A. Tahirovic S. Hug N. Mayinger P. Curr. Biol. 2001; 11: 1421-1426Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Inp51p/Sjl1p, Inp52p/Sjl2p, and Inp53p/Sjl3p contain in addition to the SAC domain a C-terminal 5-phosphatase domain and thus constitute homologs of mammalian synaptojanin 1, which is essential for synaptic vesicle recycling during endocytosis. Synaptojanin 1-deficient neurons show accumulation of PtdIns(4,5)P2, leaving it open whether its SAC domain is enzymatically functional in mammalian cells (14Cremona O. Di Paolo G. Wenk M.R. Luthi A. Kim W.T. Takei K. Daniell L. Nemoto Y. Shears S.B. Flavell R.A. McCormick D.A. De Camilli P. Cell. 1999; 99: 179-188Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). Synaptojanin 2 displays similar substrate specificity, yet a wider tissue distribution than synaptojanin 1 and appears to act at an earlier step in clathrin-mediated endocytosis (15Nemoto Y. Arribas M. Haffner C. DeCamilli P. J. Biol. Chem. 1997; 272: 30817-30821Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 16Rusk N. Le P.U. Mariggio S. Guay G. Lurisci C. Nabi I.R. Corda D. Symons M. Curr. Biol. 2003; 13: 659-663Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). None of the human PtdIns phosphatases containing a SAC domain have been characterized in depth for their biological function. Information about human SAC1 (hSAC1) is hitherto restricted to an EST (KIAA0851), which maps to the C3CER1 segment on chromosome 3p21.3. C3CER1 is commonly eliminated in SCID-derived tumors (17Kiss H. Kedra D. Kiss C. Kost-Alimova M. Yang Y. Klein G. Imreh S. Dumanski J.P. Genomics. 2001; 73: 10-19Crossref PubMed Scopus (28) Google Scholar, 18Kiss H. Yang Y. Kiss C. Andersson K. Klein G. Imreh S. Dumanski J.P. Eur. J. Hum. Genet. 2002; 10: 52-61Crossref PubMed Scopus (36) Google Scholar). Human SAC2 was enzymatically characterized and found to exert 5-phosphatase activity specific for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 (19Minagawa T. Ijuin T. Mochizuki Y. Takenawa T. J. Biol. Chem. 2001; 276: 22011-22015Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Yeast strains with mutations in SAC1 exhibit an array of phenotypes including inositol auxotrophy (20Whitters E.A. Cleves A.E. McGee T.P. Skinner H.B. Bankaitis V.A. J. Cell Biol. 1993; 122: 79-94Crossref PubMed Scopus (135) Google Scholar), cold sensitivity (21Novick P. Osmond B.C. Botstein D. Genetics. 1989; 121: 659-674Crossref PubMed Google Scholar), secretory defects in chitin deposition (13Schorr M. Then A. Tahirovic S. Hug N. Mayinger P. Curr. Biol. 2001; 11: 1421-1426Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), multiple drug sensitivity (22Hughes W.E. Pocklington M.J. Orr E. Paddon C.J. Yeast. 1999; 15: 1111-1124Crossref PubMed Scopus (18) Google Scholar), and ATP transport deficiencies into the ER (23Kochendörfer K.U. Then A.R. Kearns B.G. Bankaitis V.A. Mayinger P. EMBO J. 1999; 18: 1506-1515Crossref PubMed Scopus (51) Google Scholar). Mutations in yeast SAC1 are capable of suppressing lethality of some secretory pathway (SEC) mutants, like SEC14 deficiency (24Cleves A.E. Novick P.J. Bankaitis V.A. J. Cell Biol. 1989; 109: 2939-2950Crossref PubMed Scopus (190) Google Scholar). SEC14, a phosphatidylinositol/phosphatidylcholine transport protein, is responsible for establishing a critical phospholipid composition in yeast Golgi membranes and is required for secretory competence (25Rivas M.P. Kearns B.G. Xie Z. Guo S. Sekar M.C. Hosaka K. Kagiwada S. York J.D. Bankaitis V.A. Mol. Biol. Cell. 1999; 10: 2235-2250Crossref PubMed Scopus (115) Google Scholar). This is in line with the finding that ySac1p negatively regulates a pool of PtdIns(4)P in the yeast Golgi that is important for forward trafficking of chitin synthases to the cell periphery (13Schorr M. Then A. Tahirovic S. Hug N. Mayinger P. Curr. Biol. 2001; 11: 1421-1426Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In eukaryotic cells, three vesicle-based protein cargo trafficking systems have evolved, which are each defined by a special set of membrane-covering protein coats. Clathrin-coated vesicles allow transport from the plasma membrane to the trans-Golgi network and from the trans-Golgi network to endosomes. Coatomer II (COPII)-coated vesicles have been assigned to ER to Golgi anterograde transport (26Kirchhausen T. Nat. Rev. Mol. Cell. Biol. 2000; 1: 187-198Crossref PubMed Scopus (419) Google Scholar, 27Barlowe C. Biochim. Biophys. Acta. 1998; 1404: 67-76Crossref PubMed Scopus (101) Google Scholar). In contrast, coatomer (COPI)-coated vesicles are involved in membrane traffic mainly between Golgi and ER or intra-Golgi compartments (28Lowe M. Kreis T.E. Biochim. Biophys. Acta. 1998; 1404: 53-66Crossref PubMed Scopus (81) Google Scholar, 29Barlowe C. Traffic. 2000; 1: 371-377Crossref PubMed Scopus (85) Google Scholar). The COPI coatomer consists of seven proteins (α, β, β′, γ, δ, ϵ, and ζ). Formation of the COPI coat depends on the GTPase ARF1 (ADP-ribosylation factor 1), which in its GTP-bound form inserts into the target membrane and attracts coatomer components prior to vesicle budding. GTP hydrolysis by ARF correlates with uncoating of COPI proteins followed by vesicle fusion with the target membrane (26Kirchhausen T. Nat. Rev. Mol. Cell. Biol. 2000; 1: 187-198Crossref PubMed Scopus (419) Google Scholar, 30Nickel W. Brugger B. Wieland F.T. J. Cell Sci. 2002; 115: 3235-3240Crossref PubMed Google Scholar). ySac1p is described as an integral protein of ER and Golgi membranes with the SAC domain pointing toward the cytoplasm. There is still controversy about the number of functional transmembrane (TM) domains and thus the topology of SAC1, since ySac1p was demonstrated to utilize its two C-terminal TM domains (31Konrad G. Schlecker T. Faulhammer F. Mayinger P. J. Biol. Chem. 2002; 277: 10547-10554Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), whereas rSAC1 apparently only uses the first one (11Nemoto Y. Kearns B.G. Wenk M.R. Chen H. Mori K. Alb Jr., J.G. De Camilli P. Bankaitis V.A. J. Biol. Chem. 2000; 275: 34293-34305Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In addition to this controversy, the subcellular localization of endogenous mammalian SAC1 is still open, and it is not clear whether it is involved in the secretory pathway to the same extent as in yeast, which of its functions might depend on its enzymatic activity, or which binding proteins might aid in its biological functions. To address these questions, we characterized human SAC1 and show that it localizes to ER and Golgi. We generated a PtdIns phosphatase inactive variant by mutation of the core CX5R(T/S) motif (hSAC1-C/S) and find it accumulated in the Golgi. We also demonstrate for the first time that wild type hSAC1 interacts with members of the coatomer I complex and with itself. Mutation of a putative COPI-interaction motif, K(X)KXX, in hSAC1 causes a loss in COPI binding and accumulation in the Golgi. Interestingly, although the hSAC1-C/S mutant has an unaltered COPI interaction motif, it fails to efficiently bind to coatomer I. Our findings suggest that the localization of hSAC1 might not only depend on the mere presence of an intact COPI interaction motif and that the PtdIns phosphatase function of hSAC1 is needed to achieve appropriate usage of the K(X)KXX motif. Molecular Cloning of hSAC1 and Plasmids—The EST KIAA0851 was found to contain an open reading frame that is highly homologous to the yeast enzyme SAC1 (31.8% identity, 46.1% similarity). Human SAC1 cDNA was amplified from brain mRNA (Invitrogen) by reverse transcriptase-PCR using the following primers: primer a, 5′-GAG AGA GAA GGA AGG AGG TGG T-3′; primer b, 5′-TGT GGA AAA GTA TGC CTG CTA ATA GTG-3′. PCR products were subcloned into pCRII-TOPO vector (Invitrogen) generating pCRII-hSAC1 and confirmed to be identical to KIAA0851 (nucleotide coordinates 40–2272) via sequencing. The hSAC1 ORF is covered by KIAA0851 nucleotides 70–1833. The hSAC1-C/S mutation was introduced into pCRII-hSAC1 with a Transfomer™ site-directed mutagenesis kit (Clontech) using mutagenic primer 5′-GTT CCG AAG CAA TAG CAT GGA TTG TCT AG-3′ and selection primer 5′-GTG ACT GGT GAG GCC TCA ACC AAG TC-3′ according to the manufacturer's instructions. The hSAC1-KEKID mutation was generated by PCR using the following primers: primer c, 5′-GCA CAA TCC ATC TGG TGG CA-3′; mutagenic primer (primer d), 5′-A TCC TCA GTC TAT CGC TTC TGC CTG GA-3′ with the changed nucleotides underlined. The respective fragment was cloned into pCRII-TOPO, and an EcoRI fragment thereof was swapped into pGFP-hSAC1wt (see below). All mutations were confirmed by sequencing. An N-terminal FLAG tag (amino acids MDYDDDDKATAA, with hSAC1 amino acids 2–5 underlined) was fused to hSAC1wt and hSAC1-C/S coding sequences using standard PCR techniques. Insertion into the mammalian expression vector pcDNA3.1 (Invitrogen) generated pcDNA3.1-FLAG-hSAC1wt and pcDNA3.1-FLAG-hSAC1-C/S. To generate Schizosaccharomyces pombe expression vectors pESP-hSAC1wt and pESP-hSAC1-C/S, the BamHI site of pESP1 (Stratagene) was exploited to introduce the hSAC1 variants following the PCR-mediated addition of BamHI sites directly to the respective coding sequences. Products were confirmed by sequencing. BamHI-flanked ORFs from pESP-hSAC1wt and pESP-hSAC1-C/S were used to generate Cu2+-inducible hSAC1 variants in pYEX-BX (Clontech), resulting in pYEX-BX-hSAC1wt and pYEX-BX-hSAC1-C/S, respectively. To build pGFP-hSAC1wt and pGFP-hSAC1-C/S, the same BamHI-fragments were used to generate intermediates in pENTR3C prior to Gateway™-mediated shuttling (Invitrogen) into gEGFP-A30, an appropriately modified derivative of pEGFP-C2 (Clontech). Inducible Expression in Saccharomyces cerevisiae—The vectors pYEX-BX, pYEX-BX-hSAC1wt, and pYEX-BX-hSAC1-C/S were transformed into the SAC1 null strain ATY 202 (31Konrad G. Schlecker T. Faulhammer F. Mayinger P. J. Biol. Chem. 2002; 277: 10547-10554Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), streaked out onto Cu2+-containing inositol-free plates (0.5 mm CuCl2), and monitored for growth. Expression and Purification of Recombinant GST-hSAC1 Proteins in S. pombe—The vectors pESP-hSAC1wt and pESP-hSAC1-C/S, containing GST-hSAC1 fusion sequences, were transformed into the S. pombe strain SP-Q01 (Stratagene) and induced as described by the manufacturer (Stratagene). Cells were disrupted by French press in lysis buffer containing 0.3 m sorbitol, 0.1 m NaCl, 5 mm MgCl2,10mm Tris/Cl, pH 7.2, and 1% Triton X-100 followed by batch purification of GST fusion proteins with glutathione 4B FF-Sepharose (Amersham Biosciences) according to the manufacturer. Washings were done with lysis puffer, and elution of proteins occurred in 10 mm Tris/Cl, 10 mm glutathione, pH 7.5. For storage at –80 °C, glycerol was added to 25% final concentration. Identification of hSAC1 Interaction Partners—Following SDS-PAGE and Coomassie staining, respective gel pieces were excised and sent to Mobidab Molekularbiologie GmbH (Leipzig, Germany) for MALDI-TOF mass spectrometry analysis. Peptide masses corresponding to human α-COP were covered by 36 peptides (Mowse score 111, p < 0.05). 25 peptide masses matched β-COP (Mowse score 66; p < 0.05). Phosphatase Activity Assay—To measure phosphatase activity, a modified version of a malachite green assay was used (32Maehama T. Taylor G.S. Slama J.T. Dixon J.E. Anal. Biochem. 2000; 279: 248-250Crossref PubMed Scopus (99) Google Scholar). Phospho inositides were purchased from Cell Signals Inc., and phosphatidylserine was obtained from Sigma (P-1060). 1 μg of recombinant GST-hSAC1wt and GST-hSAC1-C/S in 25 μl of storage buffer were incubated (1 h, 32 °C) with 25 μl of liposomes, prepared by sonification in reaction buffer (500 μm phosphatidylserine, 100 μm diC16-PtdInsP isoform, 200 mm sodium acetate, 100 mm Tris-base, 100 mm Bis-Tris, 20 μg/ml porcine gelatin, pH 6.0). Reactions were stopped by the addition of 20 μl of 100 mmN-ethylmaleimide and centrifugation (14,000 × g, 15 min). 25 μl of the reaction supernatant was transferred to a multiwell plate, incubated with (20 min, room temperature) 50 μl of malachite green solution (33Harder K.W. Owen P. Wong L.K. Aebersold R. Clark-Lewis I. Jirik F.R. Biochem. J. 1994; 298: 395-401Crossref PubMed Scopus (183) Google Scholar) to quantify released phosphate at 600 nm. Phosphate release was calibrated to a dilution series of KH2PO4. Peptides, Immunization, and Antibodies—Peptides were synthesized according to standard procedures followed by purification on a reverse phase high pressure liquid chromatograph. Peptides used for competition experiments were hSAC1/578–587 (PRLVQKEKID), hSAC1/41–55 (TLAVKKDVPPSAVTR), and crosstide (biotin-GRPRTSSFAEG). hSAC1 antibodies were raised against recombinant GST-hSAC1 wild type protein in rabbits (Eurogentec). GST-reactive immunoglobulins were removed by adsorption to GST-Sepharose beads (serum 252 and serum 253). Additionally, polyclonal peptide antisera 69 and 7889 were raised against hSAC1 amino acids 570–587 and 41–55, respectively. The following antibodies were commercially available and used according to the manufacturer's instructions: anti-FLAG M2 (F-3165; Sigma), anti-GFP (1814460; Roche Applied Science), anti-COPA (PA1-067; Affinity Bioreagents), anti-COPB, -E, and -G, (sc-13335, sc-13345, and sc-14167; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Sec61α (sc-12322; Santa Cruz Biotechnology), anti-golgin-97 (A-21270; Molecular Probes, Inc., Eugene, OR), anti-GST (27-4577-01; Amersham Biosciences), anti-Golgi 58 K protein (G2404; Sigma), and phospho-Ser193 AFX (sc-12892; Santa Cruz Biotechnology). Immunoprecipitations and Western Blotting—Transfer of proteins to nitrocellulose was performed according to standard procedures. For hSAC1 immunoblotting, membranes were blocked with 5% skim milk in TBST (50 mm Tris/Cl, pH 7.5, 150 mm NaCl, 0.1% Tween 20) and washed with TBST. Visualization was done using suitable horseradish peroxidase-conjugated secondary antibodies and ECL. For immunoprecipitation, cell lysates were prepared by scraping subconfluent cells on ice into lysis buffer (20 mm Hepes, pH 7.9, 150 mm NaCl, 0.1% Triton X-100, Complete™), followed by passage through a 26-gauge needle (five times), clearance of the lysate by centrifugation (10 min, 6000 × g, 4 °C), and preincubation (15 min, 4 °C) with either protein A- or protein G-Sepharose 4B FF (Amersham Biosciences). Precleared lysates were incubated (2 h, 4 °C) with antibodies, followed by the addition and further incubation (45 min, 4 °C) of 50 μl of protein A- or protein G-Sepharose and five washes in lysis buffer. For oligomerization experiments, precleared lysates were incubated (32 °C, 30 min) prior to antibody reactions. Samples were boiled in 2× sample buffer (Roti®-load 1; Roth) and subjected to SDS-PAGE. Lipid Binding Assay—For assessment of phospholipid binding properties, PIP Strips™ (Echelon, Inc.) were blocked (1 h, room temperature) with TBST including 3% bovine serum albumin, incubated either with purified GST, GST-hSAC1wt, or GST-hSAC1-C/S at a concentration of 0.5 μg/ml in blocking buffer at 4 °C overnight. Following three washes in TBST, PIP Strips™ were incubated (1 h, room temperature) with anti-GST antibody in TBST followed by standard secondary antibody incubation and ECL to detect GST-tagged proteins bound to the phospholipid spots on the membrane. Cell Lines and Media—Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. All media were supplemented with 10% fetal calf serum unless stated otherwise. Human glioblastoma cells U373MG were kept in Eagle's minimum essential medium (BioWhittaker), human prostate carcinoma cells PC-3 were kept in RPMI1640 (Invitrogen), human colon carcinoma cells HCT116, HeLa (cervix carcinoma) cells were kept in Dulbecco's modified Eagle's medium with 1% penicillin/streptomycin (Invitrogen), and monkey kidney COS-7 cells were kept in Dulbecco's modified Eagle's medium (Invitrogen). Mammalian expression constructs (10 μg) were transfected using Fugene 6 (Roche Applied Science) according to the manufacturer's instructions. To generate U373MG cell lines stably expressing FLAG-hSAC1wt, FLAG-hSAC1-C/S, GFP-hSAC1wt, and GFP-hSAC1-C/S, the constructs and appropriate vector controls were transfected and selected in the presence of G418 (1 mg/ml; Calbiochem). Resistant clones were routinely kept in medium containing 200 μg/ml G418. Prior to experiments, cells were adapted for 24 h to G418-free medium. Fluorescence Microscopy—Cells grown to 70–80% confluence on chamber slides (Nalge Nunc) were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, permeabilized (10 min) with PBS containing 0.2% Triton X-100, and washed. All washes were in PBS. For detection of golgin-97, samples were blocked (1 h, room temperature) in 1× Roti®-block (Roth) and incubated (1 h, 37 °C) with antibody in Roti®-block. After washing, cells were incubated with Cy3-labeled goat anti-mouse IgG F(ab′)2 (Jackson Immunoresearch Laboratories Inc.; 1:500 in 1× Roti®-block) for 30 min. For detection of Sec61α, p58, and endogenous hSAC1, fixed cells were incubated with 0.1 m glycine in PBS (1 min, room temperature), permeabilized in 0.4% Triton X-100 in PBS (3 min, room temperature), and incubated with respective antibodies in PBS containing 1% bovine serum albumin (1 h, 37 °C). Secondary antibodies used were Cy3-conjugated goat anti-rabbit IgG (Dianova) or DTAF-labeled goat anti-rabbit IgG for hSAC1, fluorescein isothiocyanate-labeled donkey anti-goat IgG (sc-2024; Santa Cruz Biotechnology) or Cy3-labeled rabbit anti-goat IgG F(ab′)2 for Sec61α, and Cy-3 labeled goat anti-mouse IgG for p58. Slides were mounted in Mowiol (Sigma). Pictures were recorded using confocal microscopes as indicated in the figure legends. Picture processing was performed with Adobe® Photoshop 5.5 software. For reasons of simplicity, the anti-hSAC1 immunofluorescence channel is displayed in green, independent of the fluorochrome of the secondary antibody used. Identification of Human SAC1 and Generation of a PtdIns phosphatase Inactive Mutant—To clone hSAC1 cDNA, we used a set of primers flanking the ORF found in the EST KIAA0851. A cDNA fragment identical to KIAA0851 nucleotides 40–2272 was amplified via reverse transcriptase-PCR from brain mRNA (see "Experimental Procedures"). Two other human ESTs (KIAA0966 and KIAA0274) also contain a SAC1 homology domain within their sequence. KIAA0966 was recently designated hSAC2 (19Minagawa T. Ijuin T. Mochizuki Y. Takenawa T. J. Biol. Chem. 2001; 276: 22011-22015Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), whereas KIAA0274 still awaits characterization. The hSAC1 ORF (587 amino acids (aa)) exhibits 32% identity to ySac1p and 95% identity to rSAC1 (11Nemoto Y. Kearns B.G. Wenk M.R. Chen H. Mori K. Alb Jr., J.G. De Camilli P. Bankaitis V.A. J. Biol. Chem. 2000; 275: 34293-34305Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and is predicted to code for a protein of 64 kDa with a pI of 6.69. Inspection of the primary amino acid sequence reveals (Fig. 1) two potential TM domains at the C terminus (aa 521–543, aa 550–569) also present in ySAC1. A third TM domain at the N terminus of hSAC1 is not consistently predicted using different software tools and is therefore omitted in Fig. 1. A highly conserved phosphatase signature motif, CX5R(T/S), starts at aa 389 (Fig. 1). Furthermore, a putative leucine zipper (hSAC1, aa 98–126) is conserved in the mammalian SAC1 family members but absent in Caenorhabditis elegans or ySac1p (Fig. 1) (17Kiss H. Kedra D. Kiss C. Kost-Alimova M. Yang Y. Klein G. Imreh S. Dumanski J.P. Genomics. 2001; 73: 10-19Crossref PubMed Scopus (28) Google Scholar). To generate a PtdIns phosphatase inactive mutant, cysteine 389 was replaced by serine to generate hSAC1-C/S. Sequences covering the ORFs for wild type and mutant hSAC1 were subcloned into various expression constructs (see "Experimental Procedures") to analyze their functions. hSAC1, but Not hSAC1-C/S, Complements Inositol Auxotrophy of ΔSAC1 Yeast Cells—To assess whether wild type hSAC1 is functionally equivalent to ySac1p in yeast cells and to confirm that the hSAC1-C/S mutant is inactive, we inducibly expressed hSAC1wt, hSAC1-C/S, and ySAC1 in a yeast strain deleted for the SAC1 gene (ΔSAC1; see "Experimental Procedures"). Immunoblotting demonstrated that induction in this strain yielded equal protein levels for both hSAC1 variants (Fig. 2). One important phenotype of the ΔSAC1 yeast strain is inositol auxotrophy (20Whitters E.A. Cleves A.E. McGee T.P. Skinner H.B. Bankaitis V.A. J. Cell Biol. 1993; 122: 79-94Crossref PubMed Scopus (135) Google Scholar), the inability to grow on inositol-free medium. As shown in Fig. 2, the ΔSAC1 inositol auxotrophy phenotype could be rescued by Cu2+-inducible expression of hSAC1 to an extent comparable with expression of wild type ySac1p. In contrast, the hSAC1-C/S mutant was not able to complement inositol auxotrophy similar to the empty vector control. This result demonstrates (i) functional similarity between the yeast and the human SAC1 proteins and (ii) a successful impairment of hSAC1 enzymatic function through mutation of the core phosphatase motif. The hSAC1-C/S Mutant Protein Displays Reduced Lipid Phosphatase Activity—To assess whether the failure of the hSAC
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