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• W2016451610 abstract "p125, a mammalian Sec23p-interacting protein, exhibits sequence homology with bovine testis phosphatidic acid-preferring phospholipase A1. In this study, we identified and characterized a new homologue of p125, KIAA0725p. KIAA0725p exhibited remarkable sequence similarity with p125 throughout the entire sequence determined but lacked an N-terminal proline-rich, Sec23p-interacting region. In vitro binding analysis showed that KIAA0725p does not bind to Sec23p. KIAA0725p possessed phospholipase A1 activity preferentially for phosphatidic acid. We examined the effects of overexpression of KIAA0725p on the morphology of organelles. Overexpression of KIAA0725p, like that of p125, caused dispersion of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus. Different from the case of p125, overexpression of KIAA0725p resulted in dispersion of tethering proteins located in the Golgi region and caused aggregation of the endoplasmic reticulum. Our results indicate that KIAA0725p is a new member of the phosphatidic acid-preferring phospholipase A1protein family and suggest that the cellular function of KIAA0725p is different from that of p125. p125, a mammalian Sec23p-interacting protein, exhibits sequence homology with bovine testis phosphatidic acid-preferring phospholipase A1. In this study, we identified and characterized a new homologue of p125, KIAA0725p. KIAA0725p exhibited remarkable sequence similarity with p125 throughout the entire sequence determined but lacked an N-terminal proline-rich, Sec23p-interacting region. In vitro binding analysis showed that KIAA0725p does not bind to Sec23p. KIAA0725p possessed phospholipase A1 activity preferentially for phosphatidic acid. We examined the effects of overexpression of KIAA0725p on the morphology of organelles. Overexpression of KIAA0725p, like that of p125, caused dispersion of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus. Different from the case of p125, overexpression of KIAA0725p resulted in dispersion of tethering proteins located in the Golgi region and caused aggregation of the endoplasmic reticulum. Our results indicate that KIAA0725p is a new member of the phosphatidic acid-preferring phospholipase A1protein family and suggest that the cellular function of KIAA0725p is different from that of p125. Phosphatidic acid (PA) 1The abbreviations used are: PAphosphatidic acidERendoplasmic reticulumERGICendoplasmic reticulum-Golgi intermediate compartmentGSTglutathione S-transferaseCOPcoat proteinPA-PLA1phosphatidic acid-preferring phospholipase A1PCphosphatidylcholinePEphosphatidylethanolaminePLA1phospholipase A1PNSpostnuclear supernatantPSphosphatidylserineRACErapid amplification of cDNA ends 1The abbreviations used are: PAphosphatidic acidERendoplasmic reticulumERGICendoplasmic reticulum-Golgi intermediate compartmentGSTglutathione S-transferaseCOPcoat proteinPA-PLA1phosphatidic acid-preferring phospholipase A1PCphosphatidylcholinePEphosphatidylethanolaminePLA1phospholipase A1PNSpostnuclear supernatantPSphosphatidylserineRACErapid amplification of cDNA endsplays a key role in the regulation of several cellular processes. In response to hormones, growth factors, and cytokines, the PA level increases rapidly (1.Hodgkin M.N. Pettitt T.R. Martin A. Michell R.H. Pemberton A.J. Wakelam M.J.O. Trends Biochem. Sci. 1998; 23: 200-204Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). PA activates a variety of proteins involved in signal transduction pathways (2.Ghosh S. Strum J.C. Sciorra V.A. Daniel L. Bell R.M. J. Biol. Chem. 1996; 271: 8472-8480Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 3.Limatola C. Schaap D. Moolenaar W.H. van Blitterswijk W.J. Biochem. J. 1994; 304: 1001-1008Crossref PubMed Scopus (284) Google Scholar, 4.Tomic S. Greiser U. Lammers R. Kharitonenkov A. Imyanitov E. Ullrich A. Böhmer F.-D. J. Biol. Chem. 1995; 270: 21277-21284Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 5.Némoz G. Sette C. Conti M. Mol. Pharmacol. 1997; 51: 242-249Crossref PubMed Scopus (59) Google Scholar, 6.Jones G.A. Carpenter G. J. Biol. Chem. 1993; 268: 20845-20850Abstract Full Text PDF PubMed Google Scholar, 7.De Moritz A. Graan P.N.E. Gispen W.H. Wirtz K.W.A. J. Biol. Chem. 1992; 267: 7207-7210Abstract Full Text PDF PubMed Google Scholar, 8.Tsai M.H. Yu C.L. Wei F.S. Stacey D.W. Science. 1989; 243: 522-526Crossref PubMed Scopus (232) Google Scholar). In addition to the regulatory function, PA is known to cause actin polymerization (9.Ha K.S. Exton J.H. J. Cell Biol. 1993; 123: 1789-1796Crossref PubMed Scopus (154) Google Scholar, 10.Cross M.J. Roberts S. Ridley A.J. Hodgkin M.N. Stewart A. Claesson-Welsh L. Wakelam M.J.O. Curr. Biol. 1996; 6: 588-597Abstract Full Text Full Text PDF PubMed Google Scholar).Several lines of evidence suggest that PA is also involved in vesicle-mediated transport and Golgi organization (11.Ktistakis N.T. Brown H.A. Waters M.G. Sternweis P.C. Roth M.G. J. Cell Biol. 1996; 134: 295-306Crossref PubMed Scopus (328) Google Scholar, 12.Chen Y.-G. Siddhanta A. Austin C.D. Hammond S.M. Sung T.-C. Frohman M.A. Morris A.J. Shields D. J. Cell Biol. 1997; 138: 495-504Crossref PubMed Scopus (240) Google Scholar, 13.Auger R. Robin P. Camier B. Vial G. Rossignol B. Tenu J.-P. Raymond M.-N. J. Biol. Chem. 1999; 274: 28652-28659Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 14.Siddhanta A. Backer J.M. Shields D. J. Biol. Chem. 2000; 275: 12023-12031Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Recent elegant studies demonstrated that the conversion of lyso-PA, an inverted cone-shaped lipid, to PA, a cone-shaped lipid, induces fission of vesicles from the plasma membrane (15.Schmidt A. Wolde M. Thiele C. Fest W. Kratzin H. Podtelejnikov A.V. Witke W. Huttner W.B. Söling H.-D. Nature. 1999; 401: 133-141Crossref PubMed Scopus (448) Google Scholar) or tubulation of the Golgi apparatus (16.Weigert R. Silletta M.G. Spano S. Turacchio G. Cericola C. Colanzi A. Senatore S. Mancini R. Polishchuk E.V. Salmona M. Facchiano F. Burger K.N.J. Mironov A. Luini A. Corda D. Nature. 1999; 402: 429-433Crossref PubMed Scopus (277) Google Scholar). This change in lipid structure most likely induces the high curvature required for the fission of vesicles (15.Schmidt A. Wolde M. Thiele C. Fest W. Kratzin H. Podtelejnikov A.V. Witke W. Huttner W.B. Söling H.-D. Nature. 1999; 401: 133-141Crossref PubMed Scopus (448) Google Scholar, 16.Weigert R. Silletta M.G. Spano S. Turacchio G. Cericola C. Colanzi A. Senatore S. Mancini R. Polishchuk E.V. Salmona M. Facchiano F. Burger K.N.J. Mironov A. Luini A. Corda D. Nature. 1999; 402: 429-433Crossref PubMed Scopus (277) Google Scholar). In addition to this structural contribution, PA may play a regulatory role by interacting with proteins involved in vesicular transport processes. It binds to ADP-ribosylation factor, N-ethylmaleimide-sensitive factor, and kinesin (17.Manifava M. Thuring J.W.J.F. Lim Z.Y. Packman L. Holmes A.B. Ktistakis N.T. J. Biol. Chem. 2001; 276: 8987-8994Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), all of which are components necessary for vesicular transport. Thus, the analysis of PA metabolism may provide insight into the mechanisms underlying vesicle-mediated transport and Golgi organization.We previously identified p125 as a protein that interacts with mammalian Sec23p via its N-terminal proline-rich region (18.Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 19.Mizoguchi T. Nakajima K. Hatsuzawa K. Nagahama M. Hauri H.-P. Tagaya M. Tani K. Biochem. Biophys. Res. Commun. 2000; 279: 144-149Crossref PubMed Scopus (26) Google Scholar). Sec23p is a component of the COPII coat that functions in the production of vesicles from the ER (20.Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1033) Google Scholar). p125 is localized in the ERGIC and/or cis-Golgi, and its overexpression causes the dispersion of these membrane compartments, suggesting its involvement in the early secretory pathway (18.Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Interestingly, the central and C-terminal regions of p125 exhibit significant sequence homology with PA-PLA1. PA-PLA1 preferentially cleaves PA and is predominantly expressed in testis (21.Higgs H.N. Han M.H. Johnson G.E. Glomset J.A. J. Biol. Chem. 1998; 273: 5468-5477Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar).In the present study, we searched data bases using the amino acid sequence of p125 and identified a homologous protein that is encoded by human expressed sequence tag clone KIAA0725. This protein exhibits PLA1 activity preferentially for PA. Consistent with the fact that KIAA0725p does not possess an N-terminal proline-rich region, it does not bind to Sec23p. Overexpression of KIAA0725p affects the morphology of various organelles including the ER.EXPERIMENTAL PROCEDUREScDNA Cloning of KIAA0725pThe KIAA0725 clone was obtained from the Kazusa DNA Research Institute, Japan. To amplify the region upstream of KIAA0725, 5′-RACE was carried out using a MarathonTM cDNA amplification kit (CLONTECH) according to the manufacturer's instructions. Marathon-ReadyTM cDNA from human placenta and a synthetic oligonucleotide (5′-CCTGCTCCATGGGTGTTGAACCCCA) were used as the template and antisense primer for PCR, respectively. The antisense primer was complementary to nucleotides 128–152 of the reported KIAA0725 sequence.To obtain the full-length cDNA of KIAA0725p, a DNA fragment comprising from the putative initiation codon to the SalI site of KIAA0725 was amplified by PCR, and the resultant fragment was replaced with the DNA fragment comprising from the 5′-end to the SalI site of the KIAA0725 clone.Northern Blot AnalysisThe cDNA fragment (the AflII-FbaI fragment) corresponding to amino acid residues 586–706 of KIAA0725p was used as a probe. Human multiple tissues blots of poly(A)+ RNA (CLONTECH) were incubated with the32P-labeled probe in an ExpressHyb hybridization solution (CLONTECH) overnight at 68 °C. The blots were washed for 40 min in 2× SSC containing 0.05% SDS at room temperature and then for 40 min in 0.1× SSC containing 0.1% SDS at 50 °C. Radioactivity was detected with a Fuji Bioimage analyzer BAS2000.AntibodiesThe full-length KIAA0725p with the N-terminal histidine tag was expressed in Escherichia coli cells using expression vector pQE30 (Qiagen). Since the expressed protein was located in insoluble inclusion bodies, it was isolated as follows. E. coli cells expressing the histidine-tagged KIAA0725p were suspended in phosphate-buffered saline containing protease inhibitors and then passed through a French pressure cell (Aminco, SLM Instruments Inc.) three times at 8,000 p.s.i. The resulting suspension was centrifuged at 27,000 × g for 10 min, and the insoluble fraction obtained was subjected to SDS-PAGE. After staining with 0.05% Coomassie Brilliant Blue R-250, the portion of the gel corresponding to the histidine-tagged KIAA0725p band was excised. The gel fragment was homogenized in phosphate-buffered saline with a Teflon homogenizer and then directly injected into rabbits. The antibody was affinity-purified by using the antigen immobilized on nitrocellulose filters.The monoclonal anti-ERGIC53 antibody was a generous gift from Dr. H.-P. Hauri of the University of Basel. The polyclonal anti-β-COP antibody was prepared in this laboratory. The anti-FLAG antibody and anti-GST antibody were obtained from Sigma and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively.Cell CultureVero cells and 293T cells (22.Tanaka M. Gupta R. Mayer B.J. Mol. Cell. Biol. 1995; 15: 6829-6837Crossref PubMed Scopus (220) Google Scholar) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 50 IU/ml penicillin, 50 μg/ml streptomycin, and 10% fetal calf serum.Plasmid Construction, Transfection, and in Vitro Binding AssayMammalian expression plasmid pFLAG-CMV-2 (Eastman Kodak Co.) was used to express proteins fused with the N-terminal FLAG epitope. The cDNA fragment encoding full-length KIAA0725p was inserted into pFLAG-CMV-2. Mammalian expression plasmids for GST-Sec23p and FLAG-tagged p125 were prepared as described previously (18.Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).For the expression of fusion proteins, 293T cells plated on 35-mm dishes were transfected with 1–2 μg of expression plasmids using the LipofectAMINE PLUS reagent (Invitrogen) according to the manufacturer's instructions. At 24 h after transfection, the cells were lysed in lysis buffer (0.35 ml/dish) consisting of 25 mm Hepes-KOH, pH 7.2, 1% Triton X-100, 150 mmKCl, 0.5 μg/ml leupeptin, 2 μm pepstatin, 2 μg/ml aprotinin, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 1 mm dithiothreitol. The lysate was clarified by centrifugation for 10 min at 17,000 × g. The supernatant was used for the in vitro binding assay as described previously (18.Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).Site-directed MutagenesisThe PstI fragment encoding amino acid residues 286–470 of KIAA0725p was isolated from pFLAG-CMV-2 carrying KIAA0725p cDNA and then inserted into the PstI site of pKF19k DNA (TaKaRa). Oligonucleotide-directed mutagenesis was carried out using Mutan-Super Express Km (TaKaRa) according to the manufacturer's instructions. For the replacement of Ser351 with alanine, a synthetic oligonucleotide, 5′-TGCGGTCATGCTTTAGGTTCG, was used. The replacement was confirmed by DNA sequencing. The PstI fragment carrying the mutation was replaced with the corresponding fragment of pFLAG-CMV-2 carrying KIAA0725p cDNA.Preparation of 14C-Labeled Substrates1-[1-14C]Palmitoyl-2-oleoyl-PC was prepared from 1-[1-14C]palmitoyl-lyso-PC as described previously (23.Arai H. Inoue K. Natori Y. Banno Y. Nozawa Y. Nojima S. J. Biochem. (Tokyo). 1985; 97: 1525-1532Crossref PubMed Scopus (34) Google Scholar) with a slight modification. A solution comprising 0.18 μmol of 1-[1-14C]palmitoyl-lyso-PC (PerkinElmer Life Sciences) and 0.27 μmol of oleic acid was dried by evaporation, and then the residual materials were suspended in 100 mmpotassium phosphate buffer, pH 7.4. To this suspension were added 5 μmol of ATP, 5 μmol of MgCl2, 0.15 μmol of coenzyme A, and 100 μg of bovine liver microsomal proteins. The total reaction volume was 480 μl. After a 1-h incubation at 37 °C with shaking, lipids were extracted from the reaction mixture twice by the method of Bligh and Dyer (24.Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42136) Google Scholar). The extract was spotted in a line onto a preparative thin layer chromatography plate, and then the plate was developed in 50:30:8:4 (v/v) chloroform/methanol/acetic acid/water. The14C-labeled PC band was scraped off from the plate, and 14C-labeled PC was extracted from the silica gels.1-[1-14C]Palmitoyl-2-oleoyl-PA, 1-[1-14C]palmitoyl-2-oleoyl-PS, and 1-[1-14C]palmitoyl-2-oleoyl-PE were prepared by transphosphatidylation catalyzed by Actinomaduraphospholipase D as described previously (25.Comfurius P. Zwaal R.F.A. Biochim. Biophys. Acta. 1977; 488: 36-42Crossref PubMed Scopus (500) Google Scholar, 26.Horigome K. Hayakawa M. Inoue K. Nojima S. J. Biochem. (Tokyo). 1987; 101: 53-61Crossref PubMed Scopus (93) Google Scholar). For the preparation of PS or PA, 14C-labeled PC was suspended in 500 μl of diethylether, and then 500 μl of a solution comprising 100 mm sodium acetate, pH 5.5, 100 mmCaCl2, and 5 ml-Ser was added. The reaction was started by the addition of 2.5 units of Actinomadura phospholipase D (Meito Sangyo, Tokyo, Japan), and 2.5 units of phospholipase D was added every 15 min. After 1 h at 45 °C with vigorous stirring, the products were extracted twice by the method of Bligh and Dyer (24.Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42136) Google Scholar). 14C-Labeled PS and PA were separated by thin layer chromatography in 65:25:8.9:1.1 (v/v/v/v) chloroform, methanol, formic acid, water. For the preparation of PE and PA, 14C-labeled PC was treated as described above except that the reaction mixture comprised 100 mm sodium acetate, pH 5.5, 100 mm CaCl2, and 200 mmethanolamine. 14C-Labeled PE and PA were separated by thin layer chromatography in 50:40:3:7 (v/v/v/v) chloroform, methanol, 25% ammonia, water.Preparation of Cell Lysates for the Phospholipase Activity Assay293T cells plated on 60-mm dishes were transfected with 3 μg of expression plasmids using the LipofectAMINE PLUS reagent. At 24 h after transfection, the cells were washed twice with phosphate-buffered saline, and then lysis buffer (200 μl) consisting of 25 mm Hepes-KOH, pH 7.2, 250 mm sucrose, 0.5 μg/ml leupeptin, 2 μm pepstatin, 2 μg/ml aprotinin, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 1 mm dithiothreitol was added to the plate. The cells were scraped off with a cell scraper, and the resulting suspension was collected. This procedure was repeated again, and the two suspensions were pooled. The cells were homogenized with 10 strokes in a stainless steel homogenizer. The homogenate was centrifuged at 1,000 ×g for 5 min to obtain a supernatant. The protein concentration was determined using the BCA protein assay reagent (Pierce). The final protein concentration of the samples was adjusted to 3 mg/ml with lysis buffer, and then the samples were used for the phospholipase A assay.Phospholipase A AssayPhospholipase A activity was measured as described previously (27.Nagai Y. Aoki J. Sato T. Amano K. Matsuda Y. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 11053-11059Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 28.Dole V.P. Meinertz H. J. Biol. Chem. 1960; 235: 2595-2599Abstract Full Text PDF PubMed Google Scholar). 14C-Labeled phospholipid was dried and suspended in water at a concentration of 200 μm. The assay was started by the addition of 50 μl of a lipid solution to 200 μl of assay buffer. The assay mixture comprised 40 μm phospholipid (2,000–10,000 cpm) and cell lysate (120 μg as protein) in 100 mm Tris-HCl, pH 7.5, and 1 mm EDTA (PA as substrate) or in 100 mm Tris-HCl (pH 7.5) and 4 mm CaCl2 (PS, PC, or PE as substrate). In some experiments, 330 μm Triton X-100 was included in the assay mixture. After 1 h at 37 °C, the reaction was stopped by the addition of 1.25 ml of Dole's reagent (78:20:2 (v/v/v) 2-propanol, n-heptane, 1 nH2SO4). The liberated fatty acid was extracted, and its radioactivity was measured with a scintillation counter.Lipase (Triacylglycerol Lipase) AssayLipase activity was measured by the method of Groener and Knauer (29.Groener J.E.M. Knauer T.E. Biochim. Biophys. Acta. 1981; 665: 306-316Crossref PubMed Scopus (13) Google Scholar) with a slight modification. Briefly, 250 nmol of 14C-labeled triolein (trioleoyl glycerol) was dried under a gentle stream of nitrogen and then suspended with a probe-type sonicator in 100 μl of water containing 0.25 mg of gum arabic. The reaction was started by the addition of this solution to assay buffer containing an appropriate amount of enzyme. The assay mixture (250 μl) comprised 100 mm Tris-maleate, pH 8.5, 1% bovine serum albumin, and 1 mm triolein. The reaction was performed at 37 °C for 1 h and stopped by the addition of 1 ml of a solution (37:18:44 (v/v/v) benzene, chloroform, methanol) containing 0.1 mmoleic acid, followed by the addition of 125 μl of 0.6 nNaOH. The mixture was then vortexed vigorously for 4–5 min, and briefly centrifuged. The resulting upper phase was taken for the determination of released 14C-oleic acid with a scintillation counter.Immunofluorescence MicroscopyImmunofluorescence microscopy was performed as described previously (30.Tagaya M. Furuno A. Mizushima S. J. Biol. Chem. 1996; 271: 466-470Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Briefly, cells plated on coverslips were fixed with 4% paraformaldehyde, followed by sequential incubation with primary antibodies and a fluorescein isothiocyanate-conjugated or Texas Red-conjugated secondary antibody. Confocal microscopic analysis was performed with an Olympus Fluoview 300 laser-scanning microscope.RESULTSIdentification of KIAA0725p Homologous to p125The expressed sequence tag data base was searched with the BLAST program using the amino acid sequence of p125. The search revealed the presence of a human expressed sequence tag clone, KIAA0725 (3,911 base pairs), that codes for a homologous protein (KIAA0725p). Clone KIAA0725 encodes 573 amino acid residues but appears to lack nucleotides corresponding to the N-terminal region. To determine the nucleotide sequence 5′-upstream of KIAA0725, the 5′-RACE method was used. We found in RACE products that a putative initiation codon resides 413 nucleotides upstream of the 5′-end of the reported sequence. Although the sequence surrounding this methionine codon did not match the Kozak consensus sequence, one stop codon was found 30 nucleotides upstream of this methionine codon. Therefore, we assumed that this codon is the initiation codon of KIAA0725p.By combining the sequence of the KIAA0725 clone with the 5′-upstream sequence obtained in the 5′-RACE experiments, we identified an open reading frame that encodes a protein of 711 amino acids with a calculated molecular weight of 81,003 (Fig. 1A). As shown in Fig. 1B, KIAA0725p exhibits strong sequence similarity with p125 throughout the entire sequence determined (52.6% identity in the 770-amino acid overlap). KIAA0725p is more similar to p125 than PA-PLA1. KIAA0725p has a consensus sequence for most lipases, Gly-X-Ser-X-Gly, where Xrepresents any amino acid (31.Hide W.A. Chan L. Li W.-H. J. Lipid Res. 1992; 33: 167-178Abstract Full Text PDF PubMed Google Scholar). Other than this consensus sequence, no significant homology was found with other phospholipases or triacylglycerol lipases. Therefore, it seemed that KIAA0725p is a new member of the PA-PLA1 protein family.KIAA0725p lacks a signal sequence or transmembrane domain, suggesting that it is a cytosolic or peripheral membrane protein. It is noteworthy that KIAA0725p, unlike p125, does not possess a proline-rich region. In addition, KIAA0725p does not have a coiled-coil region, which also exists in the C-terminal region of p125.Tissue and Subcellular Distribution of KIAA0725pAs shown in Fig. 2A, Northern blot analysis with the KIAA0725p probe revealed a 5.4-kb transcript in all tissues examined, suggesting the ubiquitous expression of KIAA0725p. A 2.8-kb transcript was also observed. At present, we do not know whether this smaller transcript is a degradation product derived from the larger one or an alternative splicing product.Figure 2Expression of KIAA0725p. A, Northern blot analysis. Poly(A)+ mRNA human multiple tissue blots were probed with 32P-labeled cDNA of KIAA0725p. B, distribution of KIAA0725p in rat tissues. C, subcellular localization of KIAA0725p. Homogenates of 293T cells were fractionated as described previously (18.Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Proteins (50 μg) in each fraction were separated by SDS-PAGE and analyzed by immunoblotting with an anti-KIAA0725p antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To examine the expression of KIAA0725p in cells, a rabbit polyclonal antibody against bacterially expressed KIAA0725p was prepared and affinity-purified. On immunoblot analysis of various tissue extracts, the affinity-purified antibody detected a protein of 90 kDa (Fig. 2B). This value is in fair agreement with the calculated mass of KIAA0725p. Indeed, this protein exhibited the same mobility on gels as an overexpressed protein in 293T cells that had been transfected with a plasmid encoding the full-length KIAA0725p (data not shown). In addition to this 90-kDa band, an 85-kDa band was observed on the blot. This might represent a degradation product derived from the 90-kDa protein or an alternative translation product in which Met-31 is used as the initiation codon.To clarify the subcellular localization of KIAA0725p, homogenates of 293T cells were fractionated by differential centrifugation. Centrifugation of homogenates at 1,000 × g for 5 min gave PNS and nuclear fractions. The PNS was then centrifuged at 9,000 × g for 10 min, giving rise to supernatant and mitochondrial fractions. From the supernatant fraction, microsomal and cytosolic fractions were obtained by centrifugation at 105,000 ×g for 1 h. The proteins in each fraction were separated by SDS-PAGE and then analyzed by immunoblotting with an anti-KIAA0725p antibody (Fig. 2C). The results showed that KIAA0725p is predominantly localized in the cytosolic fraction in 293T cells, but some was observed in the microsomal fraction. Essentially the same results were obtained for rat liver (data not shown).KIAA0725p Does Not Bind to Sec23pp125 interacts with mouse Sec23p via its N-terminal proline-rich region (18.Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Despite its high sequence similarity with p125, KIAA0725p does not have such a proline-rich region. To determine whether or not KIAA0725p interacts with Sec23p, a pull-down assay was conducted. GST-Sec23p and FLAG-tagged KIAA0725p or FLAG-tagged p125 were transiently expressed in 293T cells. A cell lysate was prepared from each transfectant and then incubated with glutathione beads. The proteins bound to the beads were analyzed by immunoblotting with an anti-FLAG antibody. As shown in Fig. 3A, FLAG-tagged KIAA0725p was not pulled down with GST-Sec23p (lane 2). Under the same conditions, FLAG-tagged p125 was pulled down with GST-Sec23p (lane 1), as observed previously (18.Tani K. Mizoguchi T. Iwamatsu A. Hatsuzawa K. Tagaya M. J. Biol. Chem. 1999; 274: 20505-20512Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). These results suggest that KIAA0725p, unlike p125, does not bind to Sec23p. This observation is consistent with the finding that the proline-rich region, but not the phospholipase-like domain, of p125 interacts with Sec23p.Figure 3KIAA0725p does not bind to Sec23p. A, 293T cells were transfected with the plasmid for GST-Sec23p (lanes 1 and 2) or GST (lanes 3 and 4) in combination with the plasmid for FLAG-p125 (lanes 1 and 3) or FLAG-KIAA0725p (lanes 2 and 4). Lysates of the transfected cells were incubated with glutathione beads, and the bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with an anti-FLAG antibody. B, for estimation of the amounts of expressed proteins in the lysates, 4% of the lysates were analyzed by immunoblotting with an antibody against FLAG (left panel) or GST (right panel). The positions of FLAG-p125, FLAG-KIAA0725p, GST-Sec23p, and GST are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)KIAA0725p Possesses PLA1 ActivityWe examined whether or not KIAA0725p possesses PLA1 activity. FLAG-tagged KIAA0725p was transiently expressed in 293T cells, and then PLA1 activity in the PNS of the transfected cells was measured essentially as described previously (27.Nagai Y. Aoki J. Sato T. Amano K. Matsuda Y. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 11053-11059Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). As shown in Fig. 4A, the PNS of KIAA0725p-expressed cells showed much higher PLA1 activity compared with the PNS of vector-transfected cells. Among the phospholipids examined, PA and PE were effectively hydrolyzed, whereas PS was weakly hydrolyzed and PC was very weakly hydrolyzed.Figure 4PLA1 activity of KIAA0725p. PLA1 activity in the lysates of cells transfected with the plasmid for FLAG (open bars), wild-type FLAG-KIAA0725p (closed bars), or a S351A mutant (hatched bars) was measured in the absence (A) or presence (B) of 330 μm Triton X-100 as described under “Experimental Procedures.” To confirm the expression of the proteins in cells, portions of cell lysates were separated by SDS-PAGE and then analyzed by immunoblotting with an anti-FLAG antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)KIAA0725p has a stretch of sequence, Gly349-His-Ser-Leu-Gly353, that is common to most lipases including PA-PLA1 (Ser-His-Ser-Leu-Gly). The seryl residue in this sequence is known to be essential for lipase activity (32.Derewenda Z.S. Derewenda U. Biochem. Cell Biol. 1991; 69: 842-851Crossref PubMed Scopus (131) Google Scholar). To determine whether or not Ser351 of KIAA0725p is also required for its PLA1 activity, we constructed a KIAA0725p mutant, S351A, in which Ser351 was replaced with alanine to eliminate the hydroxyl group. The" @default.
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