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A Phosphatidylinositol 3-Kinase and Phosphatidylinositol Transfer Protein Act Synergistically in Formation of Constitutive Transport Vesicles from the Trans-Golgi Network*

Open AccessPublished:April 24, 1998DOI:https://doi.org/10.1074/jbc.273.17.10349
      Current evidence suggests that phosphatidylinositol (PI) kinases and phosphatidylinositol transfer protein (PITP) are involved in driving vesicular traffic from yeast and mammalian trans-Golgi network (TGN). We have tested the interaction between these cytosolic proteins in an assay that measures the formation of constitutive transport vesicles from the TGN in a hepatocyte cell-free system. This reaction is dependent on a novel PI 3-kinase, and we now report that, under conditions of limiting cytosol, purified PI 3-kinase and PITP functionally cooperate to drive exocytic vesicle formation. This synergy was observed with both yeast and mammalian PITPs, and it also extended to the formation of PI 3-phosphate. These collective findings indicate that the PI 3-kinase and PITP synergize to form a pool of PI 3-phosphate that is essential for formation of exocytic vesicles from the hepatocyte TGN.
      Much effort has recently been focused on understanding the molecular mechanisms that underlie the various vesicular trafficking reactions that operate throughout the eukaryotic secretory pathway. The p62cplx is a cytosolic complex required for the formation of polymeric IgA receptor (pIgA-R)
      The abbreviations used are: pIgA-R, polymeric IgA receptor; PI, phosphatidylinositol; PITP, phosphatidylinositol transfer protein; SGF, stacked Golgi fraction; TGN, trans-Golgi network; PI(3)P, PI 3-phosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; PC, phosphatidylcholine; GTPγS, guanosine 5′-3-O-(thio)triphosphate.
      1The abbreviations used are: pIgA-R, polymeric IgA receptor; PI, phosphatidylinositol; PITP, phosphatidylinositol transfer protein; SGF, stacked Golgi fraction; TGN, trans-Golgi network; PI(3)P, PI 3-phosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; PC, phosphatidylcholine; GTPγS, guanosine 5′-3-O-(thio)triphosphate.
      containing exocytic transport vesicle from the TGN of hepatocytes. The p62cplx consists of a 62-kDa phosphoprotein and a 25-kDa GTPase and regulates the activity of a novel PI-specific PI 3-kinase (
      • Jones S.M.
      • Crosby J.R.
      • Salamero J.
      • Howell K.E.
      ,
      • Jones S.M.
      • Howell K.E.
      ). In cytosol, the p62 molecule is phosphorylated and is not associated with the PI 3-kinase catalytic subunit. Upon receipt of some unknown signal, p62 is dephosphorylated, the PI 3-kinase regulatory p62cplx and catalytic subunits assemble with the cytoplasmic domain of TGN38 (an integral membrane protein of the TGN), and exocytic vesicle formation ensues. The PI 3-kinase activity is wortmannin-sensitive at micromolar concentrations and is stimulated by the activation of the p62cplx -associated 25-kDa GTPase. Present evidence suggests that the essential function of the PI 3-kinase in exocytic vesicle formation from the TGN is the generation of a specific PI(3)P pool. Supporting evidence comes from the demonstration that both catalytic activity and vesicle formation are equally inhibited by wortmannin.
      PITPs also have been demonstrated to function in vesicle formation from the TGN in yeast and mammalian systems (
      • Bankaitis V.A.
      • Malehorn D.E.
      • Emr S.D.
      • Greene R.
      ,
      • Bankaitis V.A.
      • Aitken J.R.
      • Cleves A.E.
      • Dowhan W.
      ,

      Ohashi, M., Jan de Vries, K., Frank, R., Snoek, G., Bankaitis, V. A., Wirtz, K., and Huttner, W. B. Nature, 377,544–547.

      ). The yeast PITP (Sec14p) is required for the formation of yeast Golgi-derived secretory vesicles (
      • Bankaitis V.A.
      • Aitken J.R.
      • Cleves A.E.
      • Dowhan W.
      ), and this essential Sec14p requirement can be bypassed by modulation of metabolic flux through specific phospholipid biosynthetic pathways (
      • Cleves A.E.
      • McGee T.P.
      • Whitters E.A.
      • Champion K.M.
      • Aitken J.R.
      • Dowhan W.
      • Goebl M.
      • Bankaitis V.A.
      ,
      • McGee T.P.
      • Skinner H.B.
      • Whitters E.A.
      • Henry S.A.
      • Bankaitis V.A.
      ,
      • Kearns B.G.
      • McGee T.P.
      • Mayinger P.
      • Gedvilaite A.
      • Phillips S.E.
      • Kagiwada S.
      • Bankaitis V.A.
      ). In mammalian membrane trafficking reactions both the formation of TGN-derived transport vesicles of constitutive and regulated secretory pathways and the regulated fusion of secretory granules with the plasma membrane are stimulated by PITP (

      Ohashi, M., Jan de Vries, K., Frank, R., Snoek, G., Bankaitis, V. A., Wirtz, K., and Huttner, W. B. Nature, 377,544–547.

      ,
      • Hay J.C.
      • Martin T.F.J.
      ). Whereas PITP cooperates with at least one other unidentified cytosolic factor to stimulate TGN-derived vesicle production, the mechanism of PITP function in that reaction remains unresolved (

      Ohashi, M., Jan de Vries, K., Frank, R., Snoek, G., Bankaitis, V. A., Wirtz, K., and Huttner, W. B. Nature, 377,544–547.

      ). In the secretory granule fusion reaction, PITP synergizes with phospholipid kinases to generate PI 4,5-bisphosphate (
      • Hay J.C.
      • Fisette P.L.
      • Jenkins G.H.
      • Fukami K.
      • Takenawa T.
      • Anderson R.A.
      • Martin T.F.J.
      ). One of the mechanisms by which PITP may stimulate phosphoinositide synthesis is by presenting PI to PI kinases (
      • Cunningham E.
      • Thomas G.M.
      • Ball A.
      • Hiles I.
      • Cockcroft S.
      ,
      • Cunningham E.
      • Tan S.K.
      • Swigart P., J.
      • Hsuan J.
      • Bankaitis V.A.
      • Cockcroft S.
      ,
      • Liscovitch M.
      • Cantley L.C.
      ). This concept remains controversial (
      • Currie R.A.
      • MacLeod B.M.G.
      • Downes C.P.
      ).
      In this paper, PITP is shown to be an essential component required for the efficient, cell-free formation of pIgA-R containing exocytic vesicles from the hepatocyte TGN. PITP synergizes with the p62cplx -associated PI 3-kinase in the formation of PI(3)P, and this synergy extends to formation of exocytic transport vesicles from the TGN.

      EXPERIMENTAL PROCEDURES

      Materials

      Unless otherwise indicated, all chemicals were obtained from Sigma or Boehringer Mannheim. Phosphatidylinositol was purchased from Avanti Polar Lipids, (Alabaster, AL). Production of specific antibodies against the pIgA-R and PITP has been described (
      • Sztul E.S.
      • Howell K.E.
      • Palade G.E.
      ,
      • Skinner H.B.
      • Alb Jr., J.G.
      • Whitters E.A.
      • Helmkamp Jr., G.M.
      • Bankaitis V.A.
      ).

      Methods

      Subcellular Fractionation Procedures

      Stacked Golgi fractions (SGF) were isolated from rat liver according to Taylor et al. (
      • Taylor R.S.
      • Jones S.M.
      • Dahl R.H.
      • Nordeen M.
      • Howell K.E.
      ). Briefly, livers were removed, finely minced, and resuspended at 6 g/10 ml 0.5 m sucrose in 100 mm KPO4, pH 6.8, 5 mmMgCl2, and 1 μg/ml each of a mixture of proteolytic inhibitors: chymstatin, leupeptin, antipain, and pepstatin. All sucrose solutions contained the same buffer and proteolytic inhibitors. The homogenate was centrifuged (1500 × g for 10 min) to pellet unbroken cells, cell debris, and nuclei. This pellet contained at least 50% of the cell protein. The resulting supernatant (PNS) was loaded in the middle of a sucrose step gradient in an SW28 tube; steps of 1.3 and 0.86 m sucrose were overlaid with the PNS supernatant (0.5 m) followed by a 0.25 m layer and centrifuged for 1 h at 100,000 × g (Beckman Instruments, Palo Alto, CA). The 0.5 m sucrose soluble fraction was collected and used for the preparation of cytosol. The SII fraction (0.5/0.86 m interface) was adjusted to 1.15m sucrose with 2 m sucrose using a refractometer (Bausch & Lomb, Boston, MA). The adjusted SII was loaded into the bottom of an SW28 tube and overlaid with equal volumes of 1.0, 0.86, and 0.25 m sucrose and centrifuged for 3 h at 76,000 × g. The resulting SGF floated to the 0.25/0.86m sucrose interface. The two-dimensional gel mapping of the protein composition of the fraction is presented in Taylor et al. (
      • Taylor R.S.
      • Fialka I.
      • Jones S.M.
      • Huber L.A.
      • Howell K.E.
      ). To prepare cytosol the soluble 0.5 m sucrose fraction of the first gradient was adjusted to 0.25 msucrose with 100 mm KPO4, pH 6.8, 5 mm MgCl2 and centrifuged for 30 min at 100,000 × g to remove any pelletable material. The remaining supernatant was concentrated using an Amicon fitted with a PM10 membrane to ∼40 mg/ml (Amicon, Beverly MA). Protein assays (DC Protein Assay, Bio-Rad) were carried out on all fractions. Aliquots of these fractions were frozen in liquid nitrogen and stored at −70 °C.

      Gel Electrophoresis and Immunoblotting

      SDS-polyacrylamide gel electrophoresis was carried out using a 5–15% acrylamide gradient and the buffer system of Maizel (
      • Maizel J.V.
      ). SDS-polyacrylamide gel electrophoresis molecular weight standards were from Bio-Rad. For immunoblots, nitrocellulose filters (Schleicher & Schuell) were blocked for 1 h in 5% defatted milk/phosphate-buffered saline/0.02% sodium azide. The filters were incubated overnight in primary antibody and washed. When using a mouse primary antibody, the filters were incubated with rabbit antibodies against mouse IgG for 2 h before the blots were visualized using 125I-protein A (ICN, Costa Mesa, CA) by autoradiography and quantitated by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).

      Immunopurification of p62cplx -associated PI 3-Kinase from SGF

      Immunopurification and analysis of PI 3-kinase activity were as described (
      • Jones S.M.
      • Howell K.E.
      ). SGF (1 mg) was solubilized in 1 ml of CHAPS buffer (20 mm HEPES, pH 6.8, 100 mm KCl, 20 mm CHAPS and proteolytic inhibitors) for 1 h on ice with vortexing. Samples were centrifuged for 30 min at 14,000 ×g in a microfuge, and the soluble material was incubated overnight with an immunoaffinity column to which antibodies against p62 were covalently bound. The column was washed with three bed volumes of CHAPS buffer and seven bed volumes of phosphate-buffered saline, and the column was eluted with two bed volumes 0.2 m glycine, pH 2.8. The eluted fraction was neutralized and concentrated and dialyzed into PAN (20 mm PIPES, pH 7.0, 100 mmNaCl) for assay of enzymatic activity. The p62cplx -associated PI 3-kinase immunopurified from SGF contains dimeric TGN38 (the transmembrane receptor for the PI 3-kinase), the p62cplx (62-kDa regulatory subunit bound to a 25-kDa GTPase), and an ∼100-kDa catalytic subunit. All experiments are carried out with p62cplx -associated PI 3-kinase isolated from SGF. The p62cplx in the cytoplasm is not associated with the PI 3-kinase catalytic subunit and has no PI 3-kinase activity.

      Purification of Sec14p

      Hexahistidine-tagged Sec14p was expressed in Escherichia coli and purified essentially as described previously (
      • Skinner H.B.
      • McGee T.P.
      • McMaster C.R.
      • Fry M.R.
      • Bell R.M.
      • Bankaitis V.A.
      ). Briefly, cells were harvested, resuspended in ice-cold lysis buffer (50 mm sodium phosphate, pH 7.1, 300 mm sodium chloride, 10 mm2-mercaptoethanol, 1 mm NaN3, 0.2 mm phenylmethylsulfonyl fluoride) and disrupted in a bead beater (Biospec Products). The homogenate was serially clarified by centrifugation at 5,000 × g, 12,000 ×g, and 100,000 × g. Sec14p was precipitated at 50% saturation with ammonium sulfate, and precipitates were dissolved in lysis buffer, dialyzed exhaustively against the same, loaded onto a column of Ni+-nitrilotriacetic acid resin (Qiagen), and eluted with a linear gradient of imidazole (0–200 mm) in lysis buffer. Peak fractions were collected and dialyzed against lysis buffer. The purified Sec14p does not contain either PI or phosphatidylcholine (PC) because these two lipids are not synthesized in E. coli. Sec14p was subsequently bound either with egg PC or PI (Avanti Polar Lipids) by incubation at room temperature for 3–4 h in the presence of at least a 100:1 molar ratio of phospholipid:Sec14p with phospholipid presented in the form of unilamellar vesicles. Protein was rebound to a Ni+-nitrilotriacetic acid column and re-eluted as before. The preparation was dialyzed exhaustively against 10 mmHEPES, pH 7.0, 150 mm KCl, 10 mm2-mercaptoethanol, 1 mm NaN3, 0.2 mm phenylmethylsulfonyl fluoride. Purified Sec14p (25 μg) contained one unit of PI transter activity.

      Generation of Rat PITPα E. coli Lysate

      Hexahistidine-tagged rat PITPα was generated by cloning the rat PITPα structural gene into the pQE31 vector (Qiagen).E. coli expressing the His6-tagged PITPα were harvested, resuspended in ice cold lysis buffer, and disrupted as above. The homogenate was serially clarified at 5,000 ×g, 12,000 × g, and 100,000 ×g, and the 100,000 × g supernatant was used in the PI 3-kinase and transfer assays. The bacterial high speed supernatant (1 mg) contained one unit of PI transfer activity.

      Phosphatidylinositol Transfer Assays

      PI transfer assays have been described previously (
      • Skinner H.B.
      • McGee T.P.
      • McMaster C.R.
      • Fry M.R.
      • Bell R.M.
      • Bankaitis V.A.
      ). Briefly, rat liver microsomes were employed as [3H]PI donors in the transfer reaction, and unlabeled PC liposomes served as acceptor vesicles. Reaction mixtures (0.25 m sucrose, 1 mm EDTA, and 5 mm Tris-HCl, pH 7.4) were incubated with either purified Sec14p or lysates containing rat PITPα lysates at 37 °C. After 30 min the reactions were centrifuged at 10,000 × g for 10 min to pellet the donor microsomes, and 1 ml of the supernatant was collected for scintillation counting. Under these conditions, the PI transfer reaction is linear as long as the input concentrations of yeast or mammalian PITP sustain 20% transfer or less. One unit of activity is defined as the amount of transfer protein that catalyzes the transfer of 1% radiolabeled phospholipid in 1 min (
      • Aitken J.F.
      • van Heusden P.H.
      • Temkin M.
      • Dowhan W.
      ).

      Cell-free Assay of pIgA-R Containing Exocytic Vesicle Formation from the TGN

      The cell-free assay of budding from an immobilized SGF was carried out as described (
      • Salamero J.
      • Sztul E.S.
      • Howell K.E.
      ). Each assay contains 2.5 mg of magnetic core and shell beads with approximately 50 μg of SGF immobilized. The immobilized fraction is characterized in Ref.
      • Jones S.M.
      • Dahl R.H.
      • Ugelstad J.
      • Howell K.E.
      . For the budding reaction the immobilized fraction was incubated in 2.5 ml containing 0.70 mg/ml cytosol, 25 mm HEPES, pH 6.7, 25 mm KCl, 1.5 mm magnesium acetate, 1.0 mm ATP, an ATP regenerating system (8.0 mmcreatine phosphate, 0.043 mg/ml creatine phosphokinase), and 5 mg/ml bovine serum albumin (final concentrations). After 10 min at 37 °C the Golgi fraction remaining on the beads was retrieved with a magnet, and the budded vesicles remained in the supernatant. The high concentration of soluble protein made it impractical to carry out gel analysis on the total budded fraction. Therefore, the budded fraction was pelleted through a 0.25 m sucrose cushion (for 1 h at 100,000 × g) to reduce the large amounts of cytosolic protein and 5 mg/ml bovine serum albumin present in the budding reaction. The pellet was resuspended in gel sample buffer and resolved by SDS-polyacrylamide gel electrophoresis. The amount of exocytic vesicle budding was determined by quantitative immunoblotting using the mature, sialylated pIgA-R (116 kDa) as the marker, and budding efficiency is calculated by determining the percentage of the 116-kDa form of the pIgA-R that is present in the budded fraction with reference to the total amount in the starting immoblized SGF. The pIgA-R is a PM receptor synthesized in relatively high amounts in rat liver (
      • Sztul E.S.
      • Howell K.E.
      • Palade G.E.
      ) and is used to define a specific population of exocytic vesicles (
      • Salamero J.
      • Sztul E.S.
      • Howell K.E.
      ). Budding of this marker in the presence of the complete cell-free system is ∼70% efficient. When the ATP regenerating system and cytosol are omitted, the background budding is ∼5%. There is no detectable PITP on the SGF, therefore, in antibody inhibition studies the antibodies against PITP were added only to cytosol for 30 min on ice before addition of cytosol to the assay.

      Phosphatidylinositol 3-Kinase Assays

      PI 3-kinase assays were as described in Refs.
      • Jones S.M.
      • Howell K.E.
      and
      • Kazlauskas A.
      • Cooper J.A.
      . Isolated complexes (5 μl) in PAN were resuspended in a reaction mixture containing 20 mmHEPES, pH 7.4, 5 mm MgCl2, 0.45 mmEGTA, 10 μm ATP (∼5 μCi of [γ-32P]ATP), and 200 μg/ml PI in a final reaction volume of 20 μl and incubated for 0–20 min at 30 °C. After incubation the reaction was stopped with 100 μl of 1 mHCl, and the lipids were extracted with 200 μl of CHCl3:MeOH (1:1) followed by 80 μl of 1 mHCl:MeOH (1:1) and dried in a speed vac (Savant, Farmingdale, NY). The samples were resuspended in 10 μl of CHCl3:MeOH (1:1) and spotted onto Silica Gel 60 TLC plates (JT Baker Chromatography; Union City, CA). The TLC plates had been pretreated with 60 mmEDTA, 2% sodium tartrate, and 50% EtOH and dried in a 100 °C oven overnight. Development of the TLC plates was in CHCl3:MeOH:4 n NH4OH (9:7:2) for approximately 2 h, and then the plates were dried for PhosphorImager analysis and subsequently exposed to film for autoradiography.

      Acknowledgments

      S. M. Jones thanks his thesis committee, Paul Melançon, Marie-France Pfenninger, John Caldwell, and John Hutton for continued support and contributions to this work. We thank John Ugelstad and Ruth Schmid (SINTEF, University of Trondheim, Norway) for the shell and core magnetic beads used in the cell-free assay.

      References

        • Jones S.M.
        • Crosby J.R.
        • Salamero J.
        • Howell K.E.
        J. Cell Biol. 1993; 122: 775-788
        • Jones S.M.
        • Howell K.E.
        J. Cell Biol. 1997; 139: 339-349
        • Bankaitis V.A.
        • Malehorn D.E.
        • Emr S.D.
        • Greene R.
        J. Cell Biol. 1989; 108: 1271-1281
        • Bankaitis V.A.
        • Aitken J.R.
        • Cleves A.E.
        • Dowhan W.
        Nature. 1990; 347: 561-562
      1. Ohashi, M., Jan de Vries, K., Frank, R., Snoek, G., Bankaitis, V. A., Wirtz, K., and Huttner, W. B. Nature, 377,544–547.

        • Cleves A.E.
        • McGee T.P.
        • Whitters E.A.
        • Champion K.M.
        • Aitken J.R.
        • Dowhan W.
        • Goebl M.
        • Bankaitis V.A.
        Cell. 1991; 64: 789-800
        • McGee T.P.
        • Skinner H.B.
        • Whitters E.A.
        • Henry S.A.
        • Bankaitis V.A.
        J. Cell Biol. 1994; 124: 273-287
        • Kearns B.G.
        • McGee T.P.
        • Mayinger P.
        • Gedvilaite A.
        • Phillips S.E.
        • Kagiwada S.
        • Bankaitis V.A.
        Nature. 1997; 387: 101-105
        • Hay J.C.
        • Martin T.F.J.
        Nature. 1993; 366: 572-575
        • Hay J.C.
        • Fisette P.L.
        • Jenkins G.H.
        • Fukami K.
        • Takenawa T.
        • Anderson R.A.
        • Martin T.F.J.
        Nature. 1995; 374: 173-177
        • Cunningham E.
        • Thomas G.M.
        • Ball A.
        • Hiles I.
        • Cockcroft S.
        Curr. Biol. 1995; 5: 775-783
        • Cunningham E.
        • Tan S.K.
        • Swigart P., J.
        • Hsuan J.
        • Bankaitis V.A.
        • Cockcroft S.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6589-6593
        • Liscovitch M.
        • Cantley L.C.
        Cell. 1994; 77: 329-334
        • Currie R.A.
        • MacLeod B.M.G.
        • Downes C.P.
        Curr. Biol. 1997; 7: 184-190
        • Sztul E.S.
        • Howell K.E.
        • Palade G.E.
        J. Cell Biol. 1985; 100: 1248-1254
        • Skinner H.B.
        • Alb Jr., J.G.
        • Whitters E.A.
        • Helmkamp Jr., G.M.
        • Bankaitis V.A.
        EMBO J. 1993; 12: 4775-4784
        • Taylor R.S.
        • Jones S.M.
        • Dahl R.H.
        • Nordeen M.
        • Howell K.E.
        Mol. Biol. Cell. 1997; 8: 1911-1931
        • Taylor R.S.
        • Fialka I.
        • Jones S.M.
        • Huber L.A.
        • Howell K.E.
        Electrophoresis. 1997; 18: 2601-2612
        • Maizel J.V.
        Methods Virol. 1971; 5: 179-246
        • Skinner H.B.
        • McGee T.P.
        • McMaster C.R.
        • Fry M.R.
        • Bell R.M.
        • Bankaitis V.A.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 112-116
        • Aitken J.F.
        • van Heusden P.H.
        • Temkin M.
        • Dowhan W.
        J. Biol. Chem. 1990; 265: 4711-4717
        • Salamero J.
        • Sztul E.S.
        • Howell K.E.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7717-7721
        • Jones S.M.
        • Dahl R.H.
        • Ugelstad J.
        • Howell K.E.
        Celis J. 2nd Ed. Cell Biology: A Laboratory Handbook. II. Academic Press, San Diego, CA1997: 12-25
        • Kazlauskas A.
        • Cooper J.A.
        EMBO J. 1990; 9: 3279-3286
        • Griffiths G.
        • Simons K.
        Science. 1986; 234: 438-443
        • Ladinsky M.S.
        • Kremer J.R.
        • Furcinitti P.S.
        • McIntosh J.R.
        • Howell K.E.
        J. Cell Biol. 1994; 127: 29-38
        • Hickinson D.M.
        • Lucocq J.M.
        • Towler M.C.
        • Clough S.
        • James J.
        • James S.R.
        • Downes C.P.
        • Ponnambalam S.
        Curr. Biol. 1997; 7: 987-990