From the Department of Cellular and Structural
Biology, University of Colorado School of Medicine, Denver, Colorado
80262 and the ¶ Department of Cell Biology, University of Alabama
at Birmingham, Birmingham, Alabama 35294-0005
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ABSTRACT |
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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.
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INTRODUCTION |
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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)1 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 (1, 2). 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 (3-5). The yeast PITP (Sec14p) is required for the formation of yeast Golgi-derived secretory vesicles (4), and this essential Sec14p requirement can be bypassed by modulation of metabolic flux through specific phospholipid biosynthetic pathways (6-8). 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 (5, 9). 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 (5). In the secretory granule fusion reaction, PITP synergizes with phospholipid kinases to generate PI 4,5-bisphosphate (10). One of the mechanisms by which PITP may stimulate phosphoinositide synthesis is by presenting PI to PI kinases (11-13). This concept remains controversial (14).
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.
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EXPERIMENTAL PROCEDURES |
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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 (15, 16).
Methods
Subcellular Fractionation Procedures--
Stacked Golgi
fractions (SGF) were isolated from rat liver according to Taylor
et al. (17). 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 mM
MgCl2, 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 1.15 M 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 0.25/0.86
M sucrose interface. The two-dimensional gel mapping of the
protein composition of the fraction is presented in Taylor et
al. (18). To prepare cytosol the soluble 0.5 M sucrose
fraction of the first gradient was adjusted to 0.25 M
sucrose 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 (19). 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 (2). 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 mM NaCl) 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 (20). Briefly, cells were harvested, resuspended in ice-cold lysis buffer (50 mM sodium phosphate, pH 7.1, 300 mM sodium chloride, 10 mM 2-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 mM HEPES, pH 7.0, 150 mM KCl, 10 mM 2-mercaptoethanol, 1 mM NaN3, 0.2 mM phenylmethylsulfonyl fluoride. Purified Sec14p (25 µg) contained one unit of PI transter activity.
Generation of Rat PITP E. coli
Lysate--
Hexahistidine-tagged rat PITP
was generated by cloning
the rat PITP
structural gene into the pQE31 vector (Qiagen).
E. coli expressing the His6-tagged PITP
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 (20). 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 PITP 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 (21).
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 (22). 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. 23. 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 mM creatine 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 (15) and is used to define a specific population of exocytic vesicles (22). 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. 2 and 24. Isolated complexes (5 µl) in PAN
were resuspended in a reaction mixture containing 20 mM
HEPES, pH 7.4, 5 mM MgCl2, 0.45 mM
EGTA, 10 µM ATP (~5 µCi of
[-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 M
HCl, and the lipids were extracted with 200 µl of
CHCl3:MeOH (1:1) followed by 80 µl of 1 M
HCl: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 mM
EDTA, 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.
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RESULTS AND DISCUSSION |
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PITP Plays an Essential Role in Budding of Exocytic Vesicles from
the Hepatocyte TGN--
PITP is a cytosolic factor that plays an
essential role in secretory vesicle formation from the yeast Golgi
complex and constitutive and regulated secretory granules in
neuroendocrine cells (4-6). We have used two independent approaches to
examine whether PITP is required for cell-free formation of pIgA-R
containing vesicles from the rat hepatocyte TGN. First, the cell-free
assay was challenged with a polyclonal antibody that recognizes both
rat PITP and PITP
isoforms (16). A
concentration-dependent inhibition of vesicle formation was
observed (Fig. 1). The ~65% budding
efficiency of the control assay was reduced to ~40, 25, and 10% by
5, 10, and 15 µg of antiserum, respectively. This inhibition was
specific as in the presence of an equivalent amount of preimmune serum the budding efficiency remained at ~65%.
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Synergy between PITP and the p62cplx-associated PI
3-Kinase Extends to Synthesis of PI(3)P--
The similar wortmannin
sensitivities of p62cplx-associated PI 3-kinase activity and
vesicle formation from the TGN suggested that PI(3)P formation
underlies the p62cplx-associated PI 3-kinase requirement for
vesicle formation. If PI 3-kinase activity and PI(3)P production are
prerequisites for exocytic vesicle formation, the functional synergy
between the p62cplx-associated PI 3-kinase and PITP should
extend to PI(3)P production. Moreover, the production of PI(3)P should
be stimulated by GTP because activation of the p62-associated
small GTPase results in activation of PI 3-kinase activity (2).
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p62cplx-associated PI 3-Kinase Can Utilize PITP-bound Phospholipid as Substrate-- There are presently two general views for how PITP might stimulate p62cplx-associated PI 3-kinase activity. The "substrate presentation" model posits that PITP acts as a co-factor that presents PI to PI kinases and thereby stimulates the initial rate of the headgroup phosphorylation reaction (11-13). The "lipid transfer" model proposes that PITP merely sustains PI kinase activity by effecting transfer of PI down a chemical gradient that is itself created by depletion of PI by metabolic enzymes such as PI kinases and PI phospholipases (14).
To examine the possibility that PITP presents substrate to the p62cplx-associated PI 3-kinase, PI 3-kinase assays were performed in a membrane-free system using purified Sec14p loaded with either PC (Sec14p-PC) or PI (Sec14p-PI). These experiments allowed the interaction between a lipid kinase and PITP to be examined in a purified system. The source of PI in the kinase assays was limited to that which was stoichiometrically bound to Sec14p (20 µg/ml, and this concentration of PI was an order of magnitude lower than that present in the standard assay (i.e. 200 µg/ml) (Fig. 4A). Under these assay conditions, the p62cplx-associated PI 3-kinase alone had minimal PI 3-kinase activity. Introduction of Sec14p-PC in the assay reduced that activity to base-line levels. By contrast, Sec14p-PI stimulated the p62cplx-associated activity 5-fold. These data suggest that the p62cplx-associated PI 3-kinase is capable of directly phosphorylating PI directly bound to PITP and that this presentation of PI enhances kinase activity.
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A Role for PITP in PI 3-Kinase Activity--
The collective data
reported herein demonstrate that the p62cplx-associated PI
3-kinase is stimulated both by Sec14p and mammalian PITP. More
dramatically, activation of the small GTPase bound to p62 in the
presence of PITP
supported a synergistic activation of the
p62cplx-associated PI 3-kinase of up to 30-fold. In addition to
activating the generation of PI(3)P, the cooperation of PITP
and the
p62cplx-associated PI 3-kinase are essential for pIgA-R vesicle
formation from the TGN. We propose that a specific pool of PI93)P is
generated "on demand" at the site at which it will be utilized. In
this regard, we emphasize that the p62cplx-associated PI
3-kinase assembles with the cytosolic domain of an integral membrane
protein of the TGN (TGN38) (2). This organization suggests that
trans-Golgi proteins (perhaps even cargo proteins) provide positional
cues for assembly of this specific PI 3-kinase.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* This work was supported by National Institute of Health Grant GM 42629 (to K. E. H.) and additional support from the Cell Biology Cores of the Hepatobiliary Center (Grant P30 DK34914) and the Monoclonal Core of the Cancer Center (Grant P30 CA-46934).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Schepens Eye Research Inst., Harvard Medical School, 20 Staniford St., Boston, MA 02114.
Supported by grants GM44530 from the National Institute of
Health and BE-232 from the American Cancer Society.
** To whom correspondence should be addressed: Dept. of Cellular and Structural Biology, Box B-111, University of Colorado School of Medicine, Denver, CO 80262. Tel.: 303-315-5153; Fax: 303-315-4729; E-mail: kathryn.howell{at}uchsc.edu.
1
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; GTPS,
guanosine 5'-3-O-(thio)triphosphate.
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REFERENCES |
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