Inr. J. Biochem.
Vol. 24, No. 9, pp. 1397-1406. Printed in Great Britain. All rights reserved
0020-711X/92$5.00+ 0.00 Copyright 0 1992Pergamon Press Ltd
1992
FUNCTION OF INTRACELLULAR PHOSPHOLIPASE A, IN VECTORIAL TRANSPORT OF APOPROTEINS FROM ER TO GOLGI AMALIA SLOMIANY,*
EWA
GRZELINSKA, CHINNASWAMY KASINATHAN, KEN-ICHIRO YAMAKI, DANUTA PALECZ and BRONISLAWL. SLOMIANY
Research Center, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2400, U.S.A. [Tel. (20 1) 456-70531 (Received 13 January 1992) The cytosolic fraction required in in airro reconstituted intracellular transport of mucus glycoprotein apopeptide (apomucin) was isolated and its potential as transport supporting factor assessed by the quantitation of the gastric apomucin transferred to Golgi. 2. The experiments with the fraction promoting transport and delivery of apomucin to Golgi revealed that the active protein has the property of phospholipase A, (PLA,) which assists ER vesicles fusion with Golgi. 3. The ability of the 76 kDa PLA, to hydrolyze phospholipids and to support transport and fusion of ER vesicles with Golgi was abolished by phosphorylation and regained foIlowing dephosphorylation. 4. The data provide evidence that 76 kDa intracellular PLA, is responsible for the fusion of ER-transport vesicles with Golgi. The process of fusion is accomplished by generation of lysophospholipids in fusing membranes.
Abstract-l.
The movement
of proteins destined for the cell surface or the organelles distal to endoplasmic reticulum
(ER) is carried out by a vesicular transport (Balch, 1989; Goda and Pfeffer, 1989; Rothman and Orci, 1990). The molecular mechanism involved in this complex transport pathway has been studied in semiintact cells and cell-free systems and a number of specific protein and factors were found to be involved in the vesicle formation and fusion (Glick and Rothman, 1987; Baker er al., 1988; Block et al., 1988;
Nakamo and Muramatsu, 1989; Beckers et al., 1990; Balch, 1990). Considerable progress in the i~ vilro reconstituted transport was achieved by utilization of cytosol derived factors, which are essential for preparation of the transport vesicles for fusion (Block et al., 1988; Diaz er al., 1989). Evidence suggests that the cytosol derived fusion factors are required for the transport vesicles to lose their cytoplasmic coat, to attach to the Golgi receptor membrane and to fuse with the membrane. The involvement of these cytosol derived protein factors in the final events preceding fusion of the Golgi attached vesicles, implies that the proteins might contribute to destabilization of the membranes lipid bilayer. The investigation of the *To whom correspondence should be addressed at: Research Center, UMDNJ-NJ Dental School, University Heights, 110 Bergen Street, Newark, NJ 07103-2400, U.S.A.
cytosolic fusion promoting fraction for its phospholipid Iytic activity led us to identification of the protein which assisted transport, promoted fusion of the ER-transport vesicles with Golgi and displayed phospholipase A, (PLA,) activity. We propose that the fusion of ER transport vesictes carrying cargo to Golgi (acceptor) membranes is accomplished with the aid of 76 kDa cytosohc PLA, which locally increases the level of lysophospholipid fusogens in uniting membranes. MATERIALS
AND METHODS
Materials [5,6,8,9,11,12,14, 15-3H]Arachidonic acid (95 Cij mmol), I-palmitoyl,2-[l-‘4C]arachidonoylphosphatidylcholine (55 mCi/mmol), I-palmitoyl-,2-[l-‘4C]oleoylphosphatidylcholine (55 mCi/mmol), I-palmitoyl-2-[3H]palmitoyl phosphatidylcho~ine (53 Ci/mmol), [‘~qphosphocholine (50 mCi/mmol) were from New England Nuclear (Boston, Mass.), or from Amersham Corp. (Arlington Heights, III.). Phospholipid standards of dipalmitoyl- and I-palmitoyl2-arachidonoylphosphatidylcholine were from Avanti (Birmingham, Ala). NEM, BSA, soybean trypsin inhibitor, polyethyleneglycol 4000, creatine phosphokinase, PMSF, pepstatin, leupeptin, ATP, GTP, CTP, creatine phosphate, fatty acid CoA and sucrose were purchased from Sigma Chemicals (St Louis, MO.). Polyacrylamide gel eiectrophoresis reagents and gels for the Phastsystem were from LKBPharmacia (Piscataway, N.J.), and for mini slab gels from Bio-Rad (Rockville Centre, N.Y.). All other reagents were of highest purity and were purchased from T.T. Baker N.J.), Fisher Scientific Chemical Co. (Phillipsburg,
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AMALIASLOMIANY
(Springfield, N.J.) and VWR Scientific (Piscataway, N.J.). The antimucin monoclonal antibodies were raised in our laboratory (Sfomiany er al., 1992). In this study. the lH7 MAb recognizing apomucin protein core was empfoyed. Subcellular fractionation: preparation of endopiasmic reriru lum membranes, Golgi membranes, cytosol and generation of the transport vesicles
Crude homogenate of rat gastric mucosa was prepared using 12 strokes of tight-fitting Dounce homogenizer, whereas rat liver required 20 strokes to break 90% of the cell in 20% tissue~buffer suspensions. In standard preparation 100 g of the tissue was minced and homogenized in 400 ml of ice-cold 10 mM HEPES-KOH. pH 7.4 buffer containing 0.5 M sucrose and 5 mM EDTA. The unbroken cells and nuclei were removed by centrifugation at 3000rpm for IO min and the supernatant was layered over 1.35 M sucrose in IO mM HEPES-KOH, pH 7.4 buffer, and centrifuged at 85,000 g for 90 min. The crude membrane fraction at the interphase with the I.35 M sucrose was removed, adjusted to 1.2 M sucrose and 1 volume of this membrane fraction was overlaid with 1 volume of 1.1 M sucrose, 1 volume of 1.0 M sucrose and 0.5 volume of 0.5 M sucrose (all sucrose solutions were prepared in 1OmM HEPES-KOH, pH 7.4 buffer) and centrifuged at f~,O~g for 2.5 hr. The Golgi membranes were recovered from OS/f.0 M sucrose interface and endoplasmic reticulum from 1.2 M sucrose. Both membrane preparations were centrifuged once again in the discontinuous sucrose gradient. The Golgi membranes were subjected to the sucrose gradient described above. whereas ER membranes were diluted to 0.5 M sucrose and overlayered onto gradient consisting of 2.0, 1.5, 1.3M sucrose and centrifuged at 85,000 g for 90 min. The membranes from 1.3/0.5 M sucrose interface were withdrawn, pelleted by centrifugation at 70,000 g for 20 min and stored at - 8O’C. Thus prepared membranes were 2-3% cross contaminated with each other and did not contain mitochondria! membranes, as judged from electron microscope examination of the fractions. The purity of the Gofgi and ER were assessed using antimucin monoclonal antibodies recognizing apomucin (lH7 MA\, specific for ER contained peptides), and glycosylated mucin (3G7 MAb recognizing GlcNAcGalNAc-O-apomucin structure assembled in Golgi). The ER derived transport vesicles were generated using modified procedure described in Mafhotra et ai. (1989) in the presence of [‘4qphosphocholjne and [“Hlofeic acid (0.005 pCi,/pi). The incubation mixture consisted of 10 mg/mf rat gastric mucosa! ER membranes, 50 mg/m! rat liver cytoso! supplemented with 0.3 mM free fatty acids, 0.4mM CoA (for [“‘CJphosphocholine labeling) or with 0.3 mM phosphocholine and 0.4 mM CoA (for fatty acid labeling experiments), 10 U/ml creatine phosphokinase 2mM creatine phosphate, 0.7 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.2 mM DTT, 20mM GTP in 33 mM HEPES-KOH, pH 7.0 buffer containing 33 mM KC1 and 2.5 mM Mg acetate. After 30 min at 37°C the incubation was terminated and transport vesicles purified. The isolated vesicles were subjected to fusion with Golgi in the presence of the cytosof enriched with fusion promoting fraction or purified 76 kDa protein. The afiquots of the vesicles and Gofgi membranes recovered after fusion were extracted with chloroform/methanol (2: 1, v/v) and the radiolabeled lipids identified and quantitated. The aliquots of the delipidated residues were used for SDS-PAGE, and immunoquantitation of the apomucin present in the transport vesicles and that delivered to Gofgi.
et al.
Isolation and purt$cation of the transport and fusion promot ing fraction from gastric mucosal cells
To obtain the gastric mucosal cell cytosof, the homogenate of 10 rat stomach mucosa1 tissue in 0.25 M sucrose, 50 mM Tris-HC! pH 7.4, 25 mM KC!, 5 mM Mg acetate, 5 mM mercaptoethano! and 1 mM PMSF was centrifuged at 10,000 g for 20 min. The supernatant was filtered and centrifuged at 25,000 rpm for 60 min in 45 Ti rotor. The soluble fraction, the “cytoso!“, was admixed with 2 mM dithiothreitof and 5 mM ATP, pH of the solution adjusted to 7.0 and 50% PEG-4000 was added dropwise until 8% concentration was reached. The mixture was incubated on ice for 30 min and the precipitated protein was recovered by centrifugation at !O,OOOgfor 15 min. The pellet was solubilized in buffer consisting of 20mM PIPES-KOH, pH 7.0, IOOmM KC!. 2 mM MgClz, 2 mM DTT and 0.5 mM ATP and applied to DEAE*el!ufose (2 x 20 cm) column. The unbound fraction was fractionated further on S-Sepharose (1 x !Ocm) column. The major protein fraction eluted from S-Sepharose column in first 32236ml volume (Fr. g--9), and the fusion promoting activity was recovered with 48. 56 ml (Fr. 12-14) of the elutate. The fraction containing fusion promoting activity were adjusted to 5% with respect to glycerol and concentrated using Centricon ultrafiltration membrane with mofecuiar cut oB of 100 kDa. Final puri~~tion of the fusion promoting protein was achieved by eiectroefution from ND-PAGE gel, and determined that only 76 kDa protein displayed fusogenic activity. Fusion of the ER transport vesicles with Go/gi
Radiolabeled transport vesicles (10 pg or 10,000 cpm) and 20 pg Golgi membranes were incubated for 30 min at 37-C in the presence of 250 pg cytoso!, and suppiementa! mixture detailed under transport vesicles generation and 5 pg of the transport and fusion promoting cytosolic proteins obtained from consecutive fraction of S-Sepharose column, or 20-80ng of the purified 76 kDa protein promoting the fusion of the vesicles with Gofgi. The incubation was stopped by 1 mM NEM and the mixture was layered on discontinuous sucrose gradient consisting of 1.0 and 0.5 M sucrose in 10 mM HEPESKOH, pH 7.4 buffer, and centrifuged at 100,000g for 2.5 hr. The Golgi membranes were recovered from l.OjO.5 M interface, diluted with 25 mM HEPES-KOH, pH 7.0, 250 mM KC! and 2.5 mM Mg acetate and centrifuged at l~,~g for 30 min. The Gofgi membranes were suspended once again in this stripping buffer (to remove associated but not fused ER transport vesicles), centrifuged and then subjected to analysis for the content of radiolabeled phospholipids and the amount of transferred apomucin. In order to measure the internafization of the transport apomucin and incorporation of the vesicular membrane into membrane continuum of the Golgi, first the externally associated components present on the vesicles and the Golgi were degraded with trypsin (100 pg/m!, 2 hr at 0°C). and then the fractions were subjected to identification and quantitation of radiolabeled phospholipids and immunoquantitation of apomucin. ~huspho~~p~e assay
The standard phosphohpase assay measured the amount of radiolabeled oleic acid hydrolyzed from I-0-pafmitoyf-2[3H]o!eoyfgfycerofphosphocho!ine or radiolabeled LPC from 1,2-diacyl-sn-glycerol phospho[‘4C]chofine. Organic solvents were removed from the phospholipid substrate under a stream of Nlr resuspended in 0.2 M Tris-HCi buffer
PLA,
and fusion
(pH 7.0) and then sonicated for 5 min. The assay mixture contained 50 mM Tris-HCI buffer, pH 7.0, 0.3 FM CaCI,, 20 fig human serum albumin (delipidated) and PC substrate (100,000 cpm/tube) in a final volume of 50~1. The reaction
was started by the addition of purified PLA, (2&80ng) protein and incubated at 37°C for 30 min. The reaction was stopped with 1.1 ml of chloroform/methanol/O.5 M HCl (20: 4: 1, v/v/v) which were followed by the addition of 1ml of hexane and 0.5 ml of water. After mixing, the hexane phase was separated and subjected to free fatty acid quantitation, whereas the chloroform/methanol phase, after concentration, and addition of 5 pg lysophosphatidylchohne/sphingomyehn/phosphatidylcholine carrier mixture was applied to thin layer plate (20 x IOcm, Silica Gel G). Usually, all samples were applied on the same plate and separated from each other by scouring the silica gel and the plates were developed in chloroform/methanol/water (65:35:8, v/v/v). The amount of radioactivity was determined by radioscanning in Berthold Radioactivity Analyzer and by scraping the area corresponding to LPC, PC and FFA and subjecting the silica gel to liquid scintillation spectroscopy. PLA,
dependentfusion
Effect of PLAz on ER transport vesicles fusion was measured using NEM-treated Golgi membranes and cytosol (to inactivate the intrinsic transport and fusion activity) and adding t&4 pg/ml of the purified transport and fusion promoting 76 kDa protein. In this assay, the amount of apomucin transferred from the vesicles to the Golgi derived from liver was measured and the radiolabeled phosphatidylcholine and phosphatidylethanolamine incorporated into the Golgi membranes was quantitated. Under normal control conditions where transport activity was not inactivated and purified 76 kDa PLA, was not added, the fusion of ER transport vesicles with Golgi afforded incorporation of 850&10,000 cpm of phosphohpids/sample and transported 81-93 ng of apomucin. The mock controls with apomucin and phospholipids (equivalent to the amount present in the vesicles) used in the fusion experiments instead of ER transport vesicles gave background value of 350-700 cpm, and were subtracted from the total cpm incorporated into the presence of transport vesicles. RESULTS
Cytosolic protein dependent apomucin transport Fusion potential of the proteins isolated from the cytosol of gastric mucosal tissue was expressed by the fractions 12-14 of the S-Sepharose column (Fig. 1). On the SDS-PAGE, the active fractions consisted of 8-10 protein bands in the molecular range of 47-160 kDa. After Centriconfiltration, the 6 protein bands of 97-160 kDa were retained on the filter, while the filtrate contained 47 and 76 kDa proteins, and the total transport promoting activity of the fraction subjected to the procedure. The separation on ND-PAGE allowed to separate the 2 remaining proteins and to conclude that the transport-related activity was associated with 76 kDa protein, while the 47 kDa component was not active in the transport assays. The addition of 76 kDa protein (2 pg/ml) to the unmodified in uitro transport assay mixture increased fusion by IS-20% (Fig. 2). Upon
of transport
vesicles
1399
transport inactivation with 1 mM N-ethylmaleimide (NEM), the fusion of ER transport vesicles with Golgi was proportional to the amount of 76 kDa protein added to the incubation mixture and the maximum effect was achieved with 24pg/ml of the protein. At the optimal concentration of the fusion promoting protein up to 65% of the radiolabeled vesicles fused with Golgi. Based on the isolated proteins activity in promoting ER vesicles fusion with Golgi, the function of the 76 kDa protein was established as the one promoting fusion. Phospholipase activity and substrate specificity of 76 kDa cytosolic protein
positional
The experiments with 76 kDa fusion promoting protein incubated with I-palmitoyl,2-[3H]oleoyl-snglycerol phosphocholine revealed phospholipid lytic activity of the protein as evidenced by generation of radiolabeled free fatty acids (Fig. 3). Since there was no phosphocholine or choline generated, the measured activity was thus specific for phospholipases A. To determine the positional lytic specificity of the 76 kDa protein, the radiolabeled products formed from the hydrolysis of 1-pa1mitoyl,2-[3 HIpalmitoylphosphatidylcholine, I-palmitoy1,2-[i4C]arachidonoylphosphatidylcholine and I-palmitoyl,2[3Hloleoyl phosphatidylcholine were analyzed by HPTLC. Under the standard phospholipase assay conditions (60 ng of 76 kDa protein and 30 min incubation), the only radiolabeled products found were [3H]palmitic acid, [‘4C]arachidonic acid or [3H]oleic acid. With sn-2 fatty acid labeled phosphatidylcholine, there was no evidence of the generation of radiolabeled lysophosphatidylcholine (Fig. 4), thus suggesting that the protein has PLA, activity. Comparison of 1,2-dipalmitoyl, 1-oleoyl-2-arachidonoyl-sn-glycerolphosphocholine and I-palmitoyl2-oleoyl-sn-glycerol phosphocholine consistently showed highest activity of the 76 kDa protein against sn-2 unsaturated long chain fatty acyl residues of the substrates. Effect of NEM and phosphorylation on PLA, activity of the 74 kDa protein The studies on cytosolic 76 kDa protein and the protein dissociated by mild salt extraction from the intracellular membranes indicated that this PLA, appears in 2 forms which differ in specific lipolytic activity. The cytosol derived PLA, displays minimum phospholipid lytic activity 0.2-l .5 pmol/mg/min, whereas the PLA, dissociated from the membranes displayed specific activity in range of 33-73 pmol/ mg/min. The experiments with CAMP dependent protein kinase and [y -32P]ATP suggested that the cytosolic form might be phosphorylated, since with phosphorylation the membrane derived PLA, activity was drastically diminished, whereas dephosphorylation of PLA, generated active enzyme again (Fig. 5 and Table I). Treatment of the isolated PLA, with I mM NEM abolished completely its phospholipid
1400
AMALIA SLOMIANY et al.
lytic potential, while incorporation of NEM into the fusion incubation mixture (Fig. 2) abolished apomucin transport from ER vesicles to Golgi. This, together with the fact that NEM-inhibited transport is restored with addition of 76 kDa PLA,, strongly suggests that this intracellular phospholipase is engaged in fusion of transport vesicles with Golgi and delivery of protein cargo to Golgi cisternae. DISCUSSION
The 76 kDa protein was purified based on its ability to support the transport between the ER and Golgi compartment in which the transport of gastric apomucin to liver derived Golgi was measured. The activity of the protein was assessed by determining the amount of radiolabeled vesicles fused with Golgi and the amount of apomucin transferred to Golgi compartments. The fact that Golgi derived from liver is totahy free of mucin, allowed us to estimate the net transport by immunoquantitation of the apomucin within Golgi. The experiments conducted with mock incubates and trypsin treated Golgi preparations showed that the mucin was indeed internalized, and that the radiolabeled transport vesicles fused with Golgi membrane. Had the fusion not taken place, the recovered vesicles would have still contained their transport cargo. On the other hand, if the apomucin and radiolabeled phosphatidylcholine were added to incubation mixture in a free form, neither the apomucin nor the radiolabeled phospholipids were detectable in the Golgi membranes, and upon trypsin treatment the apomucin was degraded. Hence, our conclusion was that the isolated 76 kDa protein is capable to support transport. Although other proteins are known for their potential to assist the transport vesicles fusion, their exact function in the fusion event is still unknown. It remains unexplored whether fusion assisting proteins are capable to transform the lipid components of the involved membranes into potential fusogens such as lysophospholipids. Our data show that when fusion promoting protein is incubated with vesicles labeled in phos~~atidylcholine, the phosphol~pid is degraded and lysophosphatidyl~holine and free fatty acids are formed. The evidence obtained with sn-2 saturated, unsaturated and arachidonic acid containing phosphatidylcholine species provides strong support that the active component of the fraction promoting fusion is a PLA, enzyme. The ability of the 76 kDa protein to release the labeled fatty acids or generate lysophospholipids in the transport vesicles and lack of any changes when the fusion is abolished with NEM, suggest that the lypolytic activity of the 76 kDa protein is directly related to its fusion potential. PLA, activity of 76 kDa protein is phospho~lation sensitive, and exhibits optimum at pH 7.4. The level of PLAz activity detectable in the preparations is to a large extent dependent upon the amount of the
protein released from the membrane. Our results indicate that cytosolic PLAz prepared under the conditions minimizing its release from membranes has little or no enzymatic activity (0.2 pmol/mg/min). The activity of such preparation, however, can be restored by dephosphoryIation. Therefore, it is possible that the expression of 76 kDa protein PLA, activity is regulated by phosphorylationdephosphorylation and reversible association with the membranes. Such regulatory mechanism would require that the inactive cytosolic pool of PLA, (to prevent premature lysis of transport vesicles) should become activated upon dephosphorylation and binding to Golgi membranes. As shown by Weidman e? al. (1989), the membrane receptor and other cytosol derived components are involved in the process of transport and fusion. The presence of specific 76 kDa PLA, receptors on the Golgi membranes would thus guarantee the vectorial movement of the synthesized protein cargo from ER to Golgi, although the sequential intra-Golgi movement could not be as readily explained. Perhaps at the Golgi stage, the composition and complexity of this transport vesicle membrane may also be a directing force in the vectorial progression of the transport. Our identification of some of the components constituting the coat of ER transport vesicles (Slomiany et al., 1990) and PLA, activity of the protein supporting and promoting transport and fusion provide further insight into the ER-Golgi complex transport pathway. Generally, the results of our experiments are in total agreement with the data and the interpretation reviewed in (Balch, 1989; Rothman and Orci, 1989; Beckers et ai.. 1990). Several points, however, are extended further. For instance, the confusing effects of NEM action which were observed from the early stages of transport vesicles formation and assigned completely to protein assisting fusion are now clearly dissociated. The early events in transport vesicle formation are indeed NEM-sensitive, but as our results show they are independent of PLAz activity. At this stage, the ~ytidyltransferases responsible for the synthesis of membrane lipids and the vesicular growth are inactivated by NEM and therefore the vesicle formation is inhibited (Slomiany et al., 1990, 1991). In a Golgi-Golgi transport diagram (Rothman and Orci, 1989), the AlF; and NEM sensitivity are separated and shown as inhibitors of the steps IV and V in fusion process. In our opinion, the AlF; and NEM act on the same 76 kDa fusion promoting protein since the PLA, activity is completely abolished in presence of NaF and/or NEM. Since PLA, activity is directly related to LPC formation in the membrane, a membrane pore which is formed by local generation of LPC could result in a great increase in ~~eability of Ca’+. The infhtx of Ca’+ is known to destabilize the membranes, enhancing the preference of lipids for nonbilayer
1401
PLA2 and fusion of transport vesicles
31
21 (A)
(B)
(Cl
CD)
Fig. 1. Isolation and purification of the 76 kDa protein which promotes transport of apoprotein (apomucin) from ER to Go&i from rat gastric mucosal tissue. From right to left: (A) the SDS-PAGE of the proteins eluting in the major peak of S-Sepharose column (*) and the fraction which promotes transport (*). (B) Fraction 12-14 from the S-Sepharose column enriched with protein promoting transport. (C) The active fraction recovered after ultra~itration on ~tri~on-1~ and (D) shows the purified 76 kDa protein which promotes the apomucin transport from ER to Goigi. The proteins were separated on 10% SDS-PAGE and stained with silver.
1402
AMALIA SLOMIANYet af.
Golgi
Inactivated
PLA,
(pg/ml)
Golgi +
+ PLA,
(pg/ml)
17.0
13.6
0 r-
x
10.2
d
‘E n 0
6.8
Fig. 2. Effect of 76 kDa protein (PLA,) on the fusion of ER transport vesicles with Goigi. From left, purified transport vesicles labeled with [r4C]oleic acid (1.7 x 10e4 cpm/sample) were subjected to fusion under control optimal conditions and in presence of the additional 2 lug/ml of PLA,. The experiments depicted in the remaining cofumns were performed with inactivated Golgi membranes and cytosol(1 mM NEM at 4°C for 30 min followed by the addition of 2 mM DTT) and increasing amount (o-4 pgjml) of the purified PLA, protein which was added to Golgi membranes before the transport vesicles and cytosoi were introduced into the mixture. Results are expressed as the amount of radiolabel incorporated into Golgi membranes as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and in the amount of apomucin transferred from ER vesicles to Golgi cisterneas (bottom panel). From each experiment depicted in the figure one half of Golgi membranes was used for quantitation of phospholipids and the other half was subjected to immunoprecipitation with antimucin lH7 MAb. The precipitates of apomucin were then subjected to 10% SDS-PAGE and silver staining.
1403
PLA, and fusion of transport vesicles
i=FA-
PC-
1
2
Fig. 3. Generation of free fatty acids (FFA) during incubation of the 76 kDa protein with PC. Purified 76 kDa protein was incubated with l-U-palmitoyl-2-[~H]oieoyl-sn-glycerolphosphocholine. The left panel (1) depicts radiolabeled PC and FFA, and the right panel (2) shows the FFA in hexane extract from incubation mixture which contained 80 ng of 76 kDa protein and 100,000 cpm of PC. The standards and the hexane extract were chromatographed on HPTLC in solvent mixture consisting of chloroform/methanol/water (6.5:3.5:8, v/v/v) and then subjected to fluorography (5 days).
.
F
PC
LPC
0
Fig. 4. Analysis of the products of PC hydrolysis released from ~-~-pa~mitoyl-2-~3HJo~eoy~-s~-g~yceroiphosph~hol~ne for the presence of lysophosphatidylchol~ne (LPC). The incubatjon mixture containing 80 ng 76 kDa phospholipase and 100,000 cpm PC substrate was extracted with acidified hexane (to remove free fatty acids as in Fig. 2) and then subjected to HPTLC in solvent system consisting of chloroform/methanol/water (65: 35: 8, v/v/v) and scanning in Berthold Radioactivity Analyzer. As shown, the 76 kDa protein did not contain phospholipase A, activity.
1404
AMALIA SLOMIANY et nl.
Fig. 5. Phosphorylation of the 76 kDa PLA, with [y-‘*P]ATP in the presence of CAMP-dependent protein kinase. The lane 1 depicts 200 ng of purifwd PLA,, dephosphorylated with 20 units of alkaline phosphatase and phosphorylated with 25 units of pure c-AMP-dependent protein kinase in presence of 0.1 mM [Y-~*P]ATP for 30min at 37°C. The mixture was passed through a Q-Sepharose column to separate CAMP-dependent protein kinase and the lane 2 shows the same preparation of PLA, without prior dephosphorylation. Both samples were subjected to 10% SDS-PAGE and scanning using Berthold Radioactivity Analyzer. The number shown reflects cpm reduced by factor of 10.
PLA, and fusion of transport vesicles Table I. Phospholipase A, activity of the 76 kDa protein promoting apomucin transport and ER vesicles fusion with Golgi Source of the 76 kDa protein or the treatment I. 2. 3. 4. 5. 6. 7.
Isolated from cytosol and from membranes Isolated from cytosol Stripped from membranes Subjected to phosphorylation Dephosphorylated Treated with I mM NaF Treated with 1 mM NEM
PLA, activity (~mol~mg/min) 3.28-22.06 (IO) 0.22-1.50 (II) 32.9472.78 (6) 2.39-3.39 (3) 77.28-82.10 (2) 0 (3) O.l(M.12 (2)
The numbers in parentheses indicate the number of the experiments performed. The activity is expressed in Fmol of LPC generated from l,2-diacyl-sn~glycerol-phospho~‘4C]cholioe substrate per mg PLA, protein per min.
(hexagonal, H,,) organization (Cullis et al., 1985). This instabitity could be relieved by the formation of inverted miceller or inverted cylindrical structures with the outer monolayers of closely opposed membranes of the vesicle and the Golgi, thus initiating the fusion process. This model would be compatible with the role of phospholipases in the regulation of Ca2+ fluxes related to the membrane fusion and intraorganeIlar calcium pool only. Some evidence for the key role of Ca*+ in the finaI step of ER to Golgi transport has been provided by analysis of the transport in oitro in the presence of Ca*+ chelator EGTA (Beckers and Balch, 1989; Balch, 1990). Further experiments are. however, required to establish whether the intravesicular Ca2+ piays the pertinent role in the fusion event that follows the formation of lysophospholipids. The isolated cytosolic PLA, is of a high molecular weight as compared to known membrane bound phospholipase A2 enzymes, most of which are of 14-18 kDa (Wong and Dennis, 1990). Only few examples of phosphohpases similar in size to the fusion promoting PLA, were reported (Hazen et al., 1990; Diez and Mong, 1990; Gassama-Diagne et al., 1989; Kramer er ai., 1989, 1991). One particularly close is a - 70 kDa arachidonoyl-hydroly~ng phospholipase A, isolated from macrophages (Leslie et al., 1988). It is interesting to note that like the PLA, described here, the macrophage derived phospholipase is also highly labile. It is possible that just as the fusion promoting PLA,, the macrophage derived enzyme is inactive in its phosphorylated form and its activity is regulated by phosphorylationdephosphorylation mechanism and the reversible association with membrane. Acknowledgements-This
was supported work by ADAMHA and NIH grants from NIAAA No. AA05858-10 and from NIDKD No. DK21684-15. REFERENCES
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Goda Y. and Pfeffer S. R. (1989) Cell-free systems to study vesicular transport along the secretory and endocytic pathways. FASEB J. 3, 2488-2495. Hazen S. L., Stuppy R. J. and Gross R. W. (1990) Purification and characterization of canine myocardial cytosolic phospholipase A,. J. biol. Chem. 265, 10,622-~0,630.
Kramer R. M., Roberts E. F., Manetta J. and Putnam J. E. (1991) The Ca2+-sensitive cytosolic phospholipase A, is a 100 kDa protein inhuman monoblast U937 cells. J. biol. Chem. 266, 5268-5272.
Kramer R. M., Hession C., Johansen B., Hayes G., McGray P.. Chaw E. P., Tizard R. and Pepinsky R. B. (1989) Structure and properties of a human non-pancreatic phosphohpase A,. J. biol. Chem. 269, 5768-5775. Leslie C. C., VoeIker D. R., Channon J. Y., Wall M. M. and Zelarney P. T. (1988) Properties and purification of an arachidonyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7. B&him. biophys. Arta 963, 476492.
Malhotra V., Serafini T., Orci L., Shepherd 3. C. and Rothman J. E. (1989) Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stock. Cell 58, 329-336. Rothman J. E. and Orci L. (1990) Movement of proteins through the Golgi stock: a molecular dissection of vesicular transport. FASEB J. 4, 1460-1468.
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Slomiany B. A., Slomiany B. L. and Slomiany A. (1990) Dissection of vesicular transport in gastric epithelial cells. Mechanism and underlying basis for vesicles formation. Gastroenterology 100, A842. Slomiany A., Kasinathan C., Slomiany B. A. and Slomiany B. L. (1991) The mechanism of NSF action in fusion of ER transport vesicles with Golgi. Gastroenterology 100, A704. Slomiany A., Tamura S., Grzelinska E., Piotrowski J. and Slomiany B. L. (1992) Mucin complexes: characterization
of the “link” component of submandibular mucus glycoprotein. Int. J. Biochem. 24, 1003-1015. Weidman P. J., Melancon P., Block M. R. and Rothman J. E. (1989) Binding of an N-ethylmaleimide sensitive fusion protein to Golgi membranes requires both a soluble protein(s) and an integral membrane receptor. J. Ceil Eiol. 108, 1589-l 596. Wong P. W.-K. and Dennis E. A. (Editors) (1990) Phospholipase A,. Role and Function in InJammation, pp. l-191. Plenum Press, New York.
Vol. 24, No. 9, pp. 1397-1406. Printed in Great Britain. All rights reserved
0020-711X/92$5.00+ 0.00 Copyright 0 1992Pergamon Press Ltd
1992
FUNCTION OF INTRACELLULAR PHOSPHOLIPASE A, IN VECTORIAL TRANSPORT OF APOPROTEINS FROM ER TO GOLGI AMALIA SLOMIANY,*
EWA
GRZELINSKA, CHINNASWAMY KASINATHAN, KEN-ICHIRO YAMAKI, DANUTA PALECZ and BRONISLAWL. SLOMIANY
Research Center, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2400, U.S.A. [Tel. (20 1) 456-70531 (Received 13 January 1992) The cytosolic fraction required in in airro reconstituted intracellular transport of mucus glycoprotein apopeptide (apomucin) was isolated and its potential as transport supporting factor assessed by the quantitation of the gastric apomucin transferred to Golgi. 2. The experiments with the fraction promoting transport and delivery of apomucin to Golgi revealed that the active protein has the property of phospholipase A, (PLA,) which assists ER vesicles fusion with Golgi. 3. The ability of the 76 kDa PLA, to hydrolyze phospholipids and to support transport and fusion of ER vesicles with Golgi was abolished by phosphorylation and regained foIlowing dephosphorylation. 4. The data provide evidence that 76 kDa intracellular PLA, is responsible for the fusion of ER-transport vesicles with Golgi. The process of fusion is accomplished by generation of lysophospholipids in fusing membranes.
Abstract-l.
The movement
of proteins destined for the cell surface or the organelles distal to endoplasmic reticulum
(ER) is carried out by a vesicular transport (Balch, 1989; Goda and Pfeffer, 1989; Rothman and Orci, 1990). The molecular mechanism involved in this complex transport pathway has been studied in semiintact cells and cell-free systems and a number of specific protein and factors were found to be involved in the vesicle formation and fusion (Glick and Rothman, 1987; Baker er al., 1988; Block et al., 1988;
Nakamo and Muramatsu, 1989; Beckers et al., 1990; Balch, 1990). Considerable progress in the i~ vilro reconstituted transport was achieved by utilization of cytosol derived factors, which are essential for preparation of the transport vesicles for fusion (Block et al., 1988; Diaz er al., 1989). Evidence suggests that the cytosol derived fusion factors are required for the transport vesicles to lose their cytoplasmic coat, to attach to the Golgi receptor membrane and to fuse with the membrane. The involvement of these cytosol derived protein factors in the final events preceding fusion of the Golgi attached vesicles, implies that the proteins might contribute to destabilization of the membranes lipid bilayer. The investigation of the *To whom correspondence should be addressed at: Research Center, UMDNJ-NJ Dental School, University Heights, 110 Bergen Street, Newark, NJ 07103-2400, U.S.A.
cytosolic fusion promoting fraction for its phospholipid Iytic activity led us to identification of the protein which assisted transport, promoted fusion of the ER-transport vesicles with Golgi and displayed phospholipase A, (PLA,) activity. We propose that the fusion of ER transport vesictes carrying cargo to Golgi (acceptor) membranes is accomplished with the aid of 76 kDa cytosohc PLA, which locally increases the level of lysophospholipid fusogens in uniting membranes. MATERIALS
AND METHODS
Materials [5,6,8,9,11,12,14, 15-3H]Arachidonic acid (95 Cij mmol), I-palmitoyl,2-[l-‘4C]arachidonoylphosphatidylcholine (55 mCi/mmol), I-palmitoyl-,2-[l-‘4C]oleoylphosphatidylcholine (55 mCi/mmol), I-palmitoyl-2-[3H]palmitoyl phosphatidylcho~ine (53 Ci/mmol), [‘~qphosphocholine (50 mCi/mmol) were from New England Nuclear (Boston, Mass.), or from Amersham Corp. (Arlington Heights, III.). Phospholipid standards of dipalmitoyl- and I-palmitoyl2-arachidonoylphosphatidylcholine were from Avanti (Birmingham, Ala). NEM, BSA, soybean trypsin inhibitor, polyethyleneglycol 4000, creatine phosphokinase, PMSF, pepstatin, leupeptin, ATP, GTP, CTP, creatine phosphate, fatty acid CoA and sucrose were purchased from Sigma Chemicals (St Louis, MO.). Polyacrylamide gel eiectrophoresis reagents and gels for the Phastsystem were from LKBPharmacia (Piscataway, N.J.), and for mini slab gels from Bio-Rad (Rockville Centre, N.Y.). All other reagents were of highest purity and were purchased from T.T. Baker N.J.), Fisher Scientific Chemical Co. (Phillipsburg,
1397
1398
AMALIASLOMIANY
(Springfield, N.J.) and VWR Scientific (Piscataway, N.J.). The antimucin monoclonal antibodies were raised in our laboratory (Sfomiany er al., 1992). In this study. the lH7 MAb recognizing apomucin protein core was empfoyed. Subcellular fractionation: preparation of endopiasmic reriru lum membranes, Golgi membranes, cytosol and generation of the transport vesicles
Crude homogenate of rat gastric mucosa was prepared using 12 strokes of tight-fitting Dounce homogenizer, whereas rat liver required 20 strokes to break 90% of the cell in 20% tissue~buffer suspensions. In standard preparation 100 g of the tissue was minced and homogenized in 400 ml of ice-cold 10 mM HEPES-KOH. pH 7.4 buffer containing 0.5 M sucrose and 5 mM EDTA. The unbroken cells and nuclei were removed by centrifugation at 3000rpm for IO min and the supernatant was layered over 1.35 M sucrose in IO mM HEPES-KOH, pH 7.4 buffer, and centrifuged at 85,000 g for 90 min. The crude membrane fraction at the interphase with the I.35 M sucrose was removed, adjusted to 1.2 M sucrose and 1 volume of this membrane fraction was overlaid with 1 volume of 1.1 M sucrose, 1 volume of 1.0 M sucrose and 0.5 volume of 0.5 M sucrose (all sucrose solutions were prepared in 1OmM HEPES-KOH, pH 7.4 buffer) and centrifuged at f~,O~g for 2.5 hr. The Golgi membranes were recovered from OS/f.0 M sucrose interface and endoplasmic reticulum from 1.2 M sucrose. Both membrane preparations were centrifuged once again in the discontinuous sucrose gradient. The Golgi membranes were subjected to the sucrose gradient described above. whereas ER membranes were diluted to 0.5 M sucrose and overlayered onto gradient consisting of 2.0, 1.5, 1.3M sucrose and centrifuged at 85,000 g for 90 min. The membranes from 1.3/0.5 M sucrose interface were withdrawn, pelleted by centrifugation at 70,000 g for 20 min and stored at - 8O’C. Thus prepared membranes were 2-3% cross contaminated with each other and did not contain mitochondria! membranes, as judged from electron microscope examination of the fractions. The purity of the Gofgi and ER were assessed using antimucin monoclonal antibodies recognizing apomucin (lH7 MA\, specific for ER contained peptides), and glycosylated mucin (3G7 MAb recognizing GlcNAcGalNAc-O-apomucin structure assembled in Golgi). The ER derived transport vesicles were generated using modified procedure described in Mafhotra et ai. (1989) in the presence of [‘4qphosphocholjne and [“Hlofeic acid (0.005 pCi,/pi). The incubation mixture consisted of 10 mg/mf rat gastric mucosa! ER membranes, 50 mg/m! rat liver cytoso! supplemented with 0.3 mM free fatty acids, 0.4mM CoA (for [“‘CJphosphocholine labeling) or with 0.3 mM phosphocholine and 0.4 mM CoA (for fatty acid labeling experiments), 10 U/ml creatine phosphokinase 2mM creatine phosphate, 0.7 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.2 mM DTT, 20mM GTP in 33 mM HEPES-KOH, pH 7.0 buffer containing 33 mM KC1 and 2.5 mM Mg acetate. After 30 min at 37°C the incubation was terminated and transport vesicles purified. The isolated vesicles were subjected to fusion with Golgi in the presence of the cytosof enriched with fusion promoting fraction or purified 76 kDa protein. The afiquots of the vesicles and Gofgi membranes recovered after fusion were extracted with chloroform/methanol (2: 1, v/v) and the radiolabeled lipids identified and quantitated. The aliquots of the delipidated residues were used for SDS-PAGE, and immunoquantitation of the apomucin present in the transport vesicles and that delivered to Gofgi.
et al.
Isolation and purt$cation of the transport and fusion promot ing fraction from gastric mucosal cells
To obtain the gastric mucosal cell cytosof, the homogenate of 10 rat stomach mucosa1 tissue in 0.25 M sucrose, 50 mM Tris-HC! pH 7.4, 25 mM KC!, 5 mM Mg acetate, 5 mM mercaptoethano! and 1 mM PMSF was centrifuged at 10,000 g for 20 min. The supernatant was filtered and centrifuged at 25,000 rpm for 60 min in 45 Ti rotor. The soluble fraction, the “cytoso!“, was admixed with 2 mM dithiothreitof and 5 mM ATP, pH of the solution adjusted to 7.0 and 50% PEG-4000 was added dropwise until 8% concentration was reached. The mixture was incubated on ice for 30 min and the precipitated protein was recovered by centrifugation at !O,OOOgfor 15 min. The pellet was solubilized in buffer consisting of 20mM PIPES-KOH, pH 7.0, IOOmM KC!. 2 mM MgClz, 2 mM DTT and 0.5 mM ATP and applied to DEAE*el!ufose (2 x 20 cm) column. The unbound fraction was fractionated further on S-Sepharose (1 x !Ocm) column. The major protein fraction eluted from S-Sepharose column in first 32236ml volume (Fr. g--9), and the fusion promoting activity was recovered with 48. 56 ml (Fr. 12-14) of the elutate. The fraction containing fusion promoting activity were adjusted to 5% with respect to glycerol and concentrated using Centricon ultrafiltration membrane with mofecuiar cut oB of 100 kDa. Final puri~~tion of the fusion promoting protein was achieved by eiectroefution from ND-PAGE gel, and determined that only 76 kDa protein displayed fusogenic activity. Fusion of the ER transport vesicles with Go/gi
Radiolabeled transport vesicles (10 pg or 10,000 cpm) and 20 pg Golgi membranes were incubated for 30 min at 37-C in the presence of 250 pg cytoso!, and suppiementa! mixture detailed under transport vesicles generation and 5 pg of the transport and fusion promoting cytosolic proteins obtained from consecutive fraction of S-Sepharose column, or 20-80ng of the purified 76 kDa protein promoting the fusion of the vesicles with Gofgi. The incubation was stopped by 1 mM NEM and the mixture was layered on discontinuous sucrose gradient consisting of 1.0 and 0.5 M sucrose in 10 mM HEPESKOH, pH 7.4 buffer, and centrifuged at 100,000g for 2.5 hr. The Golgi membranes were recovered from l.OjO.5 M interface, diluted with 25 mM HEPES-KOH, pH 7.0, 250 mM KC! and 2.5 mM Mg acetate and centrifuged at l~,~g for 30 min. The Gofgi membranes were suspended once again in this stripping buffer (to remove associated but not fused ER transport vesicles), centrifuged and then subjected to analysis for the content of radiolabeled phospholipids and the amount of transferred apomucin. In order to measure the internafization of the transport apomucin and incorporation of the vesicular membrane into membrane continuum of the Golgi, first the externally associated components present on the vesicles and the Golgi were degraded with trypsin (100 pg/m!, 2 hr at 0°C). and then the fractions were subjected to identification and quantitation of radiolabeled phospholipids and immunoquantitation of apomucin. ~huspho~~p~e assay
The standard phosphohpase assay measured the amount of radiolabeled oleic acid hydrolyzed from I-0-pafmitoyf-2[3H]o!eoyfgfycerofphosphocho!ine or radiolabeled LPC from 1,2-diacyl-sn-glycerol phospho[‘4C]chofine. Organic solvents were removed from the phospholipid substrate under a stream of Nlr resuspended in 0.2 M Tris-HCi buffer
PLA,
and fusion
(pH 7.0) and then sonicated for 5 min. The assay mixture contained 50 mM Tris-HCI buffer, pH 7.0, 0.3 FM CaCI,, 20 fig human serum albumin (delipidated) and PC substrate (100,000 cpm/tube) in a final volume of 50~1. The reaction
was started by the addition of purified PLA, (2&80ng) protein and incubated at 37°C for 30 min. The reaction was stopped with 1.1 ml of chloroform/methanol/O.5 M HCl (20: 4: 1, v/v/v) which were followed by the addition of 1ml of hexane and 0.5 ml of water. After mixing, the hexane phase was separated and subjected to free fatty acid quantitation, whereas the chloroform/methanol phase, after concentration, and addition of 5 pg lysophosphatidylchohne/sphingomyehn/phosphatidylcholine carrier mixture was applied to thin layer plate (20 x IOcm, Silica Gel G). Usually, all samples were applied on the same plate and separated from each other by scouring the silica gel and the plates were developed in chloroform/methanol/water (65:35:8, v/v/v). The amount of radioactivity was determined by radioscanning in Berthold Radioactivity Analyzer and by scraping the area corresponding to LPC, PC and FFA and subjecting the silica gel to liquid scintillation spectroscopy. PLA,
dependentfusion
Effect of PLAz on ER transport vesicles fusion was measured using NEM-treated Golgi membranes and cytosol (to inactivate the intrinsic transport and fusion activity) and adding t&4 pg/ml of the purified transport and fusion promoting 76 kDa protein. In this assay, the amount of apomucin transferred from the vesicles to the Golgi derived from liver was measured and the radiolabeled phosphatidylcholine and phosphatidylethanolamine incorporated into the Golgi membranes was quantitated. Under normal control conditions where transport activity was not inactivated and purified 76 kDa PLA, was not added, the fusion of ER transport vesicles with Golgi afforded incorporation of 850&10,000 cpm of phosphohpids/sample and transported 81-93 ng of apomucin. The mock controls with apomucin and phospholipids (equivalent to the amount present in the vesicles) used in the fusion experiments instead of ER transport vesicles gave background value of 350-700 cpm, and were subtracted from the total cpm incorporated into the presence of transport vesicles. RESULTS
Cytosolic protein dependent apomucin transport Fusion potential of the proteins isolated from the cytosol of gastric mucosal tissue was expressed by the fractions 12-14 of the S-Sepharose column (Fig. 1). On the SDS-PAGE, the active fractions consisted of 8-10 protein bands in the molecular range of 47-160 kDa. After Centriconfiltration, the 6 protein bands of 97-160 kDa were retained on the filter, while the filtrate contained 47 and 76 kDa proteins, and the total transport promoting activity of the fraction subjected to the procedure. The separation on ND-PAGE allowed to separate the 2 remaining proteins and to conclude that the transport-related activity was associated with 76 kDa protein, while the 47 kDa component was not active in the transport assays. The addition of 76 kDa protein (2 pg/ml) to the unmodified in uitro transport assay mixture increased fusion by IS-20% (Fig. 2). Upon
of transport
vesicles
1399
transport inactivation with 1 mM N-ethylmaleimide (NEM), the fusion of ER transport vesicles with Golgi was proportional to the amount of 76 kDa protein added to the incubation mixture and the maximum effect was achieved with 24pg/ml of the protein. At the optimal concentration of the fusion promoting protein up to 65% of the radiolabeled vesicles fused with Golgi. Based on the isolated proteins activity in promoting ER vesicles fusion with Golgi, the function of the 76 kDa protein was established as the one promoting fusion. Phospholipase activity and substrate specificity of 76 kDa cytosolic protein
positional
The experiments with 76 kDa fusion promoting protein incubated with I-palmitoyl,2-[3H]oleoyl-snglycerol phosphocholine revealed phospholipid lytic activity of the protein as evidenced by generation of radiolabeled free fatty acids (Fig. 3). Since there was no phosphocholine or choline generated, the measured activity was thus specific for phospholipases A. To determine the positional lytic specificity of the 76 kDa protein, the radiolabeled products formed from the hydrolysis of 1-pa1mitoyl,2-[3 HIpalmitoylphosphatidylcholine, I-palmitoy1,2-[i4C]arachidonoylphosphatidylcholine and I-palmitoyl,2[3Hloleoyl phosphatidylcholine were analyzed by HPTLC. Under the standard phospholipase assay conditions (60 ng of 76 kDa protein and 30 min incubation), the only radiolabeled products found were [3H]palmitic acid, [‘4C]arachidonic acid or [3H]oleic acid. With sn-2 fatty acid labeled phosphatidylcholine, there was no evidence of the generation of radiolabeled lysophosphatidylcholine (Fig. 4), thus suggesting that the protein has PLA, activity. Comparison of 1,2-dipalmitoyl, 1-oleoyl-2-arachidonoyl-sn-glycerolphosphocholine and I-palmitoyl2-oleoyl-sn-glycerol phosphocholine consistently showed highest activity of the 76 kDa protein against sn-2 unsaturated long chain fatty acyl residues of the substrates. Effect of NEM and phosphorylation on PLA, activity of the 74 kDa protein The studies on cytosolic 76 kDa protein and the protein dissociated by mild salt extraction from the intracellular membranes indicated that this PLA, appears in 2 forms which differ in specific lipolytic activity. The cytosol derived PLA, displays minimum phospholipid lytic activity 0.2-l .5 pmol/mg/min, whereas the PLA, dissociated from the membranes displayed specific activity in range of 33-73 pmol/ mg/min. The experiments with CAMP dependent protein kinase and [y -32P]ATP suggested that the cytosolic form might be phosphorylated, since with phosphorylation the membrane derived PLA, activity was drastically diminished, whereas dephosphorylation of PLA, generated active enzyme again (Fig. 5 and Table I). Treatment of the isolated PLA, with I mM NEM abolished completely its phospholipid
1400
AMALIA SLOMIANY et al.
lytic potential, while incorporation of NEM into the fusion incubation mixture (Fig. 2) abolished apomucin transport from ER vesicles to Golgi. This, together with the fact that NEM-inhibited transport is restored with addition of 76 kDa PLA,, strongly suggests that this intracellular phospholipase is engaged in fusion of transport vesicles with Golgi and delivery of protein cargo to Golgi cisternae. DISCUSSION
The 76 kDa protein was purified based on its ability to support the transport between the ER and Golgi compartment in which the transport of gastric apomucin to liver derived Golgi was measured. The activity of the protein was assessed by determining the amount of radiolabeled vesicles fused with Golgi and the amount of apomucin transferred to Golgi compartments. The fact that Golgi derived from liver is totahy free of mucin, allowed us to estimate the net transport by immunoquantitation of the apomucin within Golgi. The experiments conducted with mock incubates and trypsin treated Golgi preparations showed that the mucin was indeed internalized, and that the radiolabeled transport vesicles fused with Golgi membrane. Had the fusion not taken place, the recovered vesicles would have still contained their transport cargo. On the other hand, if the apomucin and radiolabeled phosphatidylcholine were added to incubation mixture in a free form, neither the apomucin nor the radiolabeled phospholipids were detectable in the Golgi membranes, and upon trypsin treatment the apomucin was degraded. Hence, our conclusion was that the isolated 76 kDa protein is capable to support transport. Although other proteins are known for their potential to assist the transport vesicles fusion, their exact function in the fusion event is still unknown. It remains unexplored whether fusion assisting proteins are capable to transform the lipid components of the involved membranes into potential fusogens such as lysophospholipids. Our data show that when fusion promoting protein is incubated with vesicles labeled in phos~~atidylcholine, the phosphol~pid is degraded and lysophosphatidyl~holine and free fatty acids are formed. The evidence obtained with sn-2 saturated, unsaturated and arachidonic acid containing phosphatidylcholine species provides strong support that the active component of the fraction promoting fusion is a PLA, enzyme. The ability of the 76 kDa protein to release the labeled fatty acids or generate lysophospholipids in the transport vesicles and lack of any changes when the fusion is abolished with NEM, suggest that the lypolytic activity of the 76 kDa protein is directly related to its fusion potential. PLA, activity of 76 kDa protein is phospho~lation sensitive, and exhibits optimum at pH 7.4. The level of PLAz activity detectable in the preparations is to a large extent dependent upon the amount of the
protein released from the membrane. Our results indicate that cytosolic PLAz prepared under the conditions minimizing its release from membranes has little or no enzymatic activity (0.2 pmol/mg/min). The activity of such preparation, however, can be restored by dephosphoryIation. Therefore, it is possible that the expression of 76 kDa protein PLA, activity is regulated by phosphorylationdephosphorylation and reversible association with the membranes. Such regulatory mechanism would require that the inactive cytosolic pool of PLA, (to prevent premature lysis of transport vesicles) should become activated upon dephosphorylation and binding to Golgi membranes. As shown by Weidman e? al. (1989), the membrane receptor and other cytosol derived components are involved in the process of transport and fusion. The presence of specific 76 kDa PLA, receptors on the Golgi membranes would thus guarantee the vectorial movement of the synthesized protein cargo from ER to Golgi, although the sequential intra-Golgi movement could not be as readily explained. Perhaps at the Golgi stage, the composition and complexity of this transport vesicle membrane may also be a directing force in the vectorial progression of the transport. Our identification of some of the components constituting the coat of ER transport vesicles (Slomiany et al., 1990) and PLA, activity of the protein supporting and promoting transport and fusion provide further insight into the ER-Golgi complex transport pathway. Generally, the results of our experiments are in total agreement with the data and the interpretation reviewed in (Balch, 1989; Rothman and Orci, 1989; Beckers et ai.. 1990). Several points, however, are extended further. For instance, the confusing effects of NEM action which were observed from the early stages of transport vesicles formation and assigned completely to protein assisting fusion are now clearly dissociated. The early events in transport vesicle formation are indeed NEM-sensitive, but as our results show they are independent of PLAz activity. At this stage, the ~ytidyltransferases responsible for the synthesis of membrane lipids and the vesicular growth are inactivated by NEM and therefore the vesicle formation is inhibited (Slomiany et al., 1990, 1991). In a Golgi-Golgi transport diagram (Rothman and Orci, 1989), the AlF; and NEM sensitivity are separated and shown as inhibitors of the steps IV and V in fusion process. In our opinion, the AlF; and NEM act on the same 76 kDa fusion promoting protein since the PLA, activity is completely abolished in presence of NaF and/or NEM. Since PLA, activity is directly related to LPC formation in the membrane, a membrane pore which is formed by local generation of LPC could result in a great increase in ~~eability of Ca’+. The infhtx of Ca’+ is known to destabilize the membranes, enhancing the preference of lipids for nonbilayer
1401
PLA2 and fusion of transport vesicles
31
21 (A)
(B)
(Cl
CD)
Fig. 1. Isolation and purification of the 76 kDa protein which promotes transport of apoprotein (apomucin) from ER to Go&i from rat gastric mucosal tissue. From right to left: (A) the SDS-PAGE of the proteins eluting in the major peak of S-Sepharose column (*) and the fraction which promotes transport (*). (B) Fraction 12-14 from the S-Sepharose column enriched with protein promoting transport. (C) The active fraction recovered after ultra~itration on ~tri~on-1~ and (D) shows the purified 76 kDa protein which promotes the apomucin transport from ER to Goigi. The proteins were separated on 10% SDS-PAGE and stained with silver.
1402
AMALIA SLOMIANYet af.
Golgi
Inactivated
PLA,
(pg/ml)
Golgi +
+ PLA,
(pg/ml)
17.0
13.6
0 r-
x
10.2
d
‘E n 0
6.8
Fig. 2. Effect of 76 kDa protein (PLA,) on the fusion of ER transport vesicles with Goigi. From left, purified transport vesicles labeled with [r4C]oleic acid (1.7 x 10e4 cpm/sample) were subjected to fusion under control optimal conditions and in presence of the additional 2 lug/ml of PLA,. The experiments depicted in the remaining cofumns were performed with inactivated Golgi membranes and cytosol(1 mM NEM at 4°C for 30 min followed by the addition of 2 mM DTT) and increasing amount (o-4 pgjml) of the purified PLA, protein which was added to Golgi membranes before the transport vesicles and cytosoi were introduced into the mixture. Results are expressed as the amount of radiolabel incorporated into Golgi membranes as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and in the amount of apomucin transferred from ER vesicles to Golgi cisterneas (bottom panel). From each experiment depicted in the figure one half of Golgi membranes was used for quantitation of phospholipids and the other half was subjected to immunoprecipitation with antimucin lH7 MAb. The precipitates of apomucin were then subjected to 10% SDS-PAGE and silver staining.
1403
PLA, and fusion of transport vesicles
i=FA-
PC-
1
2
Fig. 3. Generation of free fatty acids (FFA) during incubation of the 76 kDa protein with PC. Purified 76 kDa protein was incubated with l-U-palmitoyl-2-[~H]oieoyl-sn-glycerolphosphocholine. The left panel (1) depicts radiolabeled PC and FFA, and the right panel (2) shows the FFA in hexane extract from incubation mixture which contained 80 ng of 76 kDa protein and 100,000 cpm of PC. The standards and the hexane extract were chromatographed on HPTLC in solvent mixture consisting of chloroform/methanol/water (6.5:3.5:8, v/v/v) and then subjected to fluorography (5 days).
.
F
PC
LPC
0
Fig. 4. Analysis of the products of PC hydrolysis released from ~-~-pa~mitoyl-2-~3HJo~eoy~-s~-g~yceroiphosph~hol~ne for the presence of lysophosphatidylchol~ne (LPC). The incubatjon mixture containing 80 ng 76 kDa phospholipase and 100,000 cpm PC substrate was extracted with acidified hexane (to remove free fatty acids as in Fig. 2) and then subjected to HPTLC in solvent system consisting of chloroform/methanol/water (65: 35: 8, v/v/v) and scanning in Berthold Radioactivity Analyzer. As shown, the 76 kDa protein did not contain phospholipase A, activity.
1404
AMALIA SLOMIANY et nl.
Fig. 5. Phosphorylation of the 76 kDa PLA, with [y-‘*P]ATP in the presence of CAMP-dependent protein kinase. The lane 1 depicts 200 ng of purifwd PLA,, dephosphorylated with 20 units of alkaline phosphatase and phosphorylated with 25 units of pure c-AMP-dependent protein kinase in presence of 0.1 mM [Y-~*P]ATP for 30min at 37°C. The mixture was passed through a Q-Sepharose column to separate CAMP-dependent protein kinase and the lane 2 shows the same preparation of PLA, without prior dephosphorylation. Both samples were subjected to 10% SDS-PAGE and scanning using Berthold Radioactivity Analyzer. The number shown reflects cpm reduced by factor of 10.
PLA, and fusion of transport vesicles Table I. Phospholipase A, activity of the 76 kDa protein promoting apomucin transport and ER vesicles fusion with Golgi Source of the 76 kDa protein or the treatment I. 2. 3. 4. 5. 6. 7.
Isolated from cytosol and from membranes Isolated from cytosol Stripped from membranes Subjected to phosphorylation Dephosphorylated Treated with I mM NaF Treated with 1 mM NEM
PLA, activity (~mol~mg/min) 3.28-22.06 (IO) 0.22-1.50 (II) 32.9472.78 (6) 2.39-3.39 (3) 77.28-82.10 (2) 0 (3) O.l(M.12 (2)
The numbers in parentheses indicate the number of the experiments performed. The activity is expressed in Fmol of LPC generated from l,2-diacyl-sn~glycerol-phospho~‘4C]cholioe substrate per mg PLA, protein per min.
(hexagonal, H,,) organization (Cullis et al., 1985). This instabitity could be relieved by the formation of inverted miceller or inverted cylindrical structures with the outer monolayers of closely opposed membranes of the vesicle and the Golgi, thus initiating the fusion process. This model would be compatible with the role of phospholipases in the regulation of Ca2+ fluxes related to the membrane fusion and intraorganeIlar calcium pool only. Some evidence for the key role of Ca*+ in the finaI step of ER to Golgi transport has been provided by analysis of the transport in oitro in the presence of Ca*+ chelator EGTA (Beckers and Balch, 1989; Balch, 1990). Further experiments are. however, required to establish whether the intravesicular Ca2+ piays the pertinent role in the fusion event that follows the formation of lysophospholipids. The isolated cytosolic PLA, is of a high molecular weight as compared to known membrane bound phospholipase A2 enzymes, most of which are of 14-18 kDa (Wong and Dennis, 1990). Only few examples of phosphohpases similar in size to the fusion promoting PLA, were reported (Hazen et al., 1990; Diez and Mong, 1990; Gassama-Diagne et al., 1989; Kramer er ai., 1989, 1991). One particularly close is a - 70 kDa arachidonoyl-hydroly~ng phospholipase A, isolated from macrophages (Leslie et al., 1988). It is interesting to note that like the PLA, described here, the macrophage derived phospholipase is also highly labile. It is possible that just as the fusion promoting PLA,, the macrophage derived enzyme is inactive in its phosphorylated form and its activity is regulated by phosphorylationdephosphorylation mechanism and the reversible association with membrane. Acknowledgements-This
was supported work by ADAMHA and NIH grants from NIAAA No. AA05858-10 and from NIDKD No. DK21684-15. REFERENCES
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