lo cells; this increased to 183 pg/h/107 cells upon addition of pyruvate Ian J. Cartwright, Abdel-Malik Hebbachi, and Joan A.

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY 1993 by The Americn Society for Biochemietry nd Moleculr Biology, Inc. Vol. 268, No. 28, Ieue of Octobe~ 5, pp Printed in d. S. A. Trnsit nd Sorting of Apolipoprotein B within the Endoplsmic Reticulum nd Golgi Comprtments of Isolted Heptocytes from Norml nd Orotic Acid-fed Rts* (Received for publiction, April 9, 1993, nd in revised form, My 26, 1993) In J. Crtwright, Abdel-Mlik Hebbchi, nd Jon A. HigginsS From the Deprtment of Moleculr Biology nd Biotechnology, University of Sheffield, Sheffield SI 2UH, United Kingdom Apolipoprotein B (pob) secretion by isolted rt heptocytes ws dependent on ddition of olete to the incubtion medium nd inhibited in heptocytes isolted from livers of orotic cid-fed rts (OA heptocytes). To investigte the intrcellulr trnsit of newly synthesized pob under different conditions, norml heptocytes (with or without olete) or OA heptocytes (with olete) were incubted with [S6S]methionine, nd subcellulr frctions (rough endoplsmic reticulum, smooth endoplsmic reticulum,, nd trns- Golgi nd membrne nd lumenl contents from these) were isolted t intervls. The specific ctivities nd pool sizes of pobloo nd pob48 were determined. The observtions indicte tht there re severl points t which intrcellulr trnsit of pob is regulted. Newly synthesized pob is either trnslocted to the lumen of the rough endoplsmic reticulum or remins membrne bound nd is degrded. The lumenl pob is either retined nd degrded or trnsferred to the Golgi lumen nd secreted. In OA heptocytes degrdtion of the membrne-bound form of pob is inhibited, nd the protein ccumultes in the membrnes. Although pob is trnslocted to the lumen of the rough endoplsmic reticulum in OA heptocytes, it is not pckged with lipid nd is trnsferred to the Golgi lumen only slowly. Very low density lipoproteins (VLDL), the vehicle of trnsport of endogenous tricylglycerol in the plsm, consist of core of nonpolr lipids (tricylglycerol nd cholesterol ester) stbilized by n outer shell of mphipthic lipids (phospholipids nd cholesterol) nd protein. The mjor nonexchngeble protein component of VLDL is polipoprotein B (pob). Although it is essentil for secretion, pob does not behve s typicl secreted protein. Studies of rt liver, rbbit liver, nd HEP-G2 cells hve shown tht lrge frction of pob is bound to endoplsmic reticulum (1-8) nd Golgi membrnes (7-8) rther thn trnslocted to the lumen of the secretory comprtment. This membrne-bound form of pob *This work ws supported by grnts from the British Hert Foundtion. The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked dvertisement in ccordnce with 18 U.S.C. Section 1734 solely to indicte this fct. $ To whom correspondence should be ddressed Dept. of Moleculr Biology nd Biotechnology, University of Sheffield, P., Box 594, Firth Court, Sheffield S1 2UH, United Kingdom. The bbrevitions used re: VLDL, very low-density lipoproteins; pob, polipoprotein B; OA-heptocytes, heptocytes from orotic cid-fed liver; er, endoplsmic reticulum; rer, rough endoplsmic reticulum; ser, smooth endoplsmic reticulum; PAGE, polycrylmide gel electrophoresis. is not precursor of the lumenl pool (9), nd significnt mount of pob is degrded intrcellulrly (2,6,1-14). There is lso evidence from studies of HEP-G2 cells tht pob in the lumen of the endoplsmic reticulum my exist in two pools, only one of which is secreted (9). To exmine the interreltionships between the membrne bound, lumenl, secreted, nd degrded pools, we hve followed the intrcellulr trnsit of pob through the secretory comprtment of freshly isolted rt heptocytes using [35S] methionine s trcer. Heptocytes were incubted under conditions in which pob is secreted (plus olete), not secreted (minus olete), nd inhibited (heptocytes from orotic cidfed liver (OA heptocytes) (15-17)). Under ll conditions both the rdioctivity nd the mounts of pob were mesured, nd from this dt the specific ctivities of the intrcellulr pools of pob were determined. The results from these investigtions indicte tht there re severl intrcellulr sites t which pob trnsit through the secretory comprtment my be regulted. Newly synthesized pob is either trnslocted into the rough endoplsmic reticulum lumen or incorported into the membrne. The lumenl pob is either pckged with lipid nd secreted or retined in the rough endoplsmic reticulum membrne nd degrded. EXPERIMENTAL PROCEDURES Mterik-Sheep nti-humn pob ws purchsed from Boehringer Mnnheim, nd nti-sheep IgG coupled to lkline phosphtse ws from Sigm. Donkey nti-sheep IgG nd nonimmune sheep serum were purchsed from Scottish Antibodies, Lw Hospitl Crluke, Lnrkshire, United Kingdom. [35S]Methionine(cell lbeling grde) nd 251-protein A were purchsed from Amershm. All other chemicls were purchsed from Sigm. Animk-All experiments were performed on dult mle Sprgue- Dwley rts (15-3 g of body weight). These were housed in the University Animl House nd fed either stndrd rt chow pellets or for seven dys pellets prepred by mixing 1% orotic cid with powdered rt chow. Preprtion of Isolted Heptocytes-Heptocytes were isolted from livers of untreted (norml heptocytes) or orotic cid-fed rts (OA heptocytes) by perfusion with collgense in Krebs bicrbonte sline contining 1 mm glucose (18). In initil experiments the vibility of the cells ws demonstrted by the fct tht there ws no lekge of lctic dehydrogense, potssium, or ATP during incubtion. Isolted heptocytes synthesized glucose t rte of 53 Fg/h/ lo cells; this incresed to 183 pg/h/17 cells upon ddition of pyruvte (1 mm) nd/or lctte (1 mm) nd to 484 nd 5 rg/h/lo cells on ddition of glucgon or dibutyrylcyclic AMP, respectively. In most experiments isolted heptocytes were inspected by light microscopy, nd the vibility ws checked by the bility of cells to exclude.5% trypn blue. Incubtion of Heptocytes-Heptocytes were resuspended (5% w/v) in modified methionine-free Egle s minimum essentil medium, which hd been gssed with 95% oxygen, 5% CO,. 2-ml liquots were incubted in 5 ml of siliconized flsks, which were gssed t intervls before nd during the incubtion period. In most experiments

2 2938 Intrcellulr Trnsit of Apolipoprotein Heptocytes B in TABLE I Distribution of mrker enzymes in subcellulr frctions prepred from rt heptocytes Heptocytes were prepred from untreted nd orotic cid-fed rts, subcellulr frctions were prepred, nd specific ctivities of mrker enzymes were ssyed s described under "Experimentl Procedures." The results re mens f S.D. for t lest four seprte preprtions mesured in triplicte. Protein Liver homogente Heptocytes Totl microsomes rer ser Glucose-6-phosphtse ctivity Liver homogente Heptocytes Totl microsomes rer ser NADPH cytochrome c reductse ctivity Liver homogente Heptocytes Totl microsomes rer ser UDP-glctose glctosyltrnsferse Liver homogente Heptocytes Totl microsomes rer ser Untreted OA-fed liver liver mglg liver 25.9 f f f f f f f.8 3. rt f f.9.3 f..3 f. 1.4 f.2.9 rt. nmol/min/mg 47. f rt f k f t f f t f k f t f k f f f f f f f f f.8 6. f k f f f f f f f f f f t f f f f 17.8 pci of [%]methionine ws then dded, nd fter further 5-min incubtion one of the following dditions were mde depending on the experiment: 2 ml of 2 mm oleic cid bound to 1% ftty cid-free bovine serum lbumin or 2 ml of 1% ftty cid-free lbumin. Heptocytes were then incubted up to 18 min, chilled to 4 "C, nd centrifuged t 1 g for 5 min. The cell pellets nd superntnts (medi) were tken for further nlysis. Immunoprecipittion of Cellulr nd Secreted ApoB-After seprtion of heptocytes nd medi the cell pellet ws resuspended in 5 ml of ice-cold 1 mm Tris-HC1, 15 mm NC1, ph 7.4. The medium ws recentrifuged t 1, x g for 5 min to remove ny cell debris. Protese inhibitors were dded, nd pob ws immunoprecipitted from cells nd medi s described previously (8) using nti-humn pob (1:5 dilution) s primry ntibody nd nti-sheep IgG (1:5 dilution) s secondry ntibody. Preprtion of Heptocyte Subcellulr Frctions-Subcellulr frctions were prepred from 1 g of isolted heptocytes. Cells were subjected to hypnotic shock by resuspension of pellets in 2.5 ml in ice-cold 5 mm Tris-HC1, 1 mm MgCl,, ph ml of.5 M sucrose ws dded, nd the cells were homogenized using top-drive Potter Elvehjem homogenizer. Homogeniztion ws checked by inspection of the cell suspension using light microscope. A mixture of protese inhibitors ws dded (8), nd subcellulr frctions (endoplsmic reticulum (er), rough endoplsmic reticulum (rer), smooth endoplsmic reticulum (ser), cis-enriched Golgi () nd trnsenriched Golgi ()) were prepred using the sme methods s previously described for whole liver scled down for 1 g of heptocytes (19-23). Er or Golgi frctions were prepred seprtely from 1 g of heptocytes becuse we hve found tht methods for the isoltion of ll subcellulr frctions from one homogente do not yield the best preprtions on the bsis of purity. The subcellulr frctions were ssessed by determintion of the recovery nd specific ctivities of mrker enzymes, glucose-6-phosphtse nd NADPHcytochrome c reductse for er nd UDP-glctose glctosyltrnsferse for frctions. Protein ws determined by the method of Lowry et l. (24). The membrne nd lumenl content frctions of rer, ser,, nd were prepred by tretment with sodium crbonte (19-23). In some experiments the lumenl contents from frctions from one g of heptocytes were djusted to ph 7.4 nd dilyzed ginst 15 mm NC1, 1 mm Tris-HC1, ph 7.4. The density ws djusted to 1.21 with KBr nd lyered beneth discontinuous grdient mde up of lyers of KBr d = 1.63,1.2, nd 1.6 in Beckmn SW 41 centrifuge tubes, which were centrifuged t 28, X, g for 24 h (21). The lyers were collected for further nlysis. Seprtion of Proteins by SDS-PAGE-The proteins of the lumenl content frctions were precipitted by ddition of 5 mlof ethnol/ether (3/2, v/v) followed by two chnges of ether. Those of the lumenl content subfrctions of the content frctions were dried under nitrogen. Aliquots of subcellulr frctions, precipitted lumenl content frctions, content subfrctions, nd pob immunoprecipittes from cell nd medi preprtions were dissolved in smple buffer (3 mm Tris-HC1, 3 mm EDTA, 3% SDS, 15% glycerol) followed by ddition of fl-mercptoethnol (15%, w/v) nd heted t 6 "C for 2 min. Proteins were seprted on grdient gels (3-2%, w/v) of polycrylmide s described previously (8). Quuntittion of ApoB-The mss of pobloo nd pob48 in heptocytes, subcellulr frctions, nd medi ws determined by qun- tittive immunoblotting employing 1251-protein A fter seprtion of the proteins by SDS-PAGE (8). ApoB concentrtions re expressed s pg/mg of heptocyte or subcellulr frction protein. Determintion of f5s/methionine Incorportion into A~oB-[~~S] Methionine-lbeled pobloo nd pob48 were seprted by SDS- PAGE (8). After Coomssie Blue stining, gel slices contining pobloo nd pob48 were excised nd digested with 3 ml of Hz2 t 1 "C. The digests were mixed with 1 ml of scintilltion fluid nd counted. [%]methionine incorportion ws expressed s dpm/mg of subcellulr frction or dpm/g of heptocytes. In most experiments the mss of pob ws lso determined from prllel gels nd the specific ctivity ws clculted s 35S dpm incorported per pgof pob. Pulse-Chse Studies-To follow the trnsit of pob through the secretory comprtment, heptocytes (1 g in minimum essentil medium) were incubted with [35S]methionine for 3 min s bove in the bsence of olete. The heptocytes were isolted by centrifugtion t 1, X g for 5 min, resuspended (5%, w/v) in minimum essentil medium contining 45 mm unlbeled methionine, nd incubted for up to 18 min with olete (2 mm). The ppernce of [35S]methionine in pob nd the specific ctivities of pob of heptocytes, medi, nd subcellr frctions were determined t ech time point s bove. Preliminry experiments estblished tht under these conditions incorportion of [35S]methionine into totl proteins nd pob ws rrested t the end of the 3-min pulse. RESULTS Preprtion of Subcellulr Frctions from Isolted Heptocytes-In generl, methods for preprtion of subcellulr frctions developed for whole liver yielded similr results when pplied to norml or OA heptocytes (Tble I). Yields of subcellr frctions from heptocytes were generlly lower thn those from whole liver when expressed in terms of either protein yield or enzyme recovery/g of liver (cf. Ref 8). This my reflect the reltive stbility of heptocytes to homogeniztion compred with whole liver, so tht lrger mount of mteril is lost s undisrupted or poorly disrupted cells. The specific ctivities nd enrichment of ech mrker enzyme were comprble with frctions prepred from whole liver (8). However, the specific ctivity of mrker enzymes in the heptocyte homogente ws greter thn tht of the whole liver homogente. This is probbly consequence of the removl of connective tissue proteins from the isolted heptocytes.

3 rn b 9. i * 1 DO j I 8 Intrcellulr Trnsit of Apolipoprotein B in Heptocytes r.e.r. T m loo 9 I n eo - s.e.r i (.I OL BO 4 2o 1 $ *.OS Of , FIG. 1. Distribution of pob in lumenl contents from heptocytes isolted from livers of norml or orotic cid-fed rts. Subcellulr frctions were prepred from isolted heptocytes, nd the lumenl contents were subfrctionted on discontinuous grdients s described under Experimentl Procedures. The percent distribution ofpobloo nd pob48 on the grdients is plotted: norml heptocytes pob1; H, norml heptocytes pob48; OA heptocytes pob1; Ed, OA heptocytes pob48. The results re mens of two seprte determintions. The mounts of pob in the content frctions re given in Tble 11.

4 294 Intrcellulr Trnsit of Apolipoprotein B in Heptocytes TABLE I1 Distribution of pob in subcellulr frctions prepred from heptocytes from untreted nd orotic cid-fed liver Subcellulr frctions were prepred from livers of norml nd orotic cid-fed rts. The mount of pob in ech frction ws determined by quntittive immunoblotting s described under Experimentl Procedures. The results re given s pg of pob/mg of protein of ech subcellulr frction nd mens f S.D. for four seprte preprtions. The content frctions ccount for 5% of the totl protein of the er frctions, 34% tht of the frction, nd 53% tht of the frction. RER Membrne Content SER Membrne Content Membrne Content Membrne Content pobloo pob48 Untreted OA-fed Untreted OA-fed pg1m.g frction protein.52 f.3.14 f f.4.15 f f f f f f f.5.14 f.2.18 f f f.2.14 f.2.3 f zk f f f f f f f f.9.18 f f f f f.5 APO-B1 APO { 3. 3 p. D. 2. : 3 eo eo 12 Time (min).- e e c. n c p. c E 15. b o 1o.o n U L 2 u n W * 2 n LI) eo eo 12 Time (min) FIG. 2. Secretion of pob by heptocytes isolted from livers of norml or orotic cid-fed rts. Isolted heptocytes were incubted, cells nd medi were seprted, nd the pobloo nd pob48 were determined by quntittive immunoblotting s described under Experimentl Procedures., plus olete; A, minus olete;, OA heptocytes plus olete. Closed symbols, heptocyte pob; open symbols, medi pob. Vlues plotted re expressed s pg of pob/mg of heptocyte protein nd re the verges of two seprte experiments performed in duplicte. The degree of cross-contmintion between subcellulr NADPH-cytochrome c reductse contmintion. These clfrctions bsed on mrker enzymes hs been discussed in cultions ssume n bsolute loction of mrker enzyme detil elsewhere (8,25). The mximum mount of er in trns- within single orgnelle nd ssume tht the frctions pre- Golgi nd in er is <lo% with the exception of the pred re 1% pure. Neither of these ssumptions re vlid; ser which contins bout 15% nd the however, the clcultions overestimte cross-contmintion, tht contins 24% er bsed on glucose-6-phosphtse or which is probbly considerbly less thn tht bove. The

5 APO-B1 Intrcellulr Trnsit of Apolipoprotein B in Heptocytes 2941 APO-B48 TOTAL PROTEINS 3 -r- "-1 i o too zoo FIG. 3. Incorportion of [''S]methionine into cellulr nd secreted proteins of heptocytes isolted from livers of norml nd orotic cid-fed rts. Isolted heptocytes were incubted with [36S]methionine, cells nd medi were seprted, nd the incorportion of rdioctivity into totl proteins, pob1, nd pob48 ws determined s described under "Experimentl Procedures.", plus olete; A, minus olete;, OA heptocytes plus olete. Open symbols, dpm in heptocyte; closed symbols, dpm in medi. Vlues plotted re dpm incorported/mg of heptocyte protein nd re the verges of two seprte experiments performed in duplicte. identifiction of the is bsed on morphologicl ppernce nd function (26-28). As this frction is obtined during preprtion of the we hve used it in nlysis of pob synthesis nd distribution; however, cis- Golgi does hve chrcteristics intermedite between those of the er nd the nd my contin significnt mounts of er. ApoB Distribution in Subcellulr Frctions from Norml nd OA-fed Rt Liver-There ws no significnt difference between the distribution of pob in the frctions prepred from OA or norml liver. In ll frctions the mount of pob48 ws bout four times tht of pob1. The concentrtion of pob ws gretest in the, nd significnt mount of the pob of ech subcellr frction ws membrne bound (rer, 8-9%; ser 9%;, 25-3%, nd, 15-2%). The finding tht pob is present in the lumen of both the er nd Golgi frctions of OA-fed liver is surprising s pob is not secreted by these cells, nd their Golgi frctions do not contin VLDL lipid.' However, the pob in content subfrctions from OA-fed livers remined in the d = 1.21 lyer suggesting tht it is not pckged with lipid (Fig. 1 nd Tble 11). ApoB in the lumenl contents from er of norml heptocytes ws ssocited with prticles hving rnge of densities, while in the pob tended to be in the d = 1.6 lyer, which corresponds with the density of VLDL (Fig. 1). Secretion of ApoB by Heptocytes-The pob48 content of isolted heptocytes ws pproximtely three times tht of pobloo nd ws similr in norml or OA cells (Fig. 2). Over 2-h period there ws smll nd stedy increse in the pobloo nd B48 of norml heptocytes incubted plus olete or minus olete nd OA heptocytes plus olete. Secretion of pob1 from norml heptocytes plus olete ws pproximtely 1.5 pg/mg of heptocyte protein/h nd tht of pob48 ws pproximtely 4 pg/mg of heptocyte protein/h. There ws no detectble secretion of pob by norml heptocytes minus olete or OA heptocytes. Incorportion of 35S]Methionine into Heptocyte ApoB- Incorportion of [35S]methionine into cellulr pobloo ws twice tht into pob48 nd ws similr in norml heptocytes plus nd minus olete nd OA heptocytes; however, rdio- * I. J. Crtwright, A.-M. Hebbchi, nd J. A. Higgins, unpublished observtions. lbeled pob ws only secreted by norml heptocytes plus olete (Fig. 3). Incorportion of [36S]methionine into totl proteins of heptocytes nd medi ws the sme in norml nd OA heptocytes nd ws liner for bout 2 h. The specific ctivities of the pools of pob in heptocytes incubted under the three conditions were similr, tht of pobloo ws pproximtely 15 dpm/pg, nd pob48 ws pproximtely 25 dpm/pg fter 18 min of incubtion. The specific ctivity of secreted pobloo ws 5 dpm/pg t the sme time nd tht of pob48 ws 175 dpm/pg. The rte of incorportion of [35S]methionine into pobloo is therefore bout six times fster thn into pob48, lthough the ltter is lrger pool. Incorportion of P5SlMethwnine into ApoB of Heptocyte Subcellulr Frctions-The rte of incorportion of [35S]methionine into pobloo of rer, ser,, nd ws greter thn tht into pob48 in ll subcellulr frctions; however, the lbeling ptterns were similr for both forms of pob. The most striking difference in the incorportion of [35S]methionine into pob between the three incubtion conditions used ws the Golgi frctions (Fig. 4, nd b). Incorportion into the pob of the cis- nd frction ws three nd six times greter in norml heptocytes minus olete nd OA heptocytes, respectively, thn in frctions from heptocytes plus olete. [35S]Methionine ws incorpo- rted t the sme rte into pob of rer nd ser of heptocytes under ll three conditions. Accumultion of pob in the Golgi frctions is not due to incresed synthesis therefore but consequence of decresed degrdtion. When the subcellulr frctions were seprted into membrne nd lumenl contents, complex ptterns in the incor- portion of [35S]methionine emerged (Fig. 5, nd b). In norml heptocytes plus olete [35S]methionine ws incorported to greter extent into pob in the lumen of the rer nd ser thn into pob in the membrne. In norml heptocytes minus olete nd OA heptocytes, incorportion of [36S] methionine into the pob of the membrne nd contents of the rer were similr. The incresed incorportion of [35S] methionine into pobloo nd pob48 of the Golgi frctions of norml heptocytes minus olete nd OA heptocytes ws into the membrne frction rther thn in the lumen (Fig. 5, nd b). In OA heptocytes there ws no rdiolbeled pob in the Golgi lumenl contents. In norml heptocytes plus

6 2942 Intrcellulr Trnsit of Apolipoprotein B in Heptocytes r.e.r, s.ef. 6OOOGO 6ooooo I E 9 U I 1 1 i 2 I I 1 I I time (rnin) time (rnin) ooooo i 1 P 4 - I U time time (rnin) FIG. 4. Incorportion of ['"Slmethionine into pob of subcellulr frctions of heptocytes from livers of norml or orotic cid-fed rts. Isolted heptocytes were incubted with [%]methionine, t timed intervls subcellulr frctions were prepred from the cells, nd pobloo () nd pob48 (b) were seprted nd ssyed s described under "Experimentl Procedures.", plus olete; A, minus olete;, OA heptocytes plus olete. Vlues plotted re dpm incorported into pob corrected for recovery of subcellulr frctions (Tble I) nd re the verges of two seprte experiments performed in duplicte. olete lthough [36S]methionine ws incorported into pob of the Golgi lumenl contents this ws still smll frction of tht in the membrne. Thus, even under conditions in which pob secretion tkes plce the bulk of newly synthesized pob does not find its wy through the conventionl secretory pthwy but moves into pools t other intrcellulr

7 b 4 Intrcellulr Trnsit of Apolipoprotein B in Heptocytes 2943 r.e.r. 4 s.e.r. 3 3 g ,P 3 E 2 -u 1 i / / P 3 E, 2 U 1 i / / /! /,/ / P / // / / / I FIG. 4- -continued sites, the membrne nd lumen of the rer nd the membrne of the. Pulse-Chose Experiments-To follow the trnsit nd fte of pob under different conditions, norml nd OA heptocytes were lbeled by incubtion for 3 min with [35S]methionine in the bsence of olete, followed by chse period with lrge excess of unlbeled methionine for 3 h in the presence of olete. During the chse period, in norml heptocytes, the initil rdioctivity in pobloo recovered in the subcellulr frctions fell by bout lo6 dpm nd in pob48 by bout.6 X 1. At the sme time rdiolbeled pobloo nd pob48 were secreted only by norml heptocytes plus olete nd c- counted for 9 nd 7 dpm, respectively. Loss of rdiolbeled pob is therefore due to intrcellulr degrdtion rther thn secretion. In ll subcellulr frctions the dpm in pobloo were pproximtely twice those of pob48; however, both forms of pob showed similr rdiolbeling ptters (Fig. 6, nd b). During the chse period, rdioctivity in the pob of rer, ser, nd membrnes from norml heptocytes fell considerbly. Although the rdioctivity of pob in the trns- Golgi membrne rose, this only ccounted for smll frction of tht lost from the other subcellulr frctions. Similr decreses in the rdioctivity of pob occurred in rer, ser, nd

8 2944 Intrcelluhr Trnsit of Apolipoprotein B in Heptocytes r-e-r. s.e.r i,,. +'.e.'' ; o i I l ' l ' l ~ I ~ timn (min) 7 _I I J ,,..",.-....i... ~.: - ::: =... ii I I I I time (rnin) ~ I I T I ~ l ' l FIG. 5. Incorportion of "5 into pob of lumenl content nd membrne subcellulr frctions of heptocytes from livers of norml nd orotic cid-fed rts. Isolted heptocytes were incubted s in Fig. 4 except tht the subcellulr frctions were seprted into lumenl content nd membrne frctions before nlysis of pobloo () nd pob48 (b)., plus olete; A, minus olete;, OA heptocytes plus olete; closed symbols, content frctions; open symbols; membrne frctions. The inset grphs for nd re the dt shown for content frctions reproduced on lrger scle for clrity. of OA heptocytes. However, in this cse the rdioctivity in the pob of the membrne fter 6- min chse ws equl to tht lost from the other frctions. Upon incubtion for further 12 min, the rdioctivity in pob of Golgi membrnes fell by bout 5%. These observtions suggest tht OA inhibits or slows degrdtion of membrne-bound pob1, resulting in ccumultion in the trns- Golgi membrnes. The rdioctivity in pob in the rer lumenl contents lso fell considerbly during the chse experiment in both norml nd OA heptocytes. Although there ws n increse in rdiolbeled pob ppering in the contents of other frctions, this ccounted for less thn 1% of the ctivity lost suggesting tht considerble degrdtion of lumenl pob occurs in the rer. In OA heptocytes, dpm in pob of the contents of rer lso fell suggesting tht orotic cid does not inhibit degrdtion of this pool of pob. Trnsfer of pob to the Golgi lumen ws lso inhibited in OA heptocytes. The specific ctivities of pob in ech subcellulr pool were lso determined during the pulse-chse experiments (Figs. 7,

9 b Intrcellulr Trnsit of Apolipoprotein B in Heptocytes 2945 rr. 3 t c 2 2. c.- - e 15 E! - 1 i "I - c.- - E s.e.r.! c z 2oooo o 15 E" E, \ 1 U 5 5 U o i ~ 1 ~ 1 ~ ~ ~ 1 ~ C.- c f c l P CZ.- - z c 15 W E V.- c 25 1 Q1 L i 2 / lim (m Y *Lo I ' I I I I nd b). In generl, the results were consistent with the brnes rose to the sme level s tht of the ser consistent conclusions bove; however, they lso indicte tht movement with m~umultion t this site. of pob between intrcellulr pools is not uniform. In frc- Of Rdiolbeled ApoB1oo nd in tions from both norml nd OA heptocytes the specific Subfrctions of Lumenl Contents-To investigte the districtivity of the pobloo in the rer content ws bout three bution of newly synthesized pob in the subfrctions of the lumenl contents, heptocytes were incubted for 3 min with times tht in the membrne while tht of pob48 ws eight [35SImethionine, nd the lumenl contents were isolted nd times greter thn tht in the membrne. Newly synthesized into frctions d = 1.6, d = 1.2, d = 1.63, nd d PoB, Prticulrly PoB48, is pprently Preferentilly &n- = In rer nd ser frctions from norml heptocytes, neled into the rer nd is selectively degrded. In OA hepto- pob of the highest specific ctivity ws found in the d = 1.21 cytes the specific ctivity of the pob of mem- frction, suggesting tht only smll prt of the newly syn-

10 2946 Intrcellulr Trnsit of Apolipoprotein B in Heptocytes r=e.r= I I I m 6 18 FIG. 6. Intrcellulr trnsit of [S5S]methionine-lbeled pob in heptocytes isolted from livers of norml nd orotic cidfed liver. Heptocytes were incubted for 3 min with [36SS]methionine minus olete, isolted by centrifugtion, nd reincubted with unlbeled methionine plus olete s described under Experimentl Procedures. Membrne nd contents of subcellulr frctions were prepred t intervls, nd the pobloo () nd pob48 (b) were seprted nd counted. Vlues plotted re dpm in ech subcellulr frction nd re the men of two determintions performed in duplicte. B, norml heptocyte membrnes; R, norml heptocyte contents; B, OA heptocyte membrnes; Ed, OA heptocyte contents. thesized pob is pckged with lipid in the er lumen (Fig. 8). In norml heptocytes the bulk of the newly synthesized pob In both trns- nd frctions, the pob of highest in the er lumen is not ssocited with lipids, while most of specific ctivity ws in the d = 1.6 frction. In the OA the pob trnsferred to the Golgi lumen is in prticles of the heptocytes three ws no rdioctivity in the pob in the d = sme density s VLDL. 1.6 nd d = 1.2 frctions of the er or Golgi nd very little trnsfer from er to Golgi. Thus, lthough pob is trnslocted DISCUSSION to the lumen of the er in OA heptocytes, it is not pckged Freshly isolted heptocytes hve been used successfully with lipid nd is trnsferred to the Golgi lumen very slowly. for studies of lipoprotein secretion in number of lbortories

11 b Intrcellulr Trnsit of Apolipoprotein B in Heptocytes cex. s.e.r , i I I I FIG. 6-continued (29-31) nd were used in these studies to fcilitte preprtion of subcellulr frctions. The heptocytes used in our studies were vible by severl recognized criteri nd secreted po- B1OO nd pob48 in the presence of dded olete t rte of pproximtely 1.5 nd 3.5 pg/mg of heptocyte protein/h, respectively. This compres fvorbly with reported levels of pob secretion by HEP-G2 cells (.3-.4 pg of pob secreted/ h/mg of cell protein) (32, 33) or by primry cultures of rt heptocytes (.7 pg of pob secreted/h/mg of cell protein) (13). In these studies we hve used orotic cid s tool to perturb VLDL ssembly. Orotic cid selectively blocks the secretion of pob contining VLDL by the liver without ffecting secretion of high density lipoproteins (15-17). This is not due to inhibition of pob synthesis but to modifiction of the pckging of pob with lipid t or before the comprtment. In OA-fed rt liver levels of purine nucleotides increse nd those of pyrimidine nucleotides including gunine nucleotides decrese, nd the effect of orotic cid is reversed by denine (34-37). The specific effect of orotic cid

12 2948 Intrcellulr Trnsit of Apolipoprotein B in Heptocytes r.e.r. sex o ml LC E U l- m 8 m \ E 4 El ; BO 6 Q CI) 4 E 2 2 EO 1. I I I FIG. 7. Intrcellulr trnsit of [ S]methionine-lbeled pob in heptocytes isolted from livers of norml nd orotic cidfed liver. Heptocytes were incubted for 3 min with [35S]methionine minus olete, isolted by centrifugtion nd reincubted with unlbeled methionine plus olete s described under Experimentl Procedures. Membrne nd contents of subcellulr frctions were prepred t intervls, nd the pobloo nd pob48 were seprted nd specific ctivities determined. Vlues plotted re dpm in ech subcellulr frction nd re the men of two determintions performed in duplicte. W, norml heptocyte membrnes; B, norml heptocyte contents; B, OA heptocyte membrnes; E4, OA heptocyte contents. on VLDL ssembly my thus be on GTP or ATP requiring step in the secretory pthwy. As secretion of other proteins, including polipoprotein Al, is not ffected this effect must be specific for VLDL nd/or pob trnsit. In prllel study, which will be reported elsewhere, we hve exmined the lipids of liver subcellulr frctions from norml nd OA-fed rts. Consistent with results from other lbortories we found tht tricylglycerol ccumulted in the lumen of the er but not in the Golgi frction. Subcellulr frctions prepred from OAfed livers or heptocytes behved in similr wy to those from untreted livers with the exception tht when the er frctions were further seprted by centrifugtion on density grdients the frctions from OA livers, which contined tricylglycerol, tended to distribute t lower density thn those

13 2 15 b Intrcellulr Trnsit of Apolipoprotein B in Heptocytes r.e=r= s.e-r I 4i 3 1 *O I i I 6 16 FIG. 7"continued 6 1 from untreted livers. Hy et l. (17) hve isolted "liposome" frction from OA-fed liver by flottion from postmitochondril superntnt. In our study, lthough there ws n increse in "floting ft" in the livers of OA-fed rts, this ws not membrne-bound nd ll of the glucose-6-phosphtsend NADPH-cytochrome c reductse-contining elements pelleted with the microsoml frction. This discrepncy my be consequence of differences in the mount of tricylglycerol ccumulted nd in the subcellulr frctiontion procedures used. Our observtions do gree with those of Hy et l. (17) in tht pob synthesis is not inhibited, nd the protein is present in significnt mounts in ll subcellulr frctions prepred from orotic cid-treted liver. Studies from other lbortories hve minly followed the incorportion of rdioctivity into pob pools of heptocytes or Hep-G2 cells. However, s the pools of pob my vry under different metbolic conditions it is essentil to know the specific ctivities's well s the rdioctivity incorported with time. The results reported here show quite clerly tht the pools of pob in the heptocyte re not in equilibrium. Thus, more thn 9% of the pob in the rer nd ser re membrne-bound; however, newly synthesized pob is pref-

14 295 Intrcellulr Trnsit of Apolipoprotein B in Heptocytes r.e-r. s.e.r. io r 25 oo 6 4 f? Q J e -2 n i..s *.* I m i, CL Q 91 c z. ZI o OOL FIG. 8. Specific ctivities of pob in frctions.,., of pob in lumenl contents prepred from livers of norml nd orotic cidfed rts. Subcellulr frctions were prepred from isolted heptocytes, nd the lumenl contents were subfrctionted on discontinuous grdients s described under Experimentl Procedures. The pob48 nd pobloo of ech frction were seprted, nd the specific ctivities were determined s described under Experimentl Procedures. Vlues plotted re specific ctivities of pobloo nd pob48: norml heptocytes pob1;, norml heptocytes pob48; OA heptocytes pob1; EQ, OA heptocytes pob48. The results re mens of two seprte determintions. erentilly chnneled into the lumenl pool. The membrne pool is thus degrded more slowly thn the lumenl pools, nd simply mesuring disintegrtions/min incorported would demonstrte not this. A number of investigtions hve shown tht pob is degrded intrcellulrly (2, 6, 9-14) nd tht the rte of degrdtion is modulted by provision of ftty cids (11, 14) or insulin (13). Our results indicte tht most of the totl nd newly synthesized pob in heptocytes is not secreted. It is possible tht regultion of pob ssembly into VLDL is impired in isolted heptocytes the tht nd mount of intrcellulr degrdtion is exggerted compred with tht in norml liver. However, the sizes of pob pools in subcellulr frctions from heptocytes nd whole liver re very similr,

15 suggesting tht the pthwys of trnsit re the sme, lthough the throughput of pob differs under conditions tht fvor secretion. Our observtions indicte tht there re t lest two intrcellulr sites t which pob trnsit my be regulted. The first is the rer t which newly synthesized pob is either incorported into the membrne or is trnslocted into the lumen. The second is the lumen of the rer in which pob is either degrded or pckged with lipid nd trnsferred to the Golgi lumen for secretion. Pulse-chse experiments indicte tht in norml heptocytes there is trnsfer of membrnebound pob from the rer to the membrnes, nd in OA heptocytes the increse in rdiolbeled pob in the is equivlent to tht lost from the rer membrne. Thus, orotic cid ppers to inhibit degrdtion of the membrne-bound form of pob. These observtions do not necessrily pinpoint the Golgi membrnes s the site of degrdtion of membrne-bound pob. Proteins move from the er to the Golgi nd re retrieved (37), nd orotic cid my interfere with this process. Moreover there is evidence for structurl motif in the trnsmembrne region of proteins, which trgets them for degrdtion in the er (38). Studies of Hep-G2 cells hve suggested tht degrdtion of pob occurs in the er (11, 12). This is bsed lrgely on the observtion tht brefeldin A, Golgi perturbnt, does not block degrdtion of pob. However, the ction of brefeldin A is not simple; it my cuse redistribution of Golgi components bck into the er thus perturbing the equilibrium between these orgnelles (37). In rbbit frctions lrge prt of the membrne-bound pool of pob in the er nd ll tht of the re ccessible to monoclonl ntibodies t the cytosolic side of the membrne (25), in rt er pob is cytosolic (5), nd in chicken heptocytes pob hs lso been shown by cytochemistry to be on the cytosolic side of the er (39). Membrnebound pob ccounts for the mjor prt of the totl cellulr pob. Although the current models ssume tht this is pob, which hs been synthesized in excess nd not recruited for VLDL ssembly, it is possible tht it my be involved in crosstlk between cytosolic fctors or the LDL receptor nd the secretory comprtment. This is t present specultive but merits further investigtion. It hs been suggested tht pob is co-trnsltionlly pckged with lipid to be secreted (9), nd synthesis of lipid is essentil for VLDL secretion. However, concomitnt lipid synthesis is not the fctor tht determines trnsloction of pob to the lumen of the rer. In OA heptocytes pob is trnslocted but not pckged with lipid nd not secreted; while in norml heptocytes lrge prt of the lumenl pob hs density of 1.21 indicting tht it hs no or very little ssocited lipid. The enzymes for tricylglycerol synthesis re locted t the cytosolic side of the er, nd tricylglycerol cn be trnsferred to the cytosol or cross the er membrne. In cells tht secrete lipoproteins, tricylglycerol trnsfer into the lumen of the er my be vectorl process tht is bsent from other cells. This is suggested by the observtion tht pob53 cdna-trnsfected Chinese hmster ovry cells will synthesis pob53, but the protein remins membrne-bound nd is rpidly degrded (4). The presence of pproprite lipids in the lumen of the rer my be one of the fctors tht determines whether pob is ssembled into VLDL nd secreted (9). The requirement for proteins to be properly folded to be secretion-competent nd the role of moleculr chperones in this process re well estblished (41-43). The pckging of pob for secretion my thus involve chperones nd/or lipid or tricylglycerol-trnsfer proteins (44-46). When this process is impired then the Intrcellulr Trnsit of Apolipoprotein B in Heptocytes 2951 excess lumenl pob is degrded. In OA heptocytes newly synthesized pob is not pckged with lipid nd moves very slowly to the lumen of the Golgi region. However, the mount of pob in the Golgi lumen is similr to tht in norml heptocytes indicting tht the process of trnsfer is not completely blocked. Orotic cid thus ppers to ct t severl points in pob trnsit; it inhibits degrdtion of membrnebound pob nd not lumenl pob, nd it blocks ssembly of pob with lipid in the er lumen. To investigte the sequence domins involved in the ssembly with lipid nd intrcellulr trnsit of pob, number of studies hve used humn or rt heptom cell lines either trnsfected with plsmids contining truncted cdnas for pob or puromycin to rrest nd terminte pob synthesis so tht short forms of the protein re produced (9, 14, 47-5). The generl conclusion from these studies is tht short forms of pob (<pob23) re secreted without lipid nd tht cells trnsfected with lrger pob cdnas secrete prticles the size of which is directly relted to the length of the synthesized pob. Addition of olete to the incubtion medium decreses the density of the prticles secreted in the ltter group nd lso my decrese intrcellulr degrdtion of the truncted pobs. Although the pproches hve enormous potentil s yet the results hve not pinpointed ny specific domins or sequences tht regulte the trnsit of pob. This is probbly becuse the very lrge size of the molecule mkes such experiments techniclly difficult. In ddition there re cler differences between heptom cells nd norml heptocytes or liver (51). Hep-GB cells secrete pob contining triglyceride-rich prticles of similr density to low density lipoprotein. In norml heptocytes there my be second stge of mturtion of VLDL when more lipid is dded (21-23,47). 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