SYNTHESIS, INSERTION INTO THE PLASMA MEMBRANE, AND TURNOVER OF ot-bungarotoxin RECEPTORS IN CHICK SYMPATHETIC NEURONS

Transcription

1 SYNTHESIS, INSERTION INTO THE PLASMA MEMBRANE, AND TURNOVER OF t-bungarotoxin RECEPTORS IN CHICK SYMPATHETIC NEURONS SALVATORE CARBONETTO and DOUGLAS M. FAMBROUGH Frm the Department f Embrylgy, Carnegie Institutin f Washingtn, Baltimre, Maryland Dr. Carbnett's present address is the Department f Pharmaclgy, State University f New Yrk Medical Center, Syracuse, New Yrk ABSTRACT a-bungartxin was used t identify an integral membrane prtein in the plasma membrane f chick sympathetic neurns. The synthesis, insertin int the plasma membrane, and turnver f the t-bungartxin receptr were studied using istpicauy labeled amin acids (2H, 1~, 15N) t directly label receptr mlecules. Neurns incubated in medium cntaining dense amin acids cntinued t insert unlabeled receptrs frm a pl f previusly synthesized mlecules fr 2 h. Density-labeled receptrs began t appear in the plasma membrane after this 2-h perid. Synthesis f receptrs, but nt insertin int the surface, was blcked by cyclheximide (100/xg/ml). Neither clchicine (0.05/xg/ml) r actinmycin D (5 /zg/ml) has any effect n a-bungartxin receptr synthesis r insertin. Autradigraphic studies revealed that receptrs ccur n grwth cnes, axns, and cell bdies f single neurns and explanted ganglia. The rate f insertin f newly synthesized receptrs int the plasma membrane f axns extending frm explanted sympathetic ganglia was apprximately the same as that int the cell bdy prtin f the ganglin. Cytchalasin B (2/~g/ml) rapidly disrupted grwth cnes but had n effect n receptr insertin. These experiments suggested that the grwth cne is nt the ste r even the primary site fr insertin f this membrane prtein. The kinetics f turnver f the a-bungartxin receptr were a first-rder expnential with tlh -- ll h. Neurns that had their surface receptrs labeled with 12ZI-t-bungartxin prduced [12zI]idtyrsine. This prcess was inhibited by lw temperature (23~ and als by a metablic inhibitr. This is interpreted as evidence that receptrs turn ver by a mechanism in which they are internalized and then prtelytically degraded. KEY WORDS chick sympathetic neurns 9 a-bungartxin receptr, plasma membrane prtein - synthesis insertin turnver In culture, neurns grw lng prcesses in a manner that appears identical t the grwth f axns during develpment. Little is knwn abut the mechanisms f plasma membrane synthesis r turnver during this perid f rapid expansin f the cell surface. Mst f ur present infrmatin abut membrane metablism is extraplated frm electrn micrscpe investigatins, while bi- J. CELL BIOLOGY 9 The Rckefeller University Press /79/06/0555/15 $1.00 Vlume 81 June

2 chemical studies have ften been limited t neurnal cell lines that are cntinuusly dividing. In this paper we present infrmatin n the kinetics f synthesis, insertin, and turnver f a plasma membrane prtein in primary cultures f chick sympathetic neurns that are actively grwing axns. We als present evidence that membrane prteins are inserted int the plasma membrane at multiple sites alng the axn and its cell bdy and nt nly at the grwth cne f grwing axns. In these experiments we have used radiactive a-bungartxin t identify and islate a plasma membrane prtein in chick sympathetic neurns. a-bungartxin, a well-knwn ligand fr acetylchline receptrs in skeletal muscle, als binds selectively t the plasma membrane f chick sympathetic neurns (5, 13, 20, 21). Chick sympathetic neurns have acetylchline receptrs in their plasma membranes (5, 7) and several aspects f a-bungartxin binding (20, 21) suggest that it is binding t neurnal acetylchline receptrs. Hwever, a-bungartxin des nt bind t the ligand-binding site f acetylchline receptrs in sympathetic neurns (5), and may be binding t a cmpletely different mlecule (30). Thus we will cntinue t refer t this mlecule as the a-bungartxin receptr (5). a-bungartxin-receptr cmplexes can be extracted frm membranes with nn-inic detergents and crss-linked in slutin with glutaraldehyde, resulting in a cvalent labeling f the receptr (5). We have used amin acids in which the usual atms f hydrgen (1H), carbn (lzc) and nitrgen Q4N) have been replaced by heavy istpes (ZH, 1aC, and asn) t label t-bungartxin receptrs, a-bungartxin receptrs synthesized with these dense amin acids are crrespndingly mre dense than nrmal receptrs and can be distinguished by velcity centrifugatin. Cmbining the cvalent labeling and density-labeling techniques, we have fllwed the metablism f this plasma membrane prtein in neurns that are rapidly extending axns. MATERIALS AND METHODS Cell Culture Primary cultures f chick sympathetic neurns were made frm 11- t 14-day-ld embrys after the methds f Varn and Raibrn (33). Cells were grwn in Eagle's minimal essential medium (MEM) plus 2% embry extract, 10% hrse serum, 2 U/ml f nerve grwth factr (Burrughs-Wellcme C., Research Triangle Park, N. C.) and 50 mg/ml f gentamicin (Schering Crp., Kenilwrth, N. J.). Labeling f a-bungartxin Receptrs Idinated derivatives f a-bungartxin were prepared as previusly described (9). Receptrs usually were labeled by incubating intact neurns with radiactive a-bungartxin (0.1 /zg/ml) in cmplete medium at 37~ After 1 h the incubatin medium was remved and the cells were thrughly washed f unbund a- bungartxin by submersin fr 5 min int each f three small baths cntaining Hanks' salt slutin plus 0.1% bvine serum albumin, a-bungartxin-receptr cmplexes then culd be extracted frm cells with 1% Tritn X-100, 10 mm Tris HCI ph 7.8 and the a-bungartxinreceptr cmplexes in slutin stabilized with 0.1% glutaraldehyde as described previusly (5). In sme experiments a Tritn X-100 extract f cultured neurns r sympathetic ganglia frm chick embrys was incubated with radiactive a-bungartxin (0.01 t 0.05/xg/ ml) fr 1 h at 37~ and the unbund a-bungartxin was remved by gel chrmatgraphy, as described belw. Filter Assay Binding studies were dne n crude membranes prepared by hmgenizing ganglia frm the paravertebral sympathetic chain f 18- t 19-day-ld chick embrys in 10 mm Tris HCI ph 7.8. The hmgenate was centrifuged at 27,000 g fr 1 h, and the resulting membrane pellet was resuspended in phsphate-buffered saline (ph 7.2) and divided int 100-/~1 aliquts in plastic test tubes. The membranes were incubated with varius cncentratins f l~i-a-bungartxin fr 2 h at 37~ Nnspecific binding was estimated by adding large excesses f unlabeled t~-bungartxin t the radiactive a- bungartxin. The separatin f bund frm unbund a-bungartxin was dne by cllecting the membranes n cellulse acetate filters (Millipre Crp., Bedfrd, Mass.) as described by Shain et al. (32). Filters were cunted by scintillatin spectrmetry. Gel Chrmatgraphy Tw types f mlecular sieve gels were used: Bigel P-60 and Bigel P-2 (Birad Labratries, Richmnd, Calif.). Bigel P-60 clumns (1 25 cm) were equilibrated with 1% Tritn X-100, 10 mm Tris HCI, ph 7.8. Detergent extracts f chick sympathetic ganglia that had been incubated with radiactive a-bungartxin were filtered n the clumn t separate unbund a-bungartxin frm a-bungartxin-receptr cmplexes. Samples were eluted with 1% Tritn X-100, 10 mm Tris HC1, ph 7.8. A Bigel P-2 clumn ( cm) was equilibrated with 100 mm ammnium acetate ph 7.0 and calibrated by determining elutin prfiles fr mnidtyrsine 5,~ THE JOURNAL OF CELL BIOLOGY ' VOLUME 81, 1979

3 (Sigma Chemical C., St. Luis, M.), ~I-a-bungartxin, blue dextran (Pharmacia Fine Chemicals, Piscataway, N. J.), and phenl red (Sigma). This clumn was then used t analyze culture medium frm neurns that had been labeled previusly with radiactive ~bungartxin. Befre any batch f l~zi-~bungartxin was used t label cells, a preparative P-2 clumn equilibrated with MEM was used t remve breakdwn prducts f a-bungartxin that might run in the included vlume n Bigel P-2. The excluded peak frm the preparative clumn was cllected and an aliqut was chrmatgraphed n the calibrated P-2 clumn t shw that it ran as a single peak in the vid vlume. This purified ~zsi-abungartxin was then used t label receptrs n intact cells. Samples f medium frm neurns previusly labeled with a-bungartxin were adjusted t ph 7.0 and 100 mm ammnium acetate, unlabeled idtyrsine was added t give 10-5 M "carrier" idtyrsine, and the samples were analyzed by chrmatgraphy n Bigel P- 2. The recvery frm the clumn generally was 90% f the activity laded. Density-Labeling Experiments INSERTION OF RECEPTORS: Fr experiments measuring the rate f insertin f newly synthesized receptrs int the plasma membrane, a set f identical cultures f chick sympathetic neurns (usually eight t twelve 35-ram plates) were prepared frm paravertebral, sympathetic chains f 8-10 embrys. The neurns were allwed t grw fr 1 d in nrmal culture medium which was then replaced (t = 0) by "dense" medium cntaining 2H, ~ac, lsn-substituted amin acids (Merck and C., Inc., Rahway, N. J.), dialyzed embry extract (2%), dialyzed hrse serum (10%) and 2 U/ml nerve grwth factr. 1 h befre each time pint, 1 r 2 culture plates were incubated with l~zi-a-bungartxin (0.1 v.g/ml) in dense medium fr 1 h at 37~ t saturate the ppulatin f a-bungartxin receptrs in the plasma membrane. At the end f the hur the cultures were thrughly washed f unbund a-bungartxin, and the a-bungartxin-receptr cmplexes were extracted with Tritn X-100 and stabilized with glutaraldehyde as described abve. Fr each experiment it was als necessary t have light, "marker" ct-bungartxin-receptr cmplexes. These were prepared frm identical neurnal cultures that were grwn cntinuusly in nrmal medium and were incubated with ~aq-a-bungartxin. ~zsi-a-bungartxin-receptr cmplexes frm neurns grwn in dense medium plus light, marker ~aq-a-bungartxinreceptr cmplexes were layered nt 25-40% sucrsedeuterium xide gradients and centrifuged as described belw. After centrifugatin, the gradients were dripped int scintillatin vials and the radiactivity was cunted. The sedimentatin prfile f light, marker receptrs labeled with ~aq-c~-bungartxin was used t determine the psitin in the gradient f ld, light receptrs frm neurns grwn in medium cntaining dense amin acids. Thse receptrs sedimenting at a faster rate in these gradients were newly synthesized frm the dense amin acids (Fig. 4). Analysis f the number f density-labeled receptrs frm the sedimentatin prfiles f receptrs in the gradients was carried ut with the aid f a PDP-8 cmputer (Digital Equipment Crp., Maynard, Mass.). A cmputer prgram was written that culd theretically superimpse dense and light receptr peaks, each having the shape f the marker. The cmputer simultaneusly pltted the real data and the theretically cnstructed duble peak. The cmputer peratr culd repeat this prcess each time, adjusting the theretical prprtin f dense and light peaks. The best fit was btained by visual inspectin. A mre detailed descriptin f this cmputer prgram as well as an evaluatin f its accuracy has been presented elsewhere (18). TURNOVER r RECEPTORS: The turnver f a- bungartxin receptrs in the plasma membrane was measured in a pulse-chase experiment. A set f identical cultures f chick sympathetic neurns were grwn fr 1 day in nrmal medium. The cultures were then placed in dense medium fr 12 h, at the end f which 65% f the c~-bungartxin receptrs in the plasma membranes f the neurns were density~labeled. The dense medium was replaced with nrmal medium (t = 0). 1 h befre each time pint, ne r tw cultures were incubated with lzsl-a-bungartxin (0.1 tzg/ml) fr 1 h at 37~ in nrmal medium. Light "marker" 13q-a-bungartxinreceptr cmplexes were als prepared. The number f density-labeled receptrs remaining in the plasma membrane after transferring neurns t nrmal medium was determined by velcity sedimentatin and cmputer analysis f slubilized and glutaraldehyde-stabilized a- bungartxin-receptr cmplexes as described abve. Sucrse Gradient Centrifugatin Velcity sedimentatin was carried ut by layering an extract cntaining stabilized t-bungartxin-receptr cmplexes in a thin band ver a 5-20% linear sucrse gradient (5 ml) r % linear sucrse gradient in deuterium xide (11 ml) bth cntaining 1% Tritn X- 100, 1 mm phenyl methylsulfnyl fluride (Sigma) and 1 mm EDTA. 5-20% sucrse gradients were centrifuged in a Beckman SW 50.1 rtr (Beckman Instruments, Inc., Pal Alt, Calif.) at 48,000 rpm fr 5-6 h at 5~ 25-40% sucrse/deuterium xide gradients were centrifuged in a Beckman SW 41 rtr at 38,000 rpm fr 48 h at 13~ fractins were cllected and the radiactivity was cunted. In sucrse gradients f extracts frm sympathetic neurns treated with lzsi-a-bungartxin, we regularly saw a 4-6S peak that was caused by unbund r nnspecifically bund l~zi-a-bungartxin. This peak was nt blcked by incubatin f neurns in 10-4 M D- tubcurarine that cmpletely blcked binding t the 11S peak. It is imprtant t nte that the psitin f this CARBONETTO AND FAMBROUGH Neurnal Membrane Synthesis, Insertin, and Turnver 557

4 peak des nt shift in neurns grwn in dense medium (Fig. 4) where it might therwise interfere with an accurate estimatin f the number f light receptrs in a gradient. We were able t reduce the size f this peak by thrugh washing f unbund tx-bungartxin frm cells and als by filtering a-bungartxin-receptr cmplexes in slutin n Minicn A-75 filters (Amicn Crp., Lexingtn, Mass.) that retained ~70% f the cmplexes while permitting ~75% f the 4-6S binding t pass thrugh the filter. The remaining 4-6S peak separated by fractins frm the 11S receptr peak, and we have estimated its cntributin t the lls peak by simply extraplating the leading edge f the 4-6S peak. Radiistpe Cunting Samples were cunted by liquid scintillatin spectrmetry in a Packard Tricarb Scintillatin Spectrmeter (Packard Instrument C., Dwners Grves, Ill.). The scintillatin flur was ne part Tritn X-100, tw parts tluene cntaining 16 g PPO (2, 5 diphenylxazle), 0.2 g POPOP (1,4-bis[2-(5 phenylxazlyl)]benzene) per galln f tluene. In sme experiments, samples were cunted in a Packard gamma cunter. Data were crrected fr crssver between the 131I and ~2zI channels. Cell Mrphlgy Chick sympathetic neurns were cultured n cllagencated, glass cverslips, washed in Hanks' salt slutin, and fixed fr 1 h each in tw changes f 2% glutaraldehyde in 100 mm phsphate buffer, ph 7.2. The specimens were pstfixed in 1% OsO4 fr 1 h, dehydrated thrugh alchls (30%, 50%, 70%, 95%, 100% fr 5 min each) and in acetne (50% acetne/50% alchl, 100% acetne fr 5 min each) and dried in a criticalpint drying apparatus (Plarn Equipment Ltd., Watfrd, England). The specimens were then sputter-cated with gld-palladium in a Plarn SEM Cating Unit and examined in a JEOL JSM-35 scanning electrn micrscpe (JEOL Ltd., Tky, Japan). The methds used fr light micrscpe autradigraphy have been published previusly (5). RESULTS Identificatin f the a-bungartxin Receptr in Slutin Fig. 1 shws the binding f radiactive a-bungartxin t membranes prepared frm chick sympathetic neurns as a functin f c~-bungartxin cncentratin. The ttal binding (half-filled circles) has a cmpnent that saturates in 1 h at 10 nm (filled circles) in additin t nnspecific binding f the c~-bungartxin (pen circles). Nnspecific binding t hmgenates is greater than t intact neurns where it is ften <10% f the ttal a-bungartxin bund. b E v # D z I~) I [ TOXIN MOLARITY (nm) FIGURE 1 Binding f l~zi-a-bungartxin t membranes frm chick sympathetic ganglia. Crude membranes were prepared frm 18- t 19-d embrys, and the amunt f bund radiactivity was assayed after filtering n cellulse acetate filters as described in Materials and Methds. (I) I)) Ttal binding f lmi-abungartxin, (O--O) "nnspecific" binding, (~ Q) ttal binding minus nnspecific binding. The a-bungartxin receptr present in membranes frm chick sympathetic ganglia can be extracted frm these membranes by the nn-inic detergent Tritn X-100 (Fig. 2). Detergent extracts f ganglinic membranes incubated with radiactive a-bungartxin shw a peak f radiactivity that chrmatgraphs in the vid vlume n BiGel P-60 (filled circles), well-separated frm unbund a-bungartxin. If D-tubcurarine (10-4 M) is added t the detergent extract alng with radiactive a-bungartxin, the amunt f radiactivity in the excluded peak is reduced greatly, (pen circles), which helps identify this peak as the specific binding cmpnent r a- bungartxin receptr in neurns. The nn-inic detergent Tritn X-100 is mre efficient at slubilizing a-bungartxin receptr cmplexes frm the membranes f neurns than either 1 M NaCI r 1 mm EDTA (Table I), suggesting that the receptr is an integral membrane prtein. The a-bungartxin-receptr cmplexes that chrmatgraph as an excluded peak n Bigel P- 60 (Fig. 2) sediment largely as a single peak when assayed after velcity centrifugatin (Fig. 3, filled circles). Again, we identify the radiactivity in this peak as a-bungartxin-receptr cmplexes because it is absent in preparatins incubated with a-bungartxin plus 10-4 M -tubcurarine (pen circles). The a-bungartxin receptr (sedimen- 5~ THE JOURNAL OF CELL BIOLOGY' VOLUME 81, 1979

5 i5 Opue Dextran Myfube ACh receptrs ~,2 / x "~ ;e',//j Frctqn unbund G BuTX Number FIGURE 2 Chrmatgraphy f detergent extracts f chick sympathetic ganglia n Bigel P-60. Paravertebral, sympathetic ganglia were dissected frm eight 19-d embrys, hmgenized in 10 mm Tris HCl ph 7.8, and centrifuged at 27,000 g fr 45 min. The resulting membrane pellet was slubilized in 1% Tritn X-100, 10 mm Tris HC1, ph 7.8, and divided int tw equal aliquts f 500/xl. One aliqut was pre-incubated with 10 -a M n-tubcurarine fr 30 min at 37~ after which time 0.01 p,g/ml f l~zi-a-bungartxin was added t bth aliquts fr an additinal 30 min at 37~ Blue dextran 0.1% was added t the samples which were layered n a 1 x 25 cm Bigel P-60 (exclusin limit = 60,000 daltns) clumn equilibrated with 1% Tritn X- 100, 10 mm Tris, ph 7.8, and eluted with this same slutin. The psitin f chick mytube acetylchline receptrs and radiactive a-bungartxin was determined previusly. 20-drp fractins were cllected and cunted in a -/-cunter. O ~zsi-a-bungartxin; ~zsi-a-bungartxin M -tubcurarine. tatin cefficient -11S) sediments slightly faster than chick skeletal muscle acetylchline receptrs (5) which have a cefficient f 10S (12). The secnd (4-6S) peak in these gradients is als present in preparatins incubated with a-bungartxin plus D-tubcurarine and has been discussed abve (Materials and Methds). Unlike its binding t acetylchline receptrs in skeletal muscle, which is essentially irreversible, a-bungartxin dissciates frm sympathetic neurns with a half-time f -2 h at 37~ (5). Hwever, treatment f a-bungartxin-receptr cmplexes with glutaraldehyde (0.1%) "fixes" the a-bungartxin t its receptr and greatly increases the stability f the cmplexes (5). This finding is imprtant fr the density-shift studies that emply lng perids f centrifugatin t separate light frm density-labeled receptrs. Kinetics f Synthesis and Insertin f a- Bungartxin Receptrs int the Plasma Membrane T label the a-bungartxin receptr metablically, we have used a density-shift methdlgy adapted in this labratry fr the study f anther membrane prtein, the acetylchline receptr in chick skeletal muscle (11, 12, 17, 18). Neurns grwn in culture medium cntaining 2H, 1~C, I~Nsubstituted amin acids incrprate these dense amin acids int their newly synthesized prteins, rendering the prteins mre dense s that they can be separated frm ld, "light" prteins synthesized in nrmal medium cntaining 1H, ~2C, ~4N-amin acids. Treatment f intact neurns with radiactive a-bungartxin identifies the ppulatin f receptrs that is present in the plasma membrane f chick sympathetic neurns. By extracting a-bungartxin receptr cmplexes and assaying them after velcity centrifugatin n sucrse/deuterium xide gradients, we can determine hw many density-labeled and light a-bungartxin receptrs are n the surface at any time. Fig. 4 shws the sedimentatin prfiles f a- bungartxin receptr cmplexes extracted frm neurns after incubatin fr increasing lengths f time in medium cntaining dense amin acids. TABLE I Efficiency f Extractin f t-bungartxin Receptrs by Varius Slutins Slutin 150 mm NaC1 150 mm NaCI + 1 mm EDTA 150 mm NaC1 + 1% Tritn X M NaC1 Radiactivity n cells cpm Radiactivity in slutin cpm 1, , ,431 Extractin efficiency cpm in slutin ttal cpm bund\ x 100%} 65 1, Fur sets f neurnal cultures (4 plates each) were incubated with 0.1 /~g/ml f l~i-a-bungartxin fr 1 h at 37"C. They were washed thrughly t remve unbund txin and treated with 1 ml f a test slutin with cnstant swirling fr i min. The slutin was remved, centrifuged at 10,000 g fr 5 min, and an aliqut f the supemate was cunted by scintillatin spectrmetry. The activity remaining n the plates was slubilized in 1 N NaOH and als cunted. CARBONETFO AND FAMBROUGH Neurnal Membrane Synthesis, Insertin, and Turnver 559

6 x / f ~T //r ~ k~ ~'-~ IB ~ Frctin Number FIGURE 3 Sucrss velcity centrifugatin f the excluded peak frm Bigel P-60 chrmatgraphy f detergent extracts f chick sympathetic ganglia. Fur paravertebral sympathetic chains lkm 18- t 19-d embrys were incubated with lz~i-tx-bungartxin (0.05 /~g/ml) plus r minus 10-4 M -tubcurarine fr 1 h at 37~ Then they were washed f unbund tz-bungartxin, and extracted int 300 /zl f 1% Tritn 10 mm Tris HCI, ph 7.8. Blue dextran (0.1%) was added t the extracts and each was layered n a Bigel P-60 clumn. Ten-drp fractins were cllected and cunted in a?-cunter. The excluded peak fractins frm each clumn were pled, carefully layered n a 5-20% cntinuus sucrse gradient, and centrifuged fr 5 h at 5~ and 48,000 rpm in a Beckman SW 50.1 rtr. (0 O) Bigel P-60 excluded peak frm ganglia incubated in ~zi-~bungartxin; (O---O) excluded peak frm ganglia incubated with ~I-a-bungartxin plus 10-4 M -tubcurarine. The appearance f a faster sedinaenting peak by 6 h (filled circles) is due t de nv synthesis f a- bungartxin receptrs frm dense amin acids. If cyclheximide (100/zg/ml) is present frm the beginning f the incubatin perid in the medium cntaining dense amin acids, then the faster sedimenting peak des nt appear (Table II). A cmputer prgram has been used t analyze the number f light and density-labeled receptrs in each gradient. Frm these data, a curve has been cnstructed shwing the time curse f insertin f newly synthesized ct-bungartxin receptrs in the plasma membrane (Fig. 5). There is a 2-h delay befre density-labeled receptrs appear n the surface. After the delay, density-labeled receptrs appear in the plasma membrane with firstrder expnential kinetics having a half-time f ~6 hr. During the 2-h delay, the insertin f receptrs int the surface cntinues but the receptrs are supplied frm an internal pl f previusly synthesized light receptrs. The presence f this internal pl is shwn mre directly by the results f an experiment represented in Fig. 6. In this experiment, neurns are grwn fr 4 h in medium cntaining dense amin acids, by which time density-labeled receptrs are being inserted int the surface at a maximum rate (Fig. 5). Cyclheximide (100 p,g/ml) is then added and rapidly blcks synthesis f a-bungartxin receptrs (Table II) as well as prtein synthesis in general (8). In spite f the blck f prtein synthesis, density-labeled receptrs cntinue t appear n the surface fr 2 h frm a previusly synthesized internal pl (Fig. 6, pen circles), the same internal pl that must fill with newly synthesized, density-labeled receptrs befre any appear in the plasma membrane. This experiment strngly suggests that the 2-h delay is nt a result f the time required fr the amin acid pls f the cell t equilibrate with dense amin acids. We cnclude that transprt f receptrs thrugh the internal pl takes 2 h and that bth transprt and insertin int the surface are independent f prtein synthesis. We tested the effects f several drugs n the insertin f a-bungartxin receptrs int neurnal plasma membranes (Table II). As mentined previusly, cyclheximide blcks insertin nly indirectly as a result f inhibitin f new receptr synthesis. Neither clchicine, at a cncentratin that blcks axnal elngatin (8), nr actinmycin D at a cncentratin that blcks RNA synthesis (29) has any effect n the synthesis and insertin f receptrs. Turnver f a-bungartxin Receptrs The kinetics f appearance f density-labeled receptrs in the plasma membrane (Fig. 5) reflect the turnver as well as the synthesis and insertin f receptrs. The turnver rate was measured directly in a pulse-chase experiment in which a ppulatin f surface receptrs was density-labeled by a 12-hr incubatin in medium cntaining dense amin acids. The cells were transferred t nrmal medium, and the number f density labeled receptrs remaining was measured at intervals after the transfer. After 12 h in dense medium, 65% f the surface ppulatin f receptrs was labeled with dense amin acids (Fig. 5). This ppulatin decreased expnentially with a halftime f 11 h r a rate cnstant equal t 0.06/h (Fig. 7). The same turnver rate has been measured fr light receptrs in neurns grwing in dense medium (unpublished bservatins, see als 560 THE JOURNAL OF CELL BIOLOGY 9 VOLUME 81, 1979

7 I0 A. 2 hurs B. 6 hurs C. 12 hurs?, =.. 8 t ".7. 4.? 2 / /\/ //' %~! I I 2 50 grctin Number I FIGURE 4 Sucrse gradient velcity centrifugatin prfiles f c~-bungartxin receptr cmplexes frm the plasma membrane f chick sympathetic neurns cultured in medium cntaining 2H, lac, 15Nsubstituted (dense) amin acids. Neurns in medium cntaining dense amin acids were permitted t grw fr varius perids. At the end f a perid, they were incubated with ~zi-a-bungartxin (0.1 /zg/ ml) and the a-bungartxin-receptr cmplexes were extracted and prepared as described abve (Materials and Methds). Aliquts f light "marker" a-bungartxin receptrs frm neurns grwn cntinuusly in nrmal medium and labeled with 131I-a-bungartxin were layered n 25-40% sucrsedeuterium xide gradients alng with ~I-a-bungartxin receptr cmplexes frm neurns grwn in dense medium. Gradients were centrifuged at 38,000 rpm fr 48 h at 13~ in a Beckman SW 41 rtr. 25-drp fractins were cllected. The sedimentatin prfile f the light marker laq-a-bungartxinreceptr cmplexes was used t determine the psitin f light receptrs in the extracts f neurns grwn in medium cntaining dense amin acids. l~zi-a-bungartxin-receptr cmplexes frm neurns grwn in dense medium. ( ~aq-a-bungartxin receptr cmplexes frm neurns grwn in light medium. reference 18) and als can be calculated frm the difference between the rate f insertin f receptrs (8% f the surface ppulatin per h, Fig. 5) and the rate f increase f receptrs (2-3 % f the surface ppulatin per h, Fig. 10). Thus, labeling f receptrs with heavy istpes des nt seem t affect their turnver. The lag in turnver (Fig. 7) is caused by the appearance f density-labeled receptrs frm the internal pl fr 2 h after transferring the neurns int light medium and bscures the turnver f receptrs that ccurs during this perid. (The cntinued appearance f density-labeled receptrs fr 2 h after transferring neurns t light medium was shwn abve t be due t pstsynthetic transprt f receptrs t the surface membrane.) The expnential shape f the curve suggests that receptrs are lst at randm frm the surface ppulatin, and all receptrs, regardless f age, share an equal prbability f turning ver within the next unit f time. The turnver f acetylchline receptrs in chick mytubes in culture has been measured previusly by density-labeling (18) and by the decrease in binding f a-bungartxin t muscle cells treated with purmycin (9). Acetylchline receptrs in mytubes turn ver by being internalized and prtelytically degraded (9, 10). The degradatin f acetylchline receptrs has been inferred frm bservatins that radiactive c~-bungartxin bund t acetylchline receptrs is subject t prtelysis inside the cell, and that the radiactivity diffuses frm the cell and int the medium as radiactive idtyrsine. The rate f idtyrsine prductin by muscle cells matches the rate f turnver f acetylchline receptrs (2, 9). We have used a similar apprach t btain infrmatin abut the fate f a-bungartxin receptrs inserted int the plasma membrane f chick sympathetic neurns. Our data can be explained best by a mdel in which receptrs turnver by a prcess f internalizatin and subsequent prtelytic degradatin. a-bungartxin dissciates with a half-time f ~2 h frm neurns. Thus, gel chrmatgraphy f radiactivity appearing in the medium after neurnal a-bungartxin receptrs n intact neurns are saturated with ~zi-a-bungartxin shws that 70-75% f the radiactivity c-chrmatgraphs with c~-bungartxin, but that 25-30% f the radiactivity c-chrmatgraphs with the much smaller mlecule [~25I]idtyrsine (Fig. 8). The prductin f idtyrsine by neurns des nt CARBONET'rO AND FAMnROUGH Neurnal Membrane Synthesis, Insertin, and Turnver 561

8 I 6 /*+ 6 g4 ~- 40 ~3 2 I 2O clheximide Time FIGURE 5 Kinetics f insertin int the plasma membrane f newly synthesized a-bungartxin receptrs. The data pints fr this graph were btained by analysis f sedimentatin prfiles f c~-bungartxin receptr cmplexes prepared frm neurns grwn in medium cntaining dense amin acids as in Fig. 4. The number f density-labeled receptrs is pltted as a percent f the ttal number f a-bungartxin receptrs labeled in a culture; and, because neurns were plated in equal numbers, this varied little (~10%) between culture plates. The curve drawn thrugh these pints is a firstrder expnential with t ~/2 = 6 h. Nte that the halftime (6 h) falls at 8 h n the abscissa because there is a 2-h delay befre any newly synthesized receptrs appear in the surface. Symbls indicate different experimental trials. ccur if the cells are incubated with mi-a-bungartxin in the presence f 10-4 M -tubcurarine (Fig. 9), indicating that the a-bungartxin must bind t the receptr t be degraded and is nt being simply endcytsed frm the medium and degraded. Idtyrsine prductin is energy-dependent and is inhibited by treating cells with CCCP (carbnyl cyanide m-chlrphenylhydrazne), an uncupler f xidative phsphrylatin, r by maintaining cells at rm temperature (Fig. 9). Als, we find that a small amunt f idtyrsine is released (11% f the ttal radiactivity remaining n the cells 10 h after labeling with mia-bungartxin) by slubilizing cells that have been prducing idtyrsine. Accumulatin f a-bungartxin Receptrs in the Plasma Membrane Fig. 10 shws the change in the number f a- bungartxin receptrs in sympathetic neurns as a functin f time in culture (see als references 14, 26). The number f receptrs increases fr the first 2-3 d in culture, peaks at abut day 5, then slwly decreases t abut day-1 levels after 10 d. We have measured this by scintillatin {h) ( I I I Time FIGURE 6 Kinetics f inhibitin f a-bungartxin receptr synthesis by cyciheximide, Tw sets f neurnal cultures (4 plates each) plated at equal densities were transferred int medium cntaining dense amin acids and permitted t grw fr 4 h. At the erld f this time (t = 0), cyclheximide was added t ne set f cultures and the insertin f density-labeled receptrs was measured. In these experiments, mi-c~-bungartxin-teceptr cmplexes frm neurns treated with cyciheximide were centrifuged in gradients with mi-t-bungartxinreceptr cmplexes frm cntrl cultures, in rder t vercme the limitatin impsed by the six bucket rtr. The psitin f light receptrs in these samples culd be determined frm the sedimentatin prfile f light marker centrifuged in a separate gradient. (-~ 0) cntrl; ( cyclheximide (100 p.g/ml). TABLE II Effect f Varius Drugs n the Insertin f t- Bungartxin Receptrs int the Plasma Membrane Drug Treatment with drug (h) Density-labeled receptrs (% Density-laf ttal stir- beled reface recep- ceptrs (% trs) f cntrl) h Cntrl Clchicine ( g.g/ml) Cyclheximide (100/zg/ml) (cntinuus) Actinmycin D (5/zg/ml) (cntinuus) Cultures f sympathetic neurns were grwn in medium cntaining dense amin acids fr 8 h. Cultures were treated with drugs at the cncentratins and fr the perids shwn abve. After 7 h, the neurns were incubated with radiactive t-bungartxin (0.1 /~g/ml) in dense medium fr J h at 37~ The cultures were then washed f unbund a-bungaxtxin, the a-bungartxin-receptr cmplexes were extracted, and the number f density-labeled receptrs in each culture was determined. 562 THE JOURNAL OF CELL BIOLOGY-VOLUME 81, 1979

9 Time FIGURE 7 Turnver f a-bungartxin receptrs in the plasma membrane f neurns. A set f neurnal cultures (6 plates) was grwn in medium cntaining dense amin acids (2H, LaC, ~SN-amin acids) fr 12 h, at which time ~65 % f the a-bungartxin receptrs in the plasma membrane were density-labeled. The cultures then were transferred t medium cntaining light amin acids (t = 0) (~H, ~2C, ~4N-amin acids) and, at varius times thereafter, cultures were incubated with l~i-abungartxin and the number f density-labeled and light receptrs remaining in the cells was determined. The number f receptrs remaining is pltted as a percent f the density-labeled receptrs at t = 0. The data are fit by a first-rder expnential with t ~/: = 11 h r a rate f ~6% f the surface ppulatin per h. (h) each neurn by light micrscpe autradigraphy (Fig. 10, right) t shw that the differences are nt due t decreases in cell number that culd be caused by cell death. In all ur experiments n a- bungartxin receptr metablism, we have used neurnal cultures that were 1-2 d ld. During this perid, receptrs are accumulating in the plasma membrane at a rate f between 2 and 3% net additin t the surface ppulatin each hur. This rate f increase fits well with ur measurements f the rate f insertin f newly synthesized receptrs int the surface (8% f the surface ppulatin per h) and the degradatin rate f receptrs (6% f the surface ppulatin per h) r an accumulatin rate f 2%/h. Site f Insertin f a-bungartxin Receptrs int the Plasma Membrane In light micrscpe autradigraphs f sympathetic neurns that have been incubated with 12'~Ia-bungartxin, we see expsed emulsin grains ver grwth cnes and a decrease in the number f grains frm the cell bdy t the tip f the axn (Fig. 11). Althugh we did nt mentin it at the 8 k ~43~TX E ~0clyrsm e - 10 Cntrl CCCP dtc 23 ~ ~20~M~ 'IO0;~M J Freer,n Number FIGURE 8 Bigel P-2 chrmatgraphy f radiactivity in the medium f neurns labeled with ~25I-ct-bungartxin. A set f cultures (3 plates) was incubated with ~zsi-a-bungartxin t label the receptrs in the plasma membrane, washed thrughly t remve unbund ~- bungartxin, and returned t the incubatr. At the end f 10 h, aliquts f the medium were chrmatgraphed n Bigel P-2 clumn (Materials and Methds) % f the radiactivity in the medium chrmatgraphs at the same psitin as idtyrsine n Bigel P-2. cunting f the amunt f radiactive a-bungartxin specifically bund t neurnal cultures f increasing ages (Fig. 10, left). We als have estimated the amunt f a-bungartxin bund t FIGURE 9 The effect f a metablic inhibitr, lw temperature and D-tubcurarine n the prductin f idtyrsine by neurns. Fur sets f neurnal cultures (3 plates each) were incubated with lzsi-a-bungartxin. In the case f the drugs, 20 p.m CCCP (an uncupler f xidative phsphrylatin) and 100 p~m D-tubcurarine were present 30 min befre and during the incubatin f neurns with ~25I-ct-bungartxin. Cultures then were washed thrughly f unbund t~-bungartxin and three sets were returned t the incubatr. One set was maintained in CCCP while anther was kept at rm temperature in HEPES-buffered MEM, ph 7.2. At the end f 10 h, the radiactivity in the medium was analyzed by BiGel P-2 chrmatgraphy. The cells were slubilized with Tritn X-100 and the radiactivity in the extract was cunted t determine the amunt f radiactivity remaining n the cells. CARBONElq'O AND FAMBROUGH Neurnal Membrane Synthesis, Insertin, and Turnver 563

10 d: g e~a~ Dy$ in Cul)re,~0 i PIGURE 10 The number f a-bungartxin receptrs in chick sympathetic neurns during time in culture. (A) Neurnal cultures were plated at equal densities and permitted t settle and grw fr 1 d. At each time pint thereafter, three plates were incubated with l~i-t-bungartxin (0.1 p,g/ml) with r withut D-tubcurarine (10-4 M). The plates were washed f unbund a-bungartxin, slubilized in 1% Tritn X-100, 10 mm Tris, ph 7.8, and the radiactivity in the slutin was cunted. Binding nt blcked by n-tubcurarine was <10% f cntrl values and was subtracted frm cntrls. (B) Neurn cultures were made and handled as described abve, except that after neurns were incubated with l~i-a-bungartxin they were fixed in 2% glutaraldehyde, 100 mm cacdylate buffer, ph 7.4, dehydrated in alchls, and cated with Kdak NTB-2 Nuclear Track Emulsin. After 5-7 d, autradigraphs were develped in Kdak D-19 develper and fixed. Grains were cunted ver neurns, using a cmpund micrscpe with phase and brightfield ptics. time, a gradient f a-bungartxin receptrs is evident in the autradigraph shwn in ur earlier paper (5). This distributin wuld be cnsistent with varius mdels f membrane metablism and membrane flw including a mdel in which plasma membrane prtein is inserted at the grwth cne f elngating axns and mves twards the cell bdy, where it is degraded. Using the c~-bungartxin receptr as a plasma membrane marker, we have tested the hypthesis that newly synthesized membrane is added int grwth cnes (3, 4, 23, 25, 31,37) at the tips f elngating axns. In the first f these experiments, sympathetic ganglia were cultured whle rather than as dissciated neurns. After several days in culture, an extensive utgrwth r "hal" f axns surrunded each ganglin. This hal was dissected easily frm the ganglin t yield a preparatin f pure axns. The strategy f the experiment was t culture whle ganglia fr several days, transfer them t medium cntaining dense amin acids fr varius perids, then separate the ganglia frm their axns, label the a-bungartxin receptrs in the ganglia with 131I-a-bungartxin, label the # receptrs in the axns with lzh-a-bungartxin, and then analyze the number f density-labeled receptrs in the axns vs. ganglia. If newly synthesized receptrs were being incrprated at the tips f elngating axns, then the number f densitylabeled receptrs shuld be greater in the axns than the ganglia. In three separate experiments, we fund that the number f density-labeled receptrs in the ganglia was 7-27 times that f the axns. Hwever, the relative rate f insertin f newly synthesized receptrs, when nrmalized t the pre-existing surface ppulatin in the axns and ganglia during the labeling perid, was almst the same in the ganglia and axns at each time pint (Table III). It seems then that receptrs are inserted at multiple sites alng the cell and nt exclusively at the grwth cne. The cnclusin that receptrs are nt inserted predminately int grwth cne plasma membrane is strengthened by the results f experiments testing the effects f cytchalasin B. Cytchalasin B blcks axnal elngatin by disrupting the structure f the grwth cne (Fig. 12) (37). We have bserved that when grwing axns are expsed t cytchalasin B (2/xg/ml) fr as little as 3-5 min, the grwth cne filpdia cease their activity and the grwth cne takes n a clubbed appearance. This is fllwed shrtly by retractin f the fine branches f elngating neurites. The rate f receptr insertin was measured in cultures f dissciated neurns incubated cntinuusly with cytchalasin B (2 /~g/ml) r pulsed fr 3 h (5 /,~g/ml). In bth cases the insertin f receptrs was unaffected (Fig. 13), suggesting that the structure f the grwth cne is f little imprtance in this prcess. DISCUSSION Neurns in culture extend lng prcesses in a manner that appears identical t neurns grwing in viv. In ur early experiments cnfirming bservatins f Greene and his clleagues n a- bungartxin binding (7, 20, 21), we ften saw a-bungartxin receptrs ver grwth cnes in light micrscpe autradigraphs f sympathetic neurns. At the start, it seemed likely that a- bungartxin was binding t a plasma membrane prtein, and its presence ver grwth cnes was cnsistent with current cncepts that new membrane was added at the grwth cne f elngating axns. Beynd that, the presence f a unique, identifiable plasma membrane prtein in sympathetic neurns (21) gave us the ptential t learn THE JOURNAL OF CELL BIOLOGY' VOLUME 81, 1979

11 FIGURE 11 Light micrscpe autradigraphs f chick sympathetic neurns incubated with 1251-bungartxin. The upper panel shws a neurn (N) and a nn-neurnal cell (F) as viewed with phase ptics. The neurn was grwing a lng axn and has a grwth cne (g) at its tip. In the lwer panel, the same field is shwn as viewed with brightfield illuminatin, and nly the expsed silver grains are visible. Nte that the grains are restricted t the neurn and are fund alng the axn extending t the grwth cne. Bar, 20 /~m. whether the grwth cne was, indeed, the site f insertin f new membrane prteins, and we began ur studies f the metablism f a-bungartxin receptrs in chick sympathetic neurns. After transferral f cultures f neurns int medium cntaining dense amin acids, there is a 2-h delay befre density-labeled receptrs appear in the plasma membrane. During this delay, re- CARBONEITO AND FAr, ibrourh Neurnal Membrane Synthesis, Insertin, and Turnver 565

12 TABLE III Sites f Insertin f Newly Synthesized t-bungartxin Receptrs int the Plasma Membrane f Sympathetic Ganglia and Axns Ttal receptrs New receptrs Labeling Perid Axns Ganglia Axns Ganglia h cpm cpm % % 4 3,234 86, ,968 13, ,997 49, The paravertebral sympathetic chains f chick embrys (11-13 d) were islated frm the embry and cut int individual ganglia. The ganglia were cultured fr 3-5 d and prduced an extensive utgrwth r "hal" f axns. The diameter f a ganglin and its hal was ften greater than 2 mm. Individual axns, hwever, were extensively branched and their lengths are best reflected in their rates f axnal elngatin f -50 g.m/hr. 3- t 5-day-ld cultures were incubated fr varius perids in medium cntaining density-labeled amin acids. The ganglia were then separated frm their axns with the aid f a dissectin micrscpe. The ganglia were incubated with 13q-a-bungartxin and the axns with l~5ia-bungartxin, bth at a cncentratin f 0.1 /zg/ml fr 1 h at 37~ Ttal receptrs reflect cpm f l~zi-abungartxin bund (axns) r 13q-c~-bungartxin bund (ganglia) after crrectin fr the difference in specific activities f the tw istpes. The percent f new receptrs was btained after radiactive a-bungartxinreceptr cmplexes were extracted frm axns r ganglia, stabilized with glutaraldehyde, and centrifuged in sucrse-deuterium xide gradients as described abve (Materials and Methds). ceptrs are being inserted int the membrane frm a previusly synthesized pl f unlabeled receptrs. Similar delays have been bserved between bisynthesis and the appearance f secretry prteins (28) and ther integral membrane prteins at the cell surface (1, 12, 34, 38). These delays generally are attributed t the time required fr pst-translatin mdificatin, packaging and transprt t the surface, prcesses that d nt require cntinued prtein synthesis (24). An internal pl f acetylchline receptrs that are precursr t surface receptrs has been lcalized by electrn-micrscpe autradigraphy t the Glgi apparatus f muscle cells, and it appears that integral membrane prteins traverse the same cellular pathway as secretry prteins befre they appear at the surface (15). Our attempts t measure the size and turnver f the intracellular pl have met with limited success. There is a large number f a-bungartxin binding sites, ~3 times the ppulatin in the plasma membrane, that are expsed by slubilizing the cells in Tritn X-100 (unpublished bservatins). We have nt determined hw much f this ppulatin represents precursr t the plasma membrane receptrs. Our kinetic measurements indicate that nly -5% f these sites shuld represent the metablically active internal pl. The hypthesis that the grwth cne is the majr site f additin f membrane is derived frm bservatins f membrane flw and axnal elngatin in grwing neurns (3, 4, 23, 25). We have tested whether the a-bungartxin receptr reflects the hypthesized additin f new membrane at the grwth cne. In experiments cmparing the insertin f a-bungartxin receptrs int the plasma membrane f axns and whle ganglia, and als in experiments measuring the insertin f a-bungartxin receptrs in cultures f dissciated neurns treated with cytchalasin B, the grwth cne des nt seem t be the exclusive r even the primary site f insertin f this plasma membrane prtein. On the ther hand, ur results are nt cnsistent with insertin exclusively int the plasma membrane f ganglin cell bdies r axn hillcks. We fund that the newly synthesized receptrs inserted int the plasma membrane during an 8-h perid had a distributin in cell bdies and axns identical t that f the entire receptr ppulatin. Receptrs inserted int cell bdy plasma membrane culd nt pssibly diffuse far enugh in the lipid bilayer t appear in ur pure axn fractin. Maximal diffusin rates fr membrane prteins are nearly tw rders f magnitude t slw. Likewise, rapid flw f bilayer, sweeping alng the receptr mlecules, is nt a likely mechanism fr transprt f receptrs tward the grwth cne. Presently available evidence suggests sme bulk flw f membrane in the ppsite directin. There are, at present, n data suggesting bulk membrane flw rapid enugh t distribute receptrs all ver the several millimeters length f the ganglin cells. Thus, we cnclude that it is mst likely that new receptrs are inserted at multiple sites and that the spatial distributin f receptrs reflects the distributin f insertin sites. An interesting suggestin has been made by Pfenninger and Bunge (31) fr the stepwise frmatin f neurnal plasma membranes. Accrding t their mdel intra-membrane particles are ~6 TIlE JOURNAL OF CELL BIOLOGY. VOLUME 81,1979

13 ~6ul~E 12 The effect f cytchalasin B n the structure f grwth cnes f grwing neurns. Chick sympathetic neurns that were 1 d in culture were treated with cytchalasin B (2 /zg/ml) fr 5 min at 37~ fixed, and prepared fr scanning electrn micrscpy. In neurns untreated with cytchalasin B (left), elabrate grwth cnes (g) with many micrspikes are frequently fund at the tips f axns. In neurns treated with cytchalasin B the fine prcesses cmprising the grwth cne are absent, leaving a truncated structure at the tip f the axn. In the presence f cytchalasin B, prcesses extending frm a nn-neurnal cell (F) have begun t bead and als t withdraw. Bars, 10/zm. synthesized and inserted int the surface primarily at the cell bdy, resulting in a gradient f membrane particles that decreases frm the cell bdy t the tip f the grwing axn. If there are sites fr insertin f membrane prteins alng axns as well as at the cell bdy as suggested in ultrastrucrural studies by Wessels et al. (36), then this mdel fits ur data fr the a-bungartxin receptr. The intriguing pssibility remains that, in the case f neurite extensin, new lipid bilayers, relatively deficient in membrane prteins, are inserted int the grwth cne plasma membrane. In fact, it culd be that membrane expansin and insertin f specific membrane prteins invlve different cellular mechanisms in general. After cells are pulse-labeled with dense amin acids, density-labeled a-bungartxin receptrs disappear expnentially with a half-time f 11 h. This decrease is nt due t instability f receptrs synthesized with dense amin acids since receptrs synthesized in nrmal medium disappear with the same kinetics when transferred t medium cntaining dense amin acids (unpublished bservatins, see als reference 18). The first-rder kinetics f turnver suggest that, regardless f age, receptrs turn ver at the same rate by a mechanism that randmly samples the surface ppulatin. The internalizatin culd itself ccur randmly, but ther steps in the life cycle f the receptr such as mvement in the plane f the plasma membrane r randmly distributed sites f insertin culd, as well, engender randmizatin. The evidence indicates that the receptrs that disappear frm the surface are degraded. We find that a significant fractin f l~i-ce-bungartxin used t label receptrs n intact cells is metablized t idtyrsine, a prtelytic breakdwn prduct f l~zi-a-bungartxin, and we pstulate that the receptr is als being degraded. The kinetics f idtyrsine prductin have been shwn t be a reliable indicatr fr the degradatin f skeletal muscle acetylchline receptrs CARBONE'ITO AND FAMBROUGH Neurnal Membrane Synthesis, Insertin, and Turnver 567

14 bo 2 / ce6',, 12 ~6 20,/ / r yi~ i z4 t 28 l~ ~ 12 i i6 Frchn Number J I J FIGURE 13 Effect f cytchalasin B n the insertin f newly synthesized a-bungartxin receptrs int the plasma membrane f neurns. Neurns were incubated fr 5 h in cytchalasin B (2/zg/ml) in medium cntaining dense amin acids. At the end f that perid, the a- bungartxin receptrs in the plasma membrane were labeled with l~i-c~-bungartxin (Ib-----O) and were layered n 25-40% sucrse-deuterium xide gradients alng with light, "marker" 13q-t-bungartxin-receptr cmplexes (9 Analysis f the sedimentatin prfiles shws that in neurns untreated with cytchalasin B (left), 23% f the surface receptrs are densitylabeled as cmpared with 21% f the surface receptrs in neurns maintained cntinuusly in cytchalasin B (right). Nte that cytchalasin B (2 /xg/ml) has a prnunced effect n grwth cne mrphlgy after nly 5 min. labeled with l~zi-a-bungartxin (2, 9) as well as ther membrane prteins (6, 22). Unfrtunately, a-bungartxin des nt bind t neurns irreversibly as it des t muscle. Because f its dissciability, we have nt been able t cmpare quantitatively the kinetics f idtyrsine prductin with a-bungartxin receptr turnver as measured in density-labeling experiments. Several ther pieces f evidence suggest that the turnver f receptrs is accmplished by internalizatin and degradatin. The prductin f idtyrsine and n larger radiactive peptide ; (Fig. 8) requires that the a-bungartxin be degraded very thrughly t single amin acid residues r that it be cleaved specifically at the peptide bnds n either side f a single tyrsyl residue. It seems unlikely that such a specific prtease exists n the cell surface whereas degradatin thrugh enugh t digest a prtein t single amin acids wuld prbably take place within the cell in sme intracellular cmpartment. Finally, we find that there is a small amunt f idtyrsine (11% f the ttal radiactivity assciated with the cells 10 h after labeling with l~zia-bungartxin) that is released frm neurns after they are slubilized and is presumably within the cells. Further evidence fr interirizatin cmes frm experiments demnstrating that degradatin is inhibited by CCCP and is highly temperature dependent (Fig. 9). In chick mytubes, acetylchline receptrs are degraded by a first-rder prcess that is bth energy and temperature dependent (9). Ultrastructural bservatins indicate that a-bungartxin-receptr cmplexes are internalized and subsequently degraded within the cell in secndary lyssmes (10). A similar mechanism has been demnstrated recently fr the turnver f lectin receptrs in the plasma membrane f cultured neurns (19), and it seems likely that this is the cellular pathway fr the degradatin f c~-bungartxin receptrs. The number f a-bungartxin receptrs in the plasma membrane f neurns increases % between day 1 and day 3 in culture. During this perid, grwth cnes are abundant and it is ur impressin that this is the mst active perid f axnal elngatin. The increase in receptrs appears t reflect the increase in surface area that ccurs during this perid. After -5 d in culture, the number f a-bungartxin receptrs decreases either by a reductin in the synthesis r by an increase in the turnver f receptrs. This sequence f events is superficially similar t the reductin in extrajunctinal acetylchline receptrs that accmpanies innervatin f muscle fibers. Hwever, we d nt knw whether the a- bungartxin receptr in sympathetic neurns is a synaptic prtein, althugh that appears t be the case fr sme ther neurns (27, 35), nr d we knw whether the decrease in receptrs is related t synapse frmatin amng sympathetic neurns in culture. The decrease in number f receptrs als culd be a result f increased spntaneus electrical activity in the neurns, just as activity in muscles mdulates the number f acetylchline receptrs (16). We wuld like t thank Drs. Kenneth Muller, Richard Rtund, Peter Devretes, and Mr. Jhn Gardner fr their valuable suggestins thrughut the curse f these experiments. We als thank Mr. Gregry Nelsn fr his help with the scanning electrn-micrscpy and Ms. Delres Smmerville and Ms. Barbara Thmas fr their excellent technical assistance. S. Carbnett is supprted by a Natinal Institutes f Health Pstdctral Fellwship. Research in the authrs' labratries is supprted in part by a grant frm the Muscular Dystrphy Assciatin. Received fr publicatin 18 Octber 1978, and in revised frm 5 February THE JOURNAL OF CELL BIOLOGy 9 VOLUME 81, 1979

15 REFERENCES 1. A'n~NsN, P. H., S. A. Mwa, and D. F. Scm, t~ts Assembly f vesicular stmatitus virus glycprtein and matrix prtein int HeLa cell plasma membranes. J. Ml. Bil. 102: BEan, D. K., and Z. HALL Fate f t~-liungartxin bund t acetylchline receptrs f nrmal and denervated muscle. Science (Wash. D. C.). 184: BRAY, D Surface mvements during the grwth f single explanted neurns. Prc. Natl. Acnd. Sci. U. S. A. 65: BaAV, D Branching patterns f individual sympathetic neurns in culture. J. Cell Bil. $6: CARaONETrn, S. T., D. M. FAMBIOUGH, and K. J. ML~.L~t Nnequivalence f a-bungurtxin receptrs and acetylchline receptrs in chick sympathetic neurns. Prc. Natl. Acad. Sci. U. S. A. 75: C~ES'r~R, G., and S. COHEN mi-labeled human epidermal grwth factr: binding, internalizatin, and degradatin in human fibrbiasts. J. Cell Bil. 71: C'nALAZONrnS, A., L. A. GaEENE, and M. NntENE~tG Electrphysilgical characteristics f chick embry sympathetic neurns in dissciated cell culture. Brain Res. 68: D^~ELS, M. P Clchicine inhibitin f nerve fiber frmatin in vitr. J. Cell Bil. $3: DEv~rrs, P. N., and D. M. F~anuGn Acetylehline receptr turnver in membranes f develping muscle fibers. J. Cell Bil. 6S: DEvius, P. N., and D. M. F~anuGx Turnver f acetylchline receptrs in skeletal muscle. Cld Spring Harbr Symp. Quant. Bil. 40: DEv~'~s, P. N., and D. M. F~aOUGH Synthesis f acetylchline receptrs by cultured chick mytubes and denervated muse extensr digitcum lngns muscles. Prc. Nd. Acad. Sci. U. S. A. 73: DEVlt~crms, P. N., J. M. G.~mN~a, and D. M. F.,O~aOUGH Kinetics f bisynthesis f acetylchline receptr and subsequent incrpratin int plasma membrane f cultured chick skeletal muscle. Cell. 10: DvaA~, D. J., E. GluI.~, and C. K~SON Islatin f specific neurnes by affinity methds. Nature (Lnd.). 271: DVOEAK, D., E. Gmr~s, J. Lr~n, and C. KaDSOS Develpment f receptrs fr t-bungurtxin in chick embry sympathetic ganglin neurns in vitr. Life Sci. 22: Fm,~aOUGH, D. M., and P. N. D~wt~rzs Newly synthesized acetylchline receptrs are lcated in the Glgi apparatus. J. Cell Bil. 76: FAuaauGH, D. M Cntrl f acetylchline receptrs in skeletal muscle. Physil. Rev. g9: G~mN~, J. M., and D. M. F~ROUGH Prperties f acetylchline receptr turnver in cultured embrynic muscle cells. In Maturatin fneurtransmissin. A. Vernadakis, E. Giacbini, and G. Fdgam, editrs. Kurger, Basel GAIDNEa, J. M., and D. M. F~auGn Acetylchline receptr degradatin measured by density labeling: Effects f chlinergic ligands and evidence against recycling. Cell. 16: GONATXS, N. K., S. U. Kns, A. Sn~EEl~, and S. Avl~s Internalizatin f lectim in neurnal GERL. J. Cell Bil. 73: GIEENE, L. A Binding f a,-bungartxin t chick sympathetic ganglia: Prperties f the receptr and its rate f appearance during develpment. Brain Res. 111: GaEEN~, L., A. J. Srtxwsm, Z. VOGEL, and M. W. N~mtG t-bunpttxin used as a prbe fr acetylchline receptrs f cultured neurnes. Nature 245: HuB~m, A. L., and Z. A. CnN Externally dispsed plasma membrane prteins: Metablic fate f idinated plypeptides f muse L cells. J. Ceil Bil. 64: HUGHES, A The grwth f embrynic nemites-a study n cultures f chick neural tissues. J. Anat. 87: J.'O41ESON, J. D. and G. E. PALi~.OE Intracellulur transprt f secretry prteins in the pancreatic excrine cell. lii. Dissciatin f intracelluiar transprt frm prtein synthesis. J. Cell Bil. $9: KODA, L. Y., and L. M. P,~t~w Membrane marker mvement n sympathetic axns in tissue culture. J. Neurbil. 7: KOUVELAS, E. D., M. A. D:CnTe~, and L. A. GaEENE Chick sympathetic neurns develp receptrs fr a-bungarntxin in vitr, but the txin des nt blck nictinic receptrs. Brain Res. 155: LEr,rrz, T. L., and J. CHem, Lcalizatin f acetylchline receptrs in central synapses. J. Cell Bil. 75: PAL~OE, G Intracellular aspects f the prcess f prtein synthesis. Science (Wash. D. C.). 189: P~t~w, L. M., and M. G. ~EE Effects f a nervegrwth factr, embry age and metablic inhibitrs n grwth f fibres and n synthesis f ribnucleic acid and prtein in embrynic sympathetic ganglia. J. Neurchem. 18: P^Tmcx, J., and W. B. STALLCVP Immunlgical distinctin between acetylchline receptr and the a-bngartxin-binding cmpnent n sympathetic neurns. Prc. Natl. Acad. Sci. U. S. A. 74: P~N~NGe~t, K. H. and R. P. BUNCE Freeze-fracturing f nerve grwth cnes and yung fibers: a study f develping plasma membrane. J. Cell Bil. 65: SmUN, W., L. A. G~tEENE, D. CAItrEm'ea, Z. VOGEL, and A. Srrxwsm Aplysia acetylchline receptrs: blckade by and binding f ~-liungartxin. Brain Res. 72: VAION, S., and C. RAmaN Dissciatin, fractinatin and culture f chick embry sympathetic ganglinic cells. J. Neurcytl. 1: VrreTrA, E. S., and J. W. Una Immunglbulins and allantigem n the surface f lymphid cells. Bichim. Biphys. Acta. 415: VO~EL, Z., G. J. ~NE~', A. LtNG, and M. P. D~XELS Identificatin f synaptic acetylchline receptr sites in retina with perxidase-labeled a-bungartxin. Prc. Nd. Acd. Sci. U. S. A. 74: WeSSEt.LS, N. K., R. P. NU~J.L, J. T. WeeNN, and S. JOHNSON Differential labeling f the cell surface f single ciliary ganglin neurns in vitr. Prc. Nd. Acad. Sci. U. S. A. 75: YAMAD^, K. M., B. S. SPOON~ and N. K. WeSSELtS Ultrastructure and functin f grwth cnes and axns f cultured nerve cells. J. Cell Bil. 49:614~ YOUNG, R. W Bigenesis and renewal f visual cell uter segment membranes. Exp. Eye Res. 18: CA~ONETrO AND FA~mROUGH Neurnal Membrane Synthesis, Insertin, and Turnver,569