Differences in the distribution of specific glycoproteins among the regions of a single identified neuron

Brain Research, 239 (1982) 489-505

489

Elsevier Biomedical Press

DIFFERENCES IN T H E DISTRIBUTION OF SPECIFIC G L Y C O P R O T E I N S A M O N G T H E REGIONS OF A SINGLE I D E N T I F I E D N E U R O N

RICHARD T. AMBRON Department of Anatomy and Cell Biology and Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York, N Y 10032 (U.S.A.)

(Accepted October 8th, 1981) Key words': membrane glycoprotein -- Aplysia - - external surface giant neuron -- glycopeptides --

axonal transport

SUMMARY Neurons are highly differentiated cells whose various regions must differ in macromolecular composition. This is demonstrated in the present study which shows that specific membrane glycoproteins are routed to particular sites in the cell. When [3H]fucose or [3H]N-acetylgalactosamine are injected into R2, the giant neuron of Aplysia, they are incorporated into relatively few glycoproteins, several of which can be readily identified from cell to cell. Because R2's cell body is so large, its surfac~ membrane can be isolated 94 ~o free of cytosol by manual dissection. The purity of the membrane was assessed by checking the distribution of the enzyme choline acetyltransferase. R2's axon can also be analyzed separately from the cell body. At 24 h after injection, SDS polyacrylamide gel electrophoresis shows that glycoprotein-I (mol. wt. 180,000) is the major labeled component of the external membrane where it is enriched relative to the content of the other cytosolic membranes. In contrast, glycoprotein-V (mol. wt. 90,000) predominates among the membranes of the axon. The disposition of glycoprotein-I in the external membrane was indicated by exposing R2 to low concentrations of pronase in situ 24 h after injection. Labeled glycopeptides were released from R2's surface and gel filtration and high voltage electrophoresis indicated that some of these were derived from glycoprotein-I. Examination of the isolated surface membrane after proteolysis showed a reduced amount of labeled glycoprotein-I. Consistent with these findings, a glycoprotein of similar molecular weight as component-I was labeled when R2 was treated with galactose oxidase and potassium borotritide. These results indicate that the carbohydrate moieties of glycoprotein-I extend from R2's surface.

0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press

490 INTRODUCTION Cell polarity is a form of intraceltular differentiation whereby specific areas of the cell have acquired specialized functions. Secretory and absorptive epithelia are well-studied examples where the apical surface membrane has markedly different functional and structural properties than does the membrane at the basal end of the cell 26,27. Neurons are an extreme example of polarized cells and, because of the secretory face (synapse) can be at enormous distances from the cell body, they are good models to study how regional differences are established and maintained 4. It would be expected that the underlying bases for regional distinctions in structure and function are differences in macromolecular composition. Since the neuronal soma is the site of macromolecular synthesis, the maintenance of the polar character of a neuron is primarily a matter of logistics, namely the distribution of newly synthesized components to their appropriate site in the cell. One aspect of this process that has received considerable attention is the movement of membraneassociated proteins and glycoproteins via axonal transport 31. We have been studying the biosynthesis and distribution of membrane glycoproteins in R2, the giant neuron of Aplysia californica 1-6. R2 is particularly well suited for these studies because its large size permits the injection of radioactive precursors directly into the cell. Moreover, its various regions are anatomically accessible for biochemical, morphological, and electrophysiological studies. When [ZH]fucose or [3H]N-acetylgalactosamine are injected into R2, relatively few labeled membrane glycoproteins are synthesized2,3,6. Several of these can be unequivocally identified and therefore their distribution in the neuron can be followed. In this paper we have compared the newly synthesized glycoproteins that appear at the external membrane of the cell body with those found in the axon. Our results show that specific glycoproteins are directed toward given areas of the cell. We have also identified one glycoprotein that is transported to the cell surface and whose carbohydrate moieties are exposed to the outside. MATERIALS AND METHODS

Aplysia californica, weighing 50-250 g were supplied by Pacific Bio-Marine (Venice, California) and maintained at 15 °C.

Intrasomatic injection and fractionation of R2 L-[l,5,6-ZH]Fucose (4.8 Ci/mmol) or [(G.3H)]N_acetylgatactosamine (25 Ci/mmol) (New England Nuclear, Boston, MA) were prepared and injected into R2 as described 3. After injection, the nervous system was maintained at 15 °C in an artificial seawater supplemented with amino acids and vitamins 1°. Before biochemical analysis, R2 was impaled again to ensure that the cell was in good condition and then the connective tissue sheath was removed from the dorsal surface of the ganglion. R2's cell body was dissected free of its axon, added to unlabeled Aplysia nervous tissue and homogenized in a groundglass tissue grinder (Micrometric Instruments, Cleveland,

491 OH). The homogenate was centrifuged at 105,000 g to isolate a crude membrane fraction a. Soluble [aH]glycoproteins were precipitated from the supernatant with l0 trichloroacetic acid/1 ~o phosphotungstic acid. The membrane pellet was sequentially extracted at 4 °C with chloroform-methanol mixtures to remove [aH]glycolipidZ2. [ZH]glycoproteins were extracted from the pellet with sodium dodecyl sulfate (SDS), 22-mercaptoethanol (2-MSH) at 70 °C and the small amount of residual glycoprotein solubilized with 90 ~o aqueous formic acid. The sum of the radioactivity in the SDS and formic acid extracts is the particulate glycoprotein content of the neuron 3.

Dissection of R2' s cell body after injection R2's soma, free of its axon, was transferred to a 50 #l drop of supplemented seawater maintained at 4 °C. A single opening was made in the membrane using a fine tungsten needle 20. Within 10 rain the nucleus emerged, followed by the orangepigmented cytoplasm. Residual cytoplasm was removed using needles; the dissection is considered complete when the envelope is free of pigment. The envelope is then washed in a drop of 50 mM Tris-HCl (pH 7.6). In some experiments the solutions contained 0.1 ~ sodium deoxycholate to release adherent proteins. Nucleus, envelope, and cytoplasm were collected separately with micropipettes, added to carrier tissue, and fractionated as described above. When the envelope was to be examined directly by gel electrophoresis, it was placed on a 0.45 #m, millipore filter (Millipore), washed with 10 ml buffer, and extracted with hot SDS-2-MSH. Treatment of R2 with proteolytic enzymes in situ The abdominal ganglion, containing an injected R2, was pinned in a chamber and the sheath above the cell dissected away. The tissue was bathed in 2.0 ml of artifical seawater containing either 10 units of pronase (Calbiochem., San Diego, CA 450 U/rag) or 0.04~ DCC-trypsin (Miles Labs, Elkhart, 1N) for 1 h at 22 °C. R2's resting potential and elicited antidromic action potentials were monitored throughout the digestion to ensure that the membrane was intact. The bath was then carefully removed, the tissue washed 3 × with an equal volume of ice-cold seawater, and the cell body isolated and dissected. The combined bath and washes from the digestion were lyophilized, reconstituted in one-fifth volume and analyzed by gel filtration on columns of Sephadex (Pharmacia, Piscataway, N J) in 0.1 M pyridine-acetate (pH 4.7). In experiments using trypsin, soybean trypsin inhibitor was added to the combined bathing medium to a final concentration of 1 mg/ml. Likewise, all solutions used in dissecting the cell body also contained the inhibitor. Galactose oxidase-potassium borotritide. The activity of galactose oxidase (Sigma, St. Louis, MO) was assayed using a peroxidase-O-tolidine coupled system and measuring the increase in A425 resulting from the oxidation of galactose zo. We found that the enzyme had low activity in the high ionic strength solutions normally used to maintain Aplysia tissues, so all our experiments were done in 0.8 M sucrose, 50 mM Tris-HC1 (pH 7.6). Before use the enzyme was heated at 50 °C for 30 rain to destroy

492 proteolytic activity33. R2's cell body was exposed in situ and galactose-oxidase (t2 units) and potassium borotritide (0.5 mCi, Amerhsam/Searle, Arlington Heights, IL, 12.8 Ci/mmol) were added in a final volume of 0.2 ml. After 1 h at 22 °C, the tissue was washed and R2's cell body dissected from the ganglion and fractionated

Analytical procedures SDS polyacrylamide get electrophoresis. Membrane fractions containing [3H]glycoprotein were analyzed using the method of Weber and Osborn 36(gel system l) or Laemmli19 (gel system 2) as described 6. We also used 4-30 ~o polyacrylamide gradient gels (Pharmacia) run for 20 h in 0.1 M phosphate buffer (pH 7.2) containing 0.1 ~ SDS (gel system 3). Dansylated beef serum albumin was included in each sample to provide internal markers of molecular weightlL Radioactivity on gels was measured by sectioning the frozen gel into 1 mm segments, dissolving the segment with HeO2, and counting by liquid scintillation. Pronase digestion of f3H-/glycoproteins. Acetone-precipitated [3H]glycoproteins were digested with pronase for 72 h at 37 °C as described~. The pronase was incubated at 37 °C for 30 min prior to digestion to destroy any other enzyme activities that might be present. Digests were examined by gel filtration either on a column of Sephadex G-50 (1.5 × 25 cm) or Sephadex G-100 (0.9 × 104 cm) (Pharmacia) in 0.1 M pyridine acetate (pH 4.7). High voltage paper electrophoresis. [aH]glycopeptides obtained after pronase digestion were analyzed on Whatman No 3MM paper using a cooled high voltage electrophoresis apparatus at 25 V/cm for 6 h in formic acid-pyridine buffer (pHl .9) 12. The electropherogram was cut into 1 cm segments and counted. Acid hydrolysis. Samples, hydrolyzed in 4 N HC1 for 6 h at 100 °C were examined by paper chromatography on Whatman No. 1 in the descending direction using ethyl acetate-pyridine-acetic acid-water (5:5:1:3). Sugar standards were visualized with silver nitrate 2~. Isolation of component-L [ZH]component-I was isolated by extracting the lipiddepleted 105,000 g membrane pellet from an injected R2 with lithium diiodosalicytate (Eastman Kodak, Rochester, NY),L The extract was partitioned with an equal volume of 50 ~ aqueous phenol and component-I recovered from the upper (aqueous) phase after gel filtration. [3H]components II-V, found in the phenol phase, were isolated by acetone precipitation. Determination of choline acetyltransferase activity in dissected R2. Enzyme activity was measured according to a published procedurO 2. R2's cell body was dissected in 25 #1 0.2 M NaC1, 0.02 M sodium phosphate buffer (pH 7.4). The washed external envelope was added to a tube containing 25 /A buffer and the cytoplasm transferred to another tube. [3H]acetylCoA (New England Nuclear) was added at a final concentration of 0.12 mM, tbllowed by incubation at 35 °C. At 0, 15 and 30 min, 5 #1 samples were removed, spotted on Whatman 3 MM paper and electrophoresed for 30 min at pH 4.7 in pyridine-acetate buffer12. The position of [aH]acetylcholine on the electropherogram was indicated by standards. These areas were cut out and counted by liquid scintillation. Under our conditions of counting 181,000 cpm represented 1 nmol of [3H]acetylCoA.

493 Six R 2 ' s were a n a l y z e d separately a n d for each the rate o f synthesis o f [3H]acetylcholine was linear for 30 rain. W e f o u n d 6.2 ± 0 . 6 % (S.E.M.) o f the activity associated with the external envelope fraction. O n the average, each cell synthesized 2.4 ~ 0.2 nmol acetylcholine per hour. RESULTS

D&tribution of newly synthesized glycoprotein in R2 The cell body. R 2 ' s s o m a is f o u n d j u s t b e n e a t h the connective tissue sheath that covers the dorsal surface o f the a b d o m i n a l ganglion. Because o f its favorable location and large size, the s o m a is readily r e m o v e d from the ganglion (Fig. I A). If a small opening is m a d e in the isolated cell, the nucleus will gradually emerge (Fig, 1B)

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Fig. 2. SDS polyacrylamidegel electrophoresisof [aH]glycoproteinsfrom the externalenvelope(A), cytoplasmic membranes (B), and axonal membranes (C), of an R2 15 h after injection of [3H]fucose. The envelope was extracted directly with gel buffer, while the cytoplasmic rncmbranes and axonal membranes were fractionated at 105,000 g (see Materials and Methods) prior to eleetrophoresis in gel system 1. A similar enrichment of glyeoprotein-III was observed 15 h after injection Of [aH]N-acetylgalactosamine. T, trimer; D, dimer: M, monomer of beef serum albumin. followed by the cytoplasm (Fig. 1C). The external envelope can then be washed to remove additional cytoplasmic components (Fig. 1D). In the electron mierosope, the isolated envelope is seen to consist of R2's plasma membrane, with its coating of glial cells, surrounding small pockets of trapped cytoplasm and organelles. While electron micrographs indicate that most cytoplasm is removed from the envelope during dissection, this was assessed quantitatively by assaying the soluble enzyme choline acetyl transferase (see Methods)., The results from 6 individual cells indicate that, on the average, 94 ~ of R2's cytoplasm is removed from the membrane. A small number of integral membrane glycoproteins become labeled in R2 after intrasomatic injection of [all]sugar precursors. Certain of these have been consistently identified in over 60 injected R2's using a variety of gel systems and have been assigned roman numeralsa,6. To see which newly synthesized glycoproteins might be constituents of R2's external membrane, we compared by SDS polyacrylamide gel electrop-

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Fig. 3. SDS polyacrylamide gel electrophoresis of ['~H]glycoproteins from the external envelope (A), cytoplasmic membranes (B), and axonal membranes (C), of R2 24 h after injection of [3H]fucose. R2 was removed from the ganglion, dissected, and processed as described in the legend to Fig. 2. In the electropherogram from the cytoplasm, component-] appears as a shoulder on component-f1 (arrow). horesis the [3H]glycoproteins of the envelope with those of the cytoplasmic membranes*. The pattern depended upon the time after injection : in 15 h cells componentlII (tool. wt. 135,000 daltons) predominated in the envelope fraction where it was enriched relative to the composition of the cytoplasmic membranes (Fig. 2A, B). In contrast, [3H]glycoprotein-1 (tool. wt. 180,000 daltons) was the major surface component in cells 24 h after injection (Figs. 3A, B and 8A). In all these experiments, qualitatively and quantitatively similar results were obtained after injection o f either [3H]fucose or [3H]N-acetylgalactosamine. We examined the surface membrane from 7-11 individual cells at each time-period and noticed a reciprocal relationship between the amounts of labeled I and I I l : when one of the glycoproteins predominated, the other was almost always diminished (Table I). Component-V was also found in the

* The nuclear membrane was not included in these studies. The isolated, intact nucleus contained less than 2% of the total radioactivity in the cell.

496 TABLE I Distribution of radioactivity among the glycoproteins of the external envelope of R2

At the time indicated after injection, R2's cell body was isolated and dissected as described in Materials and Methods. The external envelope was either extracted directly in gel buffer, or was fractionated prior to electrophoresis. The distribution of radioactivity along the gel was plotted on graph paper as a function of the distance moved. The area under components I, III, and V, identified by standards, was cut out and compared by weighing. There was no difference in values between cells injected with fucose or N-acetylgalactosamine. For representative profiles, see Figs. 2 and 3. Time

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envelope fraction of most cells (Table I), but it was never as p r o m i n e n t as c o m p o n e n t s I a n d IIl. A biochemical analysis of the m e m b r a n e fractions is presented in Table II. A p p r o x i m a t e l y 80 ~o o f the radioactivity in the isolated surface m e m b r a n e is macromolecular, a n d more t h a n 90 ~ of this is membrane-associated glycoprotein a n d glycolipid. By a b o u t 15 h after injection, i n c o r p o r a t i o n of glycoprotein into the external m e m b r a n e fraction reaches a m a x i m u m of approximately 25 ~ of the total m e m b r a n e [aH]glycoprotein, a n a m o u n t that remains c o n s t a n t for as long as 48 h which is the longest interval examined. The axon. While specific glycoproteins are t r a n s p o r t e d to the cell surface, other glycoproteins are rapidly exported from the cell body into the axon 1. E x a m i n a t i o n of TABLE II Distribution of [ZH /-N-acetylgalactosamine in R2 dissected after injection

After injection, R2's cell body was isolated and dissected. The external envelope and the cytoplasm were fractionated separately at 105,000 g. Soluble glycoproteins were precipitated from the supernatant with TCA-PTA and the membrane pellet was extracted with chloroform-methanol to obtain pH]glycolipid and with SDS and then formic acid to obtain [3H]glycoprotein (see Materials and Methods). Previous experiments have shown that the identified glycoproteins are integral membrane componentsL The composition of the labeled lipid can be found in ref. 32. Distribution ( % of total radioactivity) 15h (n = 4)

A. Membranes Glycoprotein Glycolipid B. Soluble Glycoprotein C. %oftotalin particulate macromolecules

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497

axonal membrane glycoproteins from the dissected cells showed a predominance of the lower molecular weight components, especially component-V (Figs. 2C and 3C). Similar patterns are found at all injection sizes so that the selectivity is not due to variation in the size of precursor pools. The carbohydrate moieties of glyeoproteins I and III are exposed on the outer surface of the external membrane Glycoproteins I and IlI are integral components of R2's membranes 3,5. As surface macromolecules, we would expect that their carbohydrate moieties face the outside of the cell 28. Two approaches were used to test this hypothesis. The first involved treatment of R2 in situ with low concentrations of proteolytic enzymes. Exposure of R2's cell body to pronase. R2's cell body was exposed to pronase at 15, 24, or 48 h after injection (see Materials and Methods) in order to see if [3H]carbohydrate moieties from components I and llI could be released from the surface. After digestion, gel filtration of the bathing medium on Sephadex G-50 revealed both large and small [3H]glycopeptides, as well as radioactivity of low molecular weight (Fig. 4B). The large [3H]glycopeptides, most of which appeared in the excluded volume, were not present in the bath from the control cells (Fig. 4A). Additional treatment of these glycopeptides with pronase for as long as 72 h did not reduce their size. It is unusual for such large glycopeptides to be found after pronase digestion, and only component-I among R2's glycoproteins releases glycopeptides of this size under similar conditions of treatment 5. When the external envelope from intact R2's treated with pronase 24 h after

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injection was isolated and examined, there was a decrease in the amount of labeled component-I (compare Fig. 3A with Fig. 5). In some experiments nearly all of the high molecular weight glycoproteins were missing, including component-Ill (Fig. 5B). We have never seen a pattern like this in the envelope from untreated cells. Although pronase obviously had access to surface components, not all of the [3H]glycoprotein associated with the envelope fraction was susceptible to digestion, and in 5 experiments only 25 O//oof the total membrane [ZH]glycoprotein was released. Much of the remaining radioactivity was in component-V (Fig. 5) suggesting that this glycoprotein is not exposed on the surface. Previous experiments have shown that isolated component-V can be digested by pronase ~. Analyses of[31-Ijglycopeptides afterpronase digestion. Of all the glycoproteins in R2, component-I is the best characterized. It can be isolated using the chaotropic agent LIS (see Materials and Methods), it contains both fucose and N-acetylgalactosamine, and its glycopeptides have been partially analyzed5. Moreover, component-I would appear ro be a single species of protein since it migrates as a single band under a variety of different electrophoretic conditions. In an effort to determine which portion of the molecule is exposed on the surface of the celt, component-I was

499 isolated, treated with pronase, and its [3H]glycopeptides compared with those released from R2's surface. Also included for comparison were glycopeptides obtained by digesting a mixture of labeled components II-V (see Materials and Methods). The [3H]glycopeptides were examined by gel filtration on Sephadex G-100 (Fig. 6A). Three peaks containing large glycopeptides (arrows Fig. 6A) were present in both the digest from component-I (Fig. 6B) and the bath, but were absent in the profile from the mixture of glycoproteins (Fig. 6C). The bulk of the released [3H]glycopeptides, however, were contained in two major peaks. The largest corresponded to only a portion of the labeled glycopeptides from component-I. When the fractions from the column (bar, Fig. 6) were analyzed by high voltage electrophoresis (Fig. 7) the pattern of labeled glycopeptides from the bath was less complex than that froln either component-I or the glycoprotein mixture. Several negatively charged glycopeptides from component-I were barely detectable in the material from the bath, indicating that the pronase does not have access to the entire component-I molecule in the membrane. A negative charge at pH 1.9 is suggestive of a sulfate ester which is common among the glycoproteins of mammalian brain 24. Studies with trypsin. Treatment of R2 with trypsin also released [3H]glyco-

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Fig. 7. High voltage paper electrophoresis of [aH]glycopeptides after pronase digestion of R2 (A), component-I (B), and components I1-V (C). Fractions from the Sephadex G-100 column (Fig. 6) were pooled and electrophoresed at pH 1.9 as described in Materials and Methods. peptides into the bath, but it was not as effective as pronase : in 5 experiments on 24 h cells only about 6 ~ of the total [aH]glycoprotein associated with external membrane glycoprotein was released. Examination of the membrane after digestion showed little if any decrease in the amount of [aH]component-I (Fig. 8). This result is explained by recent experiments in which isolated component-I was found to be resistant to trypsin (unpublished). The amount of component-Ill did appear to be reduced by the trypsin, thus accounting for the glycopeptides released. The same material is released by treatment of 15 h cells also (not shown). Labeling of surface glycoconjugates using galactose oxidase. As another, independent means of determining whether or not components I and IIl have glycopeptides extending from R2's surface, we exposed 6 R2 cell bodies in situ to galactose oxidase and potassium borotritide (see Materials and Methods) 33. This treatment resulted in a two-fold increase in radioactivity incorporated into membrane glycoproteins and a three-fold increase into glycolipid when compared to controls. SDS gel electrophoresis of the labeled glycoproteins revealed at least nine major radioactive bands, one of which had a migration similiar to that of component-I, and another to component-IIl (Fig. 9). In contrast, no band corresponded to component-V, providing additional data against V being a surface glycoprotein of the cell body. Several bands that did not correspond to any of R2's gtycoproteins were also present, and these presumably are glial surface components. Radioactivity that was incorporated in the absence of the enzyme was shown by gel electrophoresis to be diffusely distributed (Fig. 9B). Acid hydrolysis of membranes labeled in the presence of the enzyme, followed

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Fig. 9. pH]glycoproteins labeled by exposure of R2 to potassium borotritide in the presence (A) or absence (B) of galactose oxidase. Lipid-depleted membranes were electrophesed on a 4-30% linear polyacrylamide gel in system 3. See Materials and Methods for details. The position of components I, lII, and V was determined by running an SDS extract from an injected R2 on a parallel gel.

502 by paper chromatography, revealed labeled galactose and galactosamine, as well as three other labeled compounds that could not be identified. This is not unexpected since sugar derivatives such as 3-0-methylgalactose are present in invertebrate glycoproteins 14 and others are likely to exist. DISCUSSION

Distribution and function of glyeoproteins in R2 We now know the immediate destination of 3 membrane glycoproteins synthesized by R2. Component-V (mol. wt. 90,000) enters the axon where it is rapidly translocated towards synapses (Figs. 2 and 3 and ref. 1). Component-III (tool. wt. 135.000) appears on the surface of the cell body, but also in the axon. and we are presently examining R2's axolemma to see if it is a constituent of the axonal surface as well. The location of component-II1 in the two regions indicates that it mediates a common function in the cell body and axon (see below). Component-l. on the other hand, is the major somatic glycoprotein of R2. Analyses of gel patterns from the cell body and axon indicate that a minimum of 85 % of the total [ZH]component-I in the neuron remains in the cell body. We do not know how component-I is diverted to the surface while at the same time component-V is exported into the axon. The mechanism underlying the process, however, is responsible for maintaining the neuron in a differentiated state. Recent evidence indicates that both components I and V are constituents of vesicles, and these presumably are responsible for the transport of the glycoproteins 5,35. One possibility being considered is the conversion of component I to component V in R2"s axon. The proteolytic processirtg of transported proteins occurs in Aplysia axons ~z and other modifications have also been reported 4. Also m support of this hypothesis is the apparent similarity in [3H]glycopeptides among R2's glycoproteins (Fig. 7}. Since axoplasm can be extruded from the right connective a2, this idea can be tested directly. There are a number of other possibilities. For example, when epithelial cells are infected with influenza or vesicular stomatitis virions, the glycoprotein of the viral coat for the influenza virus appears only at the apical surface o f the host cell while that of the VSV appears only at the basolateral region ~7. This is clearly divergence, and not proteolytic conversion, and involves separate transport pathways in the cell. Likely candidates for the molecular basis of the transport would be microtubules or microfilaments, fibrous proteins that have been associated with the movement of materials in cells 31. Agents such as colchicine 31, and the cytochalasins 2x, among others, disrupt these polymers and block their action in the cell. By employing these inhibitors in cur studies it should be possible to test for the involvement of the polymers in the transport of components I and V to their respective sites in R2.

Glyeoproteins of R2's external membrane The evidence that component-I is on the external surface of R T s cell body appears convincing. It is the predominant labeled species m the envelope fraction at 24 h after injection (Fig. 3) and a glycoprotein with the s a m e mobility on gels is found

503 after labeling surface components with galactose oxidase-KB3H4 (Fig. 9). While glycoproteins on the glial cells closely associated with R2 would also be labeled by this method, previous results have shown that they do not have a glycoprotein as large as component-I 3. Moreover, treatment of R2 with pronase after injection released [3H]glycopeptides into the bath. Under these conditions, the amount of [aH]component-I in the membrane was reduced (Fig. 5), and analyses are consistent with at least some of the released glycopeptides being derived from component-! (Figs. 6 and 7). Component-lll also seems to be a surface glycoprotein, but a transient one in that it is usually predominant at 15 h after injection and almost gone by 24 h (cf. Figs. 2 and 3). A glycoprotein similar in size to component-III is labeled by the galactoseoxidase treatment but only a relatively small amount of [H]glycopeptide attributable to component-Ill is released from 24 h R2's by trypsin or pronase. These results could be accounted for if glycoprotein III has both a high turn-over rate and a high specific activity, but quantitatively it is a minor component of the membrane. We must point out that our labeling studies actually follow only the sugar moieties of the glycoproteins. Although it is likely that the fate of the saccharide portion and that of the polypeptide are the same, this has not yet been demonstrated. The finding that components I and III are on R2's surface is an important first step in understanding the function of these glycoproteins. R2's external membrane containes receptors for acetylcholineis and glutamatO 7 and, unlike the somatic membrane of mammalian cells, is electrically excitable, meaning that it has the ion pumps and channels necessary to support an action potentiap6, 34. In particular, R2's somatic membrane has a high concentration of calcium channels. R2's axolemma, on the other hand, contains receptors for dopamine 7 and serotonin 11 as well as a high concentration of sodium channels 1~. Proteins, such as those of the potassium channel, are found in both membranes. This overall asymmetry is exactly paralleled by the 3 glycoproteins we have been studying, and it would not be unreasonable to believe for example, that component-I played some role in electrical events at R2's surface. Many important neuronal constituents, including subunits of the acetylcholine receptor ~9, Na+K + ATPase 9 and cholinesterase a7 are glycoproteins and the saxitoxin-binding component of the sodium channel may also be a glycoprotein 8. The molecular nature of these molecules in Aplysia is largely unknown. We have recently raised an antibody to component-I 2~ that can be used as a specific probe to see if binding to component-I on the surface interferes with electrical events in R2. ACKNOWLEDGEMENTS The author wants to thank Mr. David Weissman for his able technical assistance and Drs. M. D. Gershon, M. F. Maylie-Pfenninger, Karl Pfenninger and James H. Schwartz for their critical reading of the manuscript. I thank Dr. Schwartz also for helping with the acetylcholine assay. This work was supported by National Institute off Health Research Grant NS 14555 and by Research Career Development Award NS-00350.

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