Rapid axonal transport of [3H]fucosyl glycoproteins in the goldfish optic system

BRAIN RESEARCH

327

RAPID AXONAL TRANSPORT OF [aH]FUCOSYL GLYCOPROTEINS IN THE GOLDFISH OPTIC SYSTEM

DAVID S. FORMAN, BERNICE GRAFSTEIN AND BRUCE S. M c E W E N

Laboratory of Neuropharmacology, NIMH, William A. White Bldg., St. Elizabeths Hospital, Washington, D. C. 20032, Department of Physiology, Cornell University Medical College, New York, N. Y. 10021, and The Rockefeller University, New York, N. Y. 10021 (U.S.A.) (Accepted July 4th, 1972)

INTRODUCTION

Studies with radioactive amino acids have shown that most of the proteins in axons and nerve endings are synthesized in the nerve cell body and then transported to the periphery17,22,2L A general finding of these studies is that proteins are axonally transported in two main waves: the bulk of the protein moves at a 'slow' rate close to 1 mm/day, while a smaller fraction is transported at a rate more than 100 times as fast 2~,29. Fewer studies have examined the movement of specific proteins or classes of proteins. Glycoproteins are a class of proteins which are of special interest because of their strategic location at the neuron cell surface 6,34,40,43,45. When considering axonal and synaptic glycoproteins, one must ask whether the carbohydrate moieties (like the amino acids) are incorporated into the protein molecules in the perikaryon and then axonally transported, or whether carbohydrates are added to the polypeptides in the axons and endings. We have previously used [aH]glucosamine to demonstrate the rapid axonal transport of glycoproteins in the goldfish visual system21. However, there are disadvantages in using glucosamine to label glycoproteins. The metabolism of glucosamine is complex, since one expects that the labeled material will be converted to N-acetylglucosamine, N-acetylgalactosamine, and sialic acidsla; radioactivity should then be incorporated into sulfated acid mucopolysaccharides as well as into heteroglycoproteins, and into glycolipids, specifically gangliosidesl,11,20, 4a. On the other hand, the use of L-fucose as a precursor for labeling glycoproteins should circumvent the complexity presented by glucosamine50. In most tissues, including brain, fucose is incorporated into glycoproteins almost exclusively as fucosea,12,a2,41,5°. The brain does not contain measurable amounts of fucolipid to and does not incorporate labeled fucose significantly into lipidsa2,41, 50. Acid mucopolysaccharides contain very little or no fucoseg,as. In the experiments reported here, we studied the axonal transport of labeled glycoproteins in goldfish optic fibers after an intraocular injection of [aH]fucose. A preliminary report of these experiments has been published aS. Brain Research, 48 (1972) 327-342

328

D.s. I-ORMANel al.

METHODS

Two sizes of goldfish (Carassius auratus) were used in these experiments: 'small' fish (body length 4-7 cm) and 'large', pond-raised fish (10-13 cm). The large fish were selected for some experiments because they have large optic nerves and optic tracts. The fish were injected in the right eye with L-[aH]fucose (4.3 Ci/mmole, New England Nuclear) as described previously a6. In double-label experiments the injection consisted of a mixture of [3H]fucose and L-[14C]proline (209 mCi/mmole, New England Nuclear). Isotopes were injected into the small fish in 2 #l of goldfish saline, while large fish received 5/zl. The fish were killed by decapitation and dissected immediately. As in previous studies ls,19,21,2a,aS,z6, transported radioactivity was calculated by subtracting the 'background' radioactivity due to local incorporation of blood-borne precursor. The amount of background labeling is measured in the contralateral side of optic system that is connected to the eye which was not injected with isotope. Transported radioactivity is therefore calculated as L-R in the optic tracts and tecta, and R - L in the optic nerves. Early time course. Large fish were injected in the right eye with a mixture containing 2.8 #Ci of [3H]fucose and 0.7 #Ci of [14C]proline. The temperature was maintained at 21 ~ I °C 2z. Freshly dissected individual tecta, optic tracts, or optic nerves were immersed overnight in cold 10~ trichloroacetic acid (TCA) to precipitate macromolecules (2.0 ml per tectum, 0.5 ml per tract or nerve). After drying, the tecta were weighed and the tissues were solubilized in Soluene-100 (Packard Instrument Company, Downers Grove, Ill.) for liquid scintillation counting. The lengths of the optic nerves were measured before TCA fixation. Since neither fucose nor proline label significant amounts of transported lipidsZZ,41,5°, lipids were not extracted. The TCA supernatants of the tecta were extracted 3 times with diethyl ether to remove the TCA, evaporated to dryness, and redissolved in 0.5 ml of HzO for counting in Bray's scintillator s. Long-term time course ofaxonal transport. Small fish were injected with 1.2/~Ci of [3H]fucose, and sacrificed at various times after the injection. The macromolecules of individual tecta were precipitated by immersion in cold 10~ TCA overnight. Lipids were then extracted in chloroform-methanol (1 :l). The tecta were dried, weighed and solubilized in Soluene-100 for liquid scintillation counting in a toluene scintillation cocktail. TCA-soluble radioactivity was measured in Bray's scintillator. After the tecta were removed, the heads of the fish were fixed in Bouin's solution and then transferred to 70 ~o ethanol. The optic nerves were dissected out and their lengths measured, and the nerves were solubilized in Soluene-100 for scintillation counting. Experiments with acetoxycycloheximide (AXM). Small fish were injected with a mixture of 1.0/zCi of [aH]fucose and 0.2 #Ci of [14C]proline in 2/~1 of saline. At various times before or after the isotope injection, 0.05 #g of acetoxycycloheximide (AXM) was injected into the same eye. The fish were sacrificed 24 h after the isotope injection. The tecta were processed as described for the 'Early Time Course', except that left or right tecta were processed in groups of 3. Brain Research, 48 (1972) 327-342

329

AXONAL TRANSPORT OF GLYCOPROTEIN

Separation of soluble and particulate proteins. Four large fish per experiment were injected with 4.2 #Ci of [SH]fucose in the right eye. They were sacrificed either 1 or 23 days after the injection. Pooled left or right tecta or optic tracts were vigorously homogenized in 2.0 ml of ice-cold H20 in a Dounce homogenizer (Kontes Glass Company, Vineland, N. J.), and spun at 10,100 × gay for 75 min in a Spinco 40 rotor in a Spinco Model L ultracentrifuge. The supernatants were removed with Pasteur pipettes. Soluble proteins in the supernatant and particulate macromolecules in the centrifugal pellet were precipitated in 10% TCA, washed with additional 10 % TCA, and dissolved in 1.0 NNaOH. Protein was determined by the method of Lowry31, and radioactivity was measured using the Packard Model 305 Oxidizer. Examination of the incorporated radioactivity by acid hydrolysis and thin-layer chromatography. Five large goldfish per experiment were injected in both eyes with 5.0 #Ci of [aH]fucose and sacrificed 24 h later. In these experiments both left and right tecta contained transported radioactivity; the low level of background labeling was ignored (see Fig. 1). Groups of 5 tecta were homogenized in 2.0 ml ice-cold H20 and the homogenate was brought to a volume of 5.0 ml containing 10 % TCA, and then extracted twice with chloroform-methanol (3:1), twice with 95 % ethanol, and once with diethyl ether. The powdered residue was hydrolyzed under nitrogen in 0.5 ml of 0.1 N HCI at 80 °C for 3 h in a Teflon-capped vial. The hydrolysate was dried under a stream of nitrogen, and redissolved and dried several times to remove the last traces of HCI. The hydrolysates were desalted by passage through two small columns made from Pasteur pipettes containing ion-exchange resin: AG 50W-X2, and AG l-X8 (formate form), 200-400 mesh (Bio-Rad Laboratories, Richmond, Calif.). The concentrated effluent was chromatographed on Eastman Cellulose Chromagram Sheets No. 6064 in an Eastman Chromagram Developing Apparatus (Eastman Kodak, Rochester, N.Y.). Three chromatographic systems were used: (A) Ethyl acetate-pyridine-acetic acid-water (5:5:1:3) developed twice 2a. (B) n-Butanol-pyridine-0.1 N HC1 (5:3:2) developed once 5°. (C) n-Butanol-ethanol-H~O (10:1:2) developed twice 47. The chromatograms were cut into strips 2.5 mm wide which were soaked in 0.5 ml of Soluene-100 and counted in a toluene scintillator. All of the applied radioactivity could be recovered by this method. Sugar standards run in different lanes of the same sheet were visualized with a AgNOa-NaOH reagent. RESULTS

Time course of the arrival of rapidly transported glycoproteins When [aH]fucose is injected into the goldfish eye, it labels glycoproteins which are rapidly transported to the tectumaS. In order to compare the time course of the movements of transported glycoproteins with other rapidly transported proteins, large fish were injected with a mixture of [aH]fucose and [14C]proline. Fig. 1 shows the transported TCA-precipitable radioactivity found in the optic nerve, tract and tectum. The overall pattern of labeling is the same in each tissue. After a delay,

Brain Research,48 (1972) 327-342

330

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Fig. 1. Early time course of transported radioactivity after an intraocular injection of a mixture of [3H]fucose and [14C]proline. The upper 3 figures show transported TCA-precipitable radioactivity in the optic nerve, optic tract, and tecta. The lowest figure shows transported TCA-soluble radioactivity in the tecta. Bars enclose the standard error of the mean. Transported radioactivity is calculated as L-R for the optic tracts and tecta, and as R-L for the optic nerve.

proteins labeled with [14C]proline arrive r a p i d l y a n d then reach a plateau, a p a t t e r n typical for proteins labeled with a m i n o acids ls,~9,a6. T h e r e is also a delay before the [aH]fucosyl g l y c o p r o t e i n s begin to a p p e a r , b u t they then c o n t i n u e to increase for at least 18 h. These labeling p a t t e r n s a p p e a r in the optic nerve, optic tract, a n d tecta at successively later times, as one w o u l d expect o f labeled proteins which are m o v i n g d o w n the optic axons. The difference between the time o f arrival o f the earliest t r a n s p o r t e d materials in the nerve, tract, a n d tectum can be seen m o r e clearly in Brain Research, 48 (1972) 327-342

331

AXONAL TRANSPORT OF GLYCOPROTEIN

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Fig. 2. Arrival of radioactive proteins and glycoproteins in the optic nerve, optic tracts, and tecta after intraocular injections of p4C]proline and [aH]fucose. Arrows indicate the earliest arrival of transported radioactive TCA-precipitable materials.

Fig. 2. The earliest transported proteins labeled with [aH]fucose and with [14C]proline arrive in each structure at the same time, and therefore, they must be traveling at the same velocity. Using the times of arrival and the dimensions of the optic systems of large goldfish, one can estimate that this rate of transport is about 70-80 mm/day (Table I). This agrees with the rate measured by Elam and Agranoff TM. [aH]Fucose also produces some transported TCA-soluble radioactivity. The TCA-soluble component is small compared to the macromolecular one, and arrives somewhat later (Fig. 1). Fig. 3 shows the total transported TCA-precipitable radioactivity in the extraocular segments of the retinal ganglion cell axons (i.e., optic nerve 4- optic tract + tectum). Glycoproteins labeled with [SH]fucose continue to leave the eye after the export of proteins labeled with [14C]proline has stopped. Yet, despite the difference in the time course of their transport into the optic fibers, proteins labeled with the two isotopes are distributed similarly between the nerve, tract, and tectum at all times (Fig. 4). One possible explanation for the difference between the time course Brain Research, 48 (1972) 327-342

D.S. FORMAN et al.

332 TABLE 1 RATE

OF AXONAL

TRANSPORT

IN GOLDFISH

OPTIC

SYSTEM

Calculation of the rate of rapid axonal transport, based on the first arrival of radioactive macromolecules in the parts of the optic system. The same calculation applies to both proteins labeled with [14C]proline and to glycoproteins labeled with [3H]fucose, since they begin to arrive in each structure at the same time. The average length of the parts of the optic pathway in the fish in this experiment were: optic nerve, 4.6 mm; optic chiasma, 0.5 mm; and optic tract, 2.9mm.Thus, radioactive materials traveling from the end of the optic nerve nearest the eye ('proximal end') to the distal end of the optic tract ( = proximal end of the optic tectum) travel a distance of 4.6 + 0.5 + 2.9 = 8.0 ram. Materials traveling from the proximal end of the nerve to the proximal end of the tract traverse a distance of 4.6 + 0.5 = 5.1 mm. The times between the first arrival of transported radioactivity in the proximal end of each segment are calculated from Fig. 2.

Nerve ~ tectum Nerve -+ tract Tract -+ tectum

Distance (mm)

Time (h)

Rate (mm/day)

8.0 5.1 2.9

2.5 1.5 1.0

77 82 70

of [3H]fucose and [14C]proline labeling which is seen in Figs. 1 and 3 could be that although both precursors label proteins which are transported at the same rate, [3H]fucose forms a relatively long-lived precursor pool which is available for incorporation into glycoprotein after the pool of p4C]proline is depleted. Evidence for this interpretation comes from experiments with a protein synthesis inhibitor, acetoxycycloheximide.

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Brain Research, 48 (1972) 327-342

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Fig. 4. Per cent of total transported TCA-precipitableradioactivityin the optic nerve, optic tract, and tectum. The percentageswere calculatedfor the individualfishand then averaged. The ordinate shows time after injection of the isotope mixture.

Experiments with acetoxycycloheximide (AXM) When 0.05 fig of AXM is injected into the goldfish eye it profoundly inhibits protein synthesis in the retina without measurably affecting protein synthesis in the brain a6. Intraocular AXM inhibits the synthesis of transported glycoproteins labeled with [SH]glucosamine21, [aH]fucoseSS, and [asS]sulfatelg. Since AXM acts rapidly, one can determine the length of time that a labeled precursor is available for incorporation into transported macromolecules by injecting AXM at various times after the isotope injection a6. Fig. 5 shows the effect of AXM on transported radioactivity from [14C]proline and [aH]fucose, as measured in the tecta 24 h after the isotope injection. When AXM is injected before or with the isotope, it nearly abolishes the appearance of transported proteins labeled with [14C]proline (Fig. 5A). When the drug is delayed until 2 or more h after the isotope, however, a considerable amount of TCA-precipitable radioactivity is transported, representing the proteins synthesized before the AXM injection. When AXM is injected 4 or 6 h after the isotope, the amount of transported 14C-labeled protein is nearly at control levels, showing that by then the pool of labeled precursor is nearly depleted. The AXM also inhibits the

Brain Research,48 (1972) 327-342

334

D.s. FORMAN et al. Effect of Axm

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Fig. 5. Effect of intraocular AXM given at different times on the arrival of transported radioactivity in the tectum. A, Transported proteins labeled with [14C]proline. B, Transported glycoproteins labeled with [SH]fucose. C, Transported TCA-soluble radioactivity from [3H]fucose. There were 3 groups of 3 fish per time point. Lines show the standard error of the mean. The fish were killed 24 h after the isotope injection. appearance o f transported glycoproteins labeled with [SH]fucose (Fig. 5B). However, the A X M is quite effective even when it is injected 6 h after the precursor, when it still produces 55 % inhibition. This shows that the incorporation time is longer for fucose than for proline; more than half of the labeled transported glycoproteins are synthesized later than 6 h after the isotope injection. Labeled fucose also produces a relatively long-lived precursor pool in the rabbit retina 27. Brain Research, 48 (1972) 327-342

335

AXONAL TRANSPORT OF GLYCOPROTEIN

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Fig. 6. Long-term time course of radioactivity in optic nerve and tecta after an intraocular injection of [aHlfucose. 'Glycoprotein' indicates TCA-precipitabl¢ radioactivity. Lines above the bars are standard errors of the mean (N = 5, except at 63 days, where N = 4). A and B are from ref. 35. The increase in labeled glycoprotein in A between 1 and 7 days is not statistically significant. Like the t r a n s p o r t e d T C A - s o l u b l e radioactivity p r o d u c e d by [aH]glucosamine~t, the t r a n s p o r t e d T C A - s o l u b l e radioactivity from [aH]fucose is decreased by A X M , b u t to a m u c h lesser extent t h a n the m a c r o m o l e c u l a r c o m p o n e n t (Fig. 5C). The extent o f the i n h i b i t i o n o f the T C A - s o l u b l e c o m p o n e n t seems to be relatively insensitive to the time o f the A X M injection.

Long-term time course of transported radioactivity Radioactive amino acids label two main waves of transported proteins. In the

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D.S. FORMANet al.

goldfish the slow component, which contains more than twice as much radioactivity as the rapid one, moves at a rate of about 0.4 ram/day. It thus reaches the tectum about 3 weeks after an intraocular injection of labeled amino acids, producing a large increase in the amount of transported radioactivity in the rectum 21,23.24,:~. However, no increase is detectable 23 days after the [aH]fucose injection (Fig. 6A), a time when the arrival of slowly transported proteins should be evident. Slowly transported proteins labeled with radioactive amino acids can also be easily detected in the goldfish optic nerve, where the radioactivity increases greatly between one and 7 days after the injection as labeled slowly-moving proteins enter the nerve~L However, one week after an intraocular injection of [3H]fucose there is no increase in the labeling (Fig. 6C). Thus, at the level of resolution examined here there is no evidence of a slow component. Although it is possible that more sensitive methods may reveal some slow transport of glycoprotein, it appears that most (and perhaps all) glycoproteins are transported rapidly. The transported glycoproteins appear to turn over rather slowly, although some of the persistence of radioactivity is probably due to reutilization of label 5°. The amount of background labeling produced by [3H]fucose is unusually low (Fig. 6B). In this regard it is similar to amino acids such as proline and asparagine which produce very low background labeling tg. Although the background labeling produced by fucose is so low that it can be ignored for many purposes, the values of transported radioactivity presented in this paper were calculated by subtracting the background radioactivity. Separation of soluble and particulate proteins

With the simple procedure used here to separate soluble and particulate proteins, 44 % of the total protein in the tecta, and 34 % of the total protein in the optic tracts appears in the centrifugal supernatant as soluble protein. However, in both the tecta and tracts more than 90% of the transported labeled glycoproteins remain in the centrifugal pellet (Table II). They thus resemble other rapidly transported proteins, which are mainly particulate, and differ from slowly transported proteins, which are TABLE II DISTRIBUTION OF TRANSPORTED RADIOACTIVITY BETWEEN PARTICULATE AND SOLUBLE PROTEIN AFTER INTRAOCULAR INJECTION OF [3H]FuCOSE

Distribution of transported radioactivity between soluble and particulate protein. Each value is the average of two experiments. % _Particulateprotein

One day after injection Twenty-three days after injection Total protein Brain Research, 48 (1972) 327-342

Optic tecta

Optic tracts

94 97 56

91 95 66

AXONAL TRANSPORTOF GLYCOPROTEIN

337

FUCOSE

Monnose Glucose Galoctose

! •

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ORIGIN

----

] I

I00

I

200 CPM

I

I

300

400

Fig. 7. Recovery of transported TCA-precipitable radioactivity as free [aH]fucoseafter acid hydrolysis and cellulose thin-layer chromatography. Chromatographic system A (ethyl acetate-pyridine-acetic acid - water, 5:5:1:3). Spots are tracings of standard sugars. Distance along chromatogram is divided into cm. Bars show counts/min in strip of indicated position and width. distributed between the two fractions in proportions roughly similar to the distribution of total protein zl,35. If some of the slowly transported soluble proteins were glycoproteins which contained fucose, their presence might have been detected as an increase between one and 23 days in the proportion of radioactive soluble glycoproteins. However, no such increase was found (Table II), a finding which is compatible with the possibility that glycoproteins are transported only rapidly.

Nature of the transported radioactivity The relatively mild acid hydrolysis conditions used were released 91 ~o of the radioactivity in the TCA precipitate. More vigorous conditions, 24 h in 1 N HC1 at 100 °C, released 99 ~ . Virtually all of the radioactivity which was released moved in a single peak which had the mobility of free fucose in all 3 chromatographic systems; Fig. 7 shows the results of a typical experiment. If thus appears that the precursor was incorporated into macromolecules almost exclusively as fucose, confirming previous

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D.S. FORMANet al.

findings in brain27,3z,~, ~0 and other tissues 3,12. About 5 ~o of the radioactivity in the TCA precipitate could be extracted in chloroform-methanol (1:1). However, it seems likely that the radioactivity extracted by the polar lipid solvent is mainly in solubilized glycoprotein rather than in glycolipid, since the proportion of radioactivity extracted is the same or less than the proportion which can be extracted from TCA precipitates containing transported proteins labeled with radioactive amino acids (and is also the same as the proportion of extractable material measured by the Lowry method). Other laboratories have reported that radioactive fucose does not label brain lipids ~'~, 41,50, a result consistent with the absence of measurable fucolipid in brain tissue 10. The TCA-soluble radioactivity seen at 24 h contained both free fucose and other substances, some of which could be converted to fucose by mild acid treatment (and which thus may be metabolites such as GDP-fucose26,5°). By 24 h after the injection, 54 ~,] of the injected radioactivity appeared in the tank water, and 80-90 ~ of this radioactivity was volatile (and is thus apparently tritiated water32). DISCUSSION

The transported glycoproteins labeled with [aH]fucose are similar to transported glycoproteins labeled with [aH]glucosamine21: both precursors label glycoproteins which are transported rapidly at the same rate as other rapidly transported proteins, and neither labels a measurable slow component. The transported glycoproteins are mainly particulate, and their synthesis is dependent upon retinal protein synthesis. Transported sulfated acid mucopolysaccharides have similar properties ~9. These findings in the optic system of the goldfish are consistent with the results of studies of axonally transported fucosyl glycoproteins in the mouse cerebral cortex 51 and in the optic system of the chicken7 and rabbit 27. Glycoproteins labeled with [aH]fucose are distributed between the parts of the optic axons in the same proportion as proteins labeled with [14C]proline (Fig. 4). Since this relationship is maintained after the transport of 14C-labeled proteins into the optic axons has stopped (Fig. 3), the glycoproteins which enter the optic axons after that time must continue to be distributed between the nerve, tract, and tectum in the same manner as the transported proteins which arrived earlier. This finding is consistent with our conclusion that the axonal transport of glycoproteins is the same as the transport of other rapidly transported proteins, and that the difference in the time course of labeling is due only to a difference in the period of isotope incorporation. When 0.05 #g of AXM is injected into the goldfish eye it profoundly inhibits protein synthesis in the retina without measurably affecting protein synthesis in the brain a6. Since AXM does not interfere with the axonal transport process itselfa6,as,ag, 4~, intraocular AXM decreases the amount of labeled transported proteins found in the tecta by inhibiting their synthesis in the retina. The synthesis of transported, sugarlabeled glycoproteins is also dependent on retinal protein synthesis. Although protein synthesis inhibitors do not interfere with glycosyltransferase reactions, they can inhibit the incorporation of carbohydrates into glycoproteins by preventing the synBrain Research, 48 (1972) 327-342

AXONAL TRANSPORT OF GLYCOPROTEIN

339

thesis of the polypeptide acceptors. For example, AXM does not inhibit brain fucosyltransferases, but it can decrease the incorporation of [aH]fucose into glycoprotein by stopping the synthesis of the polypeptide acceptors 52. The inhibition observed when AXM is injected before or with [aH]fucose is less than the inhibition seen with amino acids (Fig. 5). This might reflect the incorporation of the [aH]fucose into a pool of polypeptide acceptors which were synthesized before the AXM was injected. If this was the case, however, one would expect more inhibition when the AXM is injected 2 h before the isotope, since that would allow time for the pool of polypeptide acceptors to be depleted. Another explanation, arising from the long life of the precursor pool, is that the transported labeled glycoproteins which appear in the presence of AXM may have been synthesized after the AXM has begun to wear off. Although AXM is a long-acting drug, protein synthesis in the goldfish brain begins to recover after 8-12 h 3°. This hypothesis would account for the slightly greater amount of transported label found when the drug is given 2 h before the [aH]fucose (Fig. 5). The presence of transported TCA-soluble radioactivity raised the possibility that small molecules might be rapidly transported and incorporated locally into macromolecules, which would then appear to have been transported. However, the TCA-soluble component is quite small compared to the macromolecular one (Fig. 1). Furthermore, it appears to lag behind the transported glycoproteins rather than preceding them, as one might expect of a precursor. The experiments with AXM also provide evidence that the transported TCA-soluble material is not a precursor of the transported macromolecules, since when the synthesis of the glycoproteins is prevented one would expect the pool of precursor to increase. Instead, it decreases somewhat (Fig. 5), suggesting that some of the transported TCA-soluble material may be produced by the breakdown of transported glycoproteins. Therefore, it seems unlikely that the local incorporation of transported TCA-soluble materials can account for the transported radioactivity found in proteins. Rather, the evidence taken together suggests that carbohydrates are incorporated into the glycoproteins in the neuron cell body and then axonally transported. This conclusion is consistent with autoradiographic evidence in a variety of cells 4,5,16,~5,a7,49, including neurons 42, that carbohydrates are incorporated in the rough endoplasmic reticulum and Golgi apparatus, organelles which are localized in the neuron cell body. Fucose, in particular, is apparently incorporated exclusively in the Golgi4,5, 25. On the other hand, the presence of glycosyltransferases in synaptosomal subcellular fractions14,15,eo,44, and other lines of evidence1, 2 have led some authors to suggest that carbohydrates are incorporated into glycoproteins in nerve endings. (Fucosyltransferases, however, are localized in microsomal fractions and are not concentrated in synaptosomes52.) Evidence that glycoproteins are axonally transported does not contradict the possibility of additional local incorporation of carbohydrate, since the methods used here are strongly biased towards labeling glycoproteins which are synthesized in the cell body and then transported. It will be important to'determine the relative contribution of axonal transport and local synthesis to the metabolism of glycoproteins in the nerve endings.

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o . s . FORMAN et al.

SUMMARY

After an intraocular injection of [3H]fucose, labeled glycoproteins are rapidly t r a n s p o r t e d to the goldfish optic tectum at a rate o f 70-80 m m / d a y at 21 °C. A small a m o u n t o f T C A - s o l u b l e m a t e r i a l is also t r a n s p o r t e d . Slow t r a n s p o r t o f labeled glycop r o t e i n s was n o t detected. M o s t o f the t r a n s p o r t e d glycoproteins are b o u n d to sedim e n t a b l e subcellular particles. Synthesis o f the t r a n s p o r t e d glycoproteins is prevented when retinal p r o t e i n synthesis is inhibited with acetoxycycloheximide. These findings agree with the results o f previous studies with labeled glucosamine, which suggested that c a r b o h y d r a t e s are i n c o r p o r a t e d into glycoproteins in the nerve cell b o d y a n d then t r a n s p o r t e d to the axons a n d endings. [3H]Fucose p r o d u c e s a relatively long-lived p o o l o f labeled prec u r s o r in the retina; m o r e t h a n h a l f o f the t r a n s p o r t e d labeled g l y c o p r o t e i n in the tectum 24 h after a [aH]fucose injection is synthesized later t h a n 6 h after the injection. ACKNOWLEDGEMENTS

This work was supported by U. S. Public Health Services grants NS-09015, NS-07080, and MH-13189. Dr. Forman was supported by a Graduate Fellowship from the National Science Foundation; his work was submitted in partial fulfillment of the requirements for a Ph. D . at The Rockefeller University. The AXM was a gift of Dr. T. J. McBride of the J. L. Smith Memorial for Cancer Research, Chas. Pfizer and Company, Maywood, N. J. and was produced there with the support of NIH contract number PH 43-64-50.

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