Subcellular localization of the 52,000 molecular weight major postsynaptic density protein

Brain ReAearch, 233 (1982) 265-286 Elsevier

BiomedicalPress

265

,

SUBCELLULAR LOCALIZATION OF THE 52,000 MOLECULAR WEIGHT MAJOR POSTSYNAPTIC DENSITY PROTEIN

P A U L T. K E L L Y a n d P A U L R. M O N T G O M E R Y

Division o f Biology, Kansas State Umversity, Manhattan, KS 66506 ( U.S.A 2 (Accepted J u n e l l t h , 1981)

Key words: p o s t s y n a p t i c densities - - a s y m m e t r i c s y n a p s e -- subcellular fract,onal~on -

pepl,de

mapping

SUMMARY We have recently reported that in isolated synaptic junctions, the quantity orthe major post-synaptic density protein (mPSDp, Mr - 52,000) increases approxmlatcly twenty-fold during the third and fourth weeks of postnatal development. In the sludy that follows, systematic analyses were cal ried out to determine the subceilular IocahJadon of this prominent synaptic protein in adult brain and non-neuronal tissues. Subcellular fractionation and SDS-gel electrophoresis were used to isolate various tissue components and identify proteins that possessed molecular weights simdar to that of the mPSDp. To unambiguously verify the molecular identity of all proteins suspected of being the mPSDp, two-dimensional peptide fingerprinting was carried out. In addition, the different subcellular fractions were examined for the presence of structures morphologically resembling the postsynaptic density. The mPSDp was found only in fractions containing identifiable asymmetric synaptic structures and/or postsynaptic densities. This protein was not found in n.~nneuronal tissues or any other fraction in which there was not a demonstrable presence of postsynaptic densities. This work strongly indicates that the major PSD protein is a molecular 'marker' specific to asymmetric synapses in the mammalian forebrain.

INTRODUCTION Most synapses in the central nervous system (CNS) are excitatory and possess a distinct morphology~.l:;J4.zl. At the point of synaptic contact, the postsynaptic membrane is distinguished by an electron opaque, fibrous, submembranous specialization. Based on its appearance this specialization has been called the postsynaptic ~306-8993/82/0000-0000/$02.75 ~ ElsevierBmmedicalPress

266 density (PSI)) t.¢, web1~, or thickening2t.4a. The presynaptic membrane frequently displays on its cytoplasmic surface a series of conical structures called presynaptic dense projectionsI. This morphological unit (i.e. the synaptic junctional complex) is characteristic of the "asymmetric', Gray Type ! synapse9.~1. In the cerebral cortex of some mammals. 80 % of all morphologically identifiable synapses are asymmetric and display a prominent PSDg, ~2. in 1974, Cotman avd co-workers developed a method to isolate PSDs, a submembranous structure presumably distinct to asymmetric synapses xs. PSD fractions prepared by this method are approximately 85-90 ~o pure in PSD structures (Fig. la). Isolated PSDs retain certain morphologicale.s.x3, histochemical6.t3.~4 and enzymaticts properties characteristic of the postsynaptic density in intact tissue. The yield of total protein associated with the PSD fraction is approximately 40 pg protein per grant of brain (rat cerebral cortex). Based upon estimates that assume nearly quantitative recovery, we calculate that PSD fractions represent about a 104-fold enrichment in these structures compared to total brain homogenates14. Other methods now exist which produce subcellular fractions that are highly enriched in PSDs and which display similar morphological composition to PSD fractions isolated by the method of Cotman et al. in which synaptic plasma membranes are treated with the ionic detergent N-I~uroyl sa~cosinate 13. Biochemical analyses of isolated PSDs have resulted in the partial characterization of a distinct, major PSD polypeptide of 52,000 molecular weight2..~,14,'~9. The major PSD polypeptide is a conspicuous component of synaptic junction (SJ) and PSD fractions, making up approximately 50% of the total protein of isolated PSDs (Fig. Ib)'-'s. Reports from different laboratories have shown that the major PSD polypeptide is largely insoluble in neutral detergents, bile salts, chelators and guanidine'-'.'~,.~-s,''~,;°,19,5~.The major PSD polypeptide has very reactive sulfhydryl groups that are easily oxidized and cause it to cross-link to itself and other proteins through the formation of inter-protein disulfide bridgese6. This cross-linking property of the major PSD polypeptide is largely responsible for its insolubility in N-!auroyl salcosinate (see Results). Although numerous reports have demonstrated that the ma,;or PSD polypeptide is highly enriched in isolated SJ and PSD fractions, systematic s~udies have not yet been carried out which examine its subcellular distribution in brain and non-neural tissues, in the studies described herein, conventional fractionation techniques have been used, together with electrophoresis under denaturing conditions, to determine the subcellular distribution of the major PSD polypeptide in different tissues. The tb,~mbiguous identification of all proteins suspected of being the major PSD polypeptide was accomplished by two-dimensional peptide fingerprinting. Additional analyses, employing electron microscopy, were used to examine various subcellular fracttons for the presence of morphologically identifiable PSDs. Our results support the contention that the major PSD protein (mPSDp) is a molecular marker specific to asymmetric synapses. A preliminary report of this work has appeared elsewhere3L

267 METHODS AND MATERIALS

Subcelhdar fractionation and electron microscopy Sprague-Dawley male and female rats (60-100 days of age) purchased from Simonsen Laboratories (Gilroy, CA) were used in these experiments. Subcellular fractions were prepared from forebrains obtained following decapitation and freehand dissection of brains rostral to the superior colliculi. Btains were homogenized in ice cold 0.32 M sucrose within 8 rain of decapitation. Purified synaptlc plasma membrane (SPM) fractions were prepared by the iodonitlotetrazolium violet (! NT) procedure from an osmotically-lysed, crude synaptosomai-mitochondrial fraction (pz)10. The only modification in past procedures was that the density gradients used to resolve SPM fractions were constructed with equal volumes of 0.8 M, 1.0 M and 1.3 M sucrose and SPM fractions were harvested from the !.0-1.3 M sucrose interface The use of 1.3 M instead of 1.2 M sucrose in the bottom step of this densRy gradient increases the yield of SPMs by approximately 10% without affecting their morphological or macromolecular composition (P. Kelly, unpublished observation). The gradients used to purify SPMs were also used to isolate purified myelin (harvested at the 0.32--0.8 M sucrose interface) and a plasma membrane fraction of hght bouyant density (harvested at the 0.8-1.0 M sucrose interface). Fractions collected from these upper intelfaces were diluted with 4-5 volumes of 0.32 M sucrose and pelleted by centrifugation at 86,000 gave for 25 min. SJ and PSD fractions were prepared from INT treated SPMs as previously described ":~. Microsomal (P~B)fractions were prepared by the methods described by l)e Bias and Mahler ~; without the use of the second step of density gradient ccnmfugation. Following the isolation of P3B fractions, they were subsequently treated with 0.5 mM iodonitrotelrazolium violet {INT) and 20 mM succinate as previously describedl:k Cytosol (Sa) fractions were prepared from two different supernatant fractions. The first Sa fraction was prepared from the postmitochondrial supernatant fraction (Sz) following centrifugation of the latter at 100,000 gay,, for 120 rain. A second and separate Sa fraction was prepared from the hypo-osmotic lysate of the crude synaptosomal pellet (P2). Following osmotic lysis of the Pe fraction as previously described ta, a supernatant fraction was obtained following centrffugation of the lysed particulates at 33,000 gave for 20 rain. This supernatant was then centrifuged at 100,000 gav~. for 120 min to produce an Sa fraction that contained, in part, soluble proteins from the synaptoplasm. Individual S~ fractions were concentrated in dialysis tubing against aquacide (Calbiochem) to a final protein concentration of approximately 5 mg/ml. Cytosolic proteins were then treated with INT and succinate as previously outlined la. Soluble proteins that became particulate dining this treatment were collected by centrifugation at 100,000 g,w. for 60 min and stored at --85 -C until analyzed by electrophoresis. Plasma membranes were prepared from rat livers by the method outlined by Hertzberg and Gilula 'a except that density gradient centrifugation was carried on a smaller scale in an SW 27 swinging bucket rotor (Beckman Instr.) instead of a zonal rotor. Following their isolation, fresh liver plasma membranes were treated with INT and succinate as described earlier.

268 Plasma membranes purified from dccapsulated lenses frc'~ adult chickens were prepared by the methods described by Blocmendal et al. 4. Gap junctions (generously provided by Dr. Larry Takemoto, Kansas Slate University, Manhattan, KS) were oblained by treating lens plasma membranes with the detergent Sarkosyl NL-97 (ref. 48). Gap junctions isolated by this method are greater than 90% pure in identifiable gap junctions 4s. SubceUular fractions treated with INT and succinate were washed free of residual salts and resuspended at a final protein concentration of 4 mg/ml in 0.32 M sucrose. Detergent treatment was carried out by the addition of two vols. of 0.4 % (v/v) Triton X-100 (Sigma), I mM HEPES (pH 7.2) to the subeellular fract'~an. The resulting mixture was incubated 10-15 min at 4 °C with occasional mixing before being layered on a 1.0 M sucrose cushion and centrifuged at 86,000 g,~ve for 120 min. The Triton-insoluble material from each fraction was collected as a pellet at the bottom of the centrifuge tube and processed for electron microscopy and gel electrophoresis. INT treated ~j fractions and lens gap junctions were not treated with Triton X-100. Subcellular fractions were examined by electron microscopy to monitor their morphology and purity. Samples were fixed in 4% (v]v) glutaraldehyde followed by I '~, (w/v) osmium tetroxide and ~tained with uranyl acetate as prewously described 10, 11. For each subcellular fraction, a minimum of 20 micrograpi',s were taken (magnification of 30,000/), At least 3 individual preparations were used for each subcellular fraction: each fraction being analyzed at both mo-phological and biochemical levels. SJ and PSD fractions u~ed in these studies were of comparable purity to fractions used in earlier studies~:~,~.~8.'-'0.

Polyaco'hmdde gel elc'ctrophoresis and / rzrJl:Con A bbuling to gels Sample~ were solubilized in sodium dodccyl sulfate (SDS} with beta-mercaptoethanol and their constituent polypeptides were resolved by slab-gel electrophorcsis a~ previously describcd'L The method of Rostas et al. '~ was used to identify Con A binding glycocomponents in slab-gels. Con A was iodinatcd and the resulting [r'sI]Con A (approximately 100/zCi/mg) was purified by affinity chromatography on Sephadex G-100 (Pharmacia). Stained gels ~ere incubated in buffer containing It'll]Con A (0.1 mg/ml) for 25 min, washed exhaustively to remove unbound lectin and then dried, under vacuum, at room temperature. Con A binding glycocomponents were visualized by autoradiography. Autoradiographic exposures were conducted at 24 °C for 24-48 h. Protein determinations were performed by the method of Lowry et al. =s and appropriate controls were included for fractions containing formazan as aforesaid la.

Tryplic peptide mapphlg of radioiodinaled proteins The method of peptide mapping of radiolabeled proteins in single polyacrylamide gel slices described by Elder et al. 18 was used with minor modifications"9. The radiolabeled tryptic peptides prepared for each protein by this method were separated on TLC plates (EM Laboratories, Inc.) by the two-dimensional electrophoresis/chromatography described earlier~a except that PPO was omitted from the chromato-

269 graphy solvent. TLC plates were analyzed by autoradiography using Kodak X-ray film. Autoradiographie exposures were carried out at --85 ~C using Cronex mtensff~ ing screens (Dupont). RESULTS

Treatment of SPM fractions with the anionic detergent N-lauroyl sarcosmate (NLS) solubilizes nearly all plasma membranes (approximately 97°,i of the protein) while leaving postsynaptic densities (PSDs) intact xz. PSD fractions prepared by this method consist of 85-95°0 PSDs (Fig. la). Isolated PSDs, when crammed by electrophoresis under denaturing conditions, contam a major polypept]de, the major PSD protein (mPSDp; Mr - 52,000) (see Fig. l b). On the basis of protein weight, the mPSDp constitutes approximately 50 % of the protein In ~solated PSD fraction- "~

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Fig. I. a: electron micrograph of a PSD fraction prepared by the method of Cotman ct ;d. 15; m ~ t show~ high magnification view of an isolated PSD (bar 0 5 !,m;. b: ,-ne-dlmen~sonal SI)S gel of a PSD fracoon (55/tg protein).

270 The procedure developed to prepare SJ and PSD fractions involves the isolation of SPM fractions, their treatment with either Triton X-100 ~ttoisolate SJs) or N-lauroyl sarcosinate (to isolate PSDs), followed by centrifugation of the mixture of solubilized and particulate material on a density gradient r°,13. Mitochondrial fragments represent a significant contaminant in SJ and PSD fractions if not removed from SPM fractions prior to detergent treatment 1°,15. Therefore, a method was employed that preferentially increases the buoyant density of mitochondria and not SPMs t°,lS. This method •;nvolves the incubation of an osmotically lysed, crude synaptosomal-mitochondrial fraction (Pz) with iodonitrotetrazolium violet (INT) and succinate. During this treatment, succinic dehydrogenase present in mitochondria enzymaticaUy converts INT to formazan, a dense precipitate that accumulates in mitochondria. Formazan increases the buoyant density of mitochondria so that they can be easily separated from SPMs by density gradient centrifugation. Since INT is the proton acceptor in the enzymatic dehydrogenation of suecinate, it will under appropriate conditions, also serve as a general oxidizing agent for other chemical groups. For example, INT will react with free cysteine (reduced form) to produce formazan. We have previously shown that substantial interprotein cross-linking resulting from disulfide bond formation is present in SJ and PSD fractions prepared by the INT procedure'z6,:a'. INT-dependent disulfide bond formation appears to greatly stabdize the PSD and preserve its morphology and macromolecular constituents. This contention is substantiated by the finding that SPM fractions, prepared from Pe fractions whose free suiphydryl groups were irreversibly blocked with N-ethyl maleimide prior to INT treatment, yield approximately 15-fold less PSD material when treated with N-lauroyl sarcosinate when compared to PSD fractions prepared from standard, INT crosslinked SPM fractions. Despite the large difference in yield of PSD associated protein, the staining profiles of these two PSD fract~ens is quite similar (results not shown). Thus, in the studies that follow we have attempted to produce similar conditions o1" interprotein cross-linking and detergent solubility for mPSDp that may be present in different subcellular fractions by treating respective fractions with INT prior to detergent extraction. The subcellular distribution of the mPSDp has been examined in fractions isolated from brain and enriched in nuclei, myelin, mitochondria, microsomes and soluble brain proteins (cytosol). in addition, a population of plasma membranes isolated from blain and possessing a lighter buoyant density than SPMs was examined as a potential source of synaptie junctional structures and mPSDp content. These ~tudle~ were carried out with both rat and bovine CNS tissues and have yielded essentially identical results (only results with rat tissues will be presented). • lnalf.sis ~ subcelhdar fractions PSDs are prominent morphological structures in S.I fractions (Fig. 2a). The mPSDp is a promment protein component of th~s same fraction [Fig. 3, lane b and Fig. 6, lane a (0], comprising about 127,~,of the total protein in SJ fractionszs. Also present in Fig. 2 are representative micrographs of subcellular fractions enriched in mitochondria (Fig. 2c), microsomes (Fig. 2d) and the Triton X-100 insoluble material

271

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(d) P3 B (microsomes)

I'=g. 2. Electron micrograph~ of varJou~ subccllular fractlon~, a. synapllc jun¢llon h a~.tJon (b J) (arrows designate PSMSs, and asterisks specify SJ, ~,lth both pre- and pt)st~ynaptlc slrUclU;C~,), reset show,, high magnification view of SJ showing PSD and overlying presynaptJc membrane (arrow,, point to synaptJc cleft region), b: Triton X-100 insoluble P.~Bfraction v, ith arrows point mg to fibrow,. PSD-hke structures; reset shows higher magnification of a PSD in thi~, fraction ~. INT-trcaled mJtochondrla, d: INT-treated P.~B (microsomes) with high magmficauon of a synapt=~, junctional complex (SJC), art )ws point to synaptic cleft reg,ons (Bar,, repre,,ent 0.5/~m)

obtained from mi~losomes (Fig. 2b). Mitochondnal fractions were quite pure and when examined by quantitative EM procedures :~-', contained less than 2",, nonmitochondrial contaminants, the most prominent of which were plasma membrane, (mltoehondria were identified on the basis of easily recognizable inner and outer membranes and eristae). The mlcrosomal fraction (PaB) used for these studies contains a rather heterogeneous population of large, plasma membrane enclosed vesicular structures with an average diameter of 125 nm. The recovery of total protein in this microsomal fraction is about 2.65 mg per gram brain weight. As shown in the inset of Fig. 2d, synaptic junctional complexes (SJCs) are frequently obsel ved in micrographs

272 o f this microsomal preparation. These SJCs are usually smaller t h a n those obse= ved in SPM fractions. These findings agree with the results o f de Bias a n d Mahler Ie, w h o developed this procedure for isolating a microsomai fraction enriched in receptor sites for a number o f potential, postsynaptic neurotransmitters. T h e y too observed these

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Fig. 3 One dJmenslonal SDS gel of various subcellular fractions, a: synaplic plasma membrane fraction (SPM) b: synaptic junmion fraction (SJ). c: postsynaptic density fraction (PSD). d: Tr,ton X100 insoluble, INT-treated P3B fraction (TX, INT-P,IB). ¢: INT-treated PaB (INT PjB). f: PaB mlcrosomal fraction (PjB) g: INT-tieatcd mitochondnal fraction (mRo.), (h) Triton X-100-insoluble, INT-treated mitochondria, (TXI mlto ). fall wells contained 55 pg protein except for the PSD fraction which contained ! 5/tg protein.)

273 rather small postsynaptic structures in their microsomal fractions lb. Electrophoretlc analysis of this microsomal fractien revealed the presence of a minor sta,ning component of 52,000 Mr (Fig. 3, lane f). Treatment of microsomal fractions (P3B) with INT has no detectable effect on its overall protein composition (compare lanes e and f, Fig. 3). INT cross-linked microsomes extracted with 0.4 % (v/v) Triton X-I00 and subjected to gradient centrifugation, gave rise to a particulate fraction (TX, INTP3B) whose morphology is shown in Fig. 2b. Most prominent in this representative micrograph are numerous structures (arrows, Fig. 2b) that resemble isolated PSDs in both size and general n-torphology (compare to Fig.!la with inset in Fig 2b) The PSDlike structures derived from P3B fractions contain little, if any, attached plasma membranes. The residual membrane contamination in thls Triton-insoluble, particulate fraction is less than 5°~ of the total cros~-sectional area occupied by electronopaque structures. Approximately 97 o~ of the protein in I NT-P.3B is solubilized during the Triton X-100 extraction. Electrophoretic analysis of th~ Trlton-lnsoluble P.~B fraction showed a significant enrichment m a 52,000 Mr band (Fig. 3: lane d, small arrow) as compared to the amount of this same polypeptide in the INT-P3B fraction (Fig. 3, lane e). Also enriched in the Triton-in',oluble P3B fraction is a 54,000 Mr component which has been idenhfied as tubuhn by two-dimensional peptlde fingerprinting (data not shown). Overall, the Triton-insoluble P3B fraction contains many polypeptides which, on the basis of Mr, are also present m SJ fractions (Fig. 3, lanes b and d); Mr regions which are most simdar m staining patterns are 15 52 K, 8 0 120 K and 200 K. When the Coomassie blue-staining material contained in the 52,000 Mr band from the Triton-insoluble P:~B fraction is subjected to radlolodination and tr~r tic digestion, the peptide fingerprint shown m Fig. 4b is obtained. The fingerprint of the 52,000 Mr band in Triton-insolubk. P:~Bfractions contains 7 highly reproducible l~';Ipeptides (labeled accordingly in Fig. 4b). The two-dimensional positams of these peptides, as well as their relative autoradiographic intensities, arc very similar to a set of 7 similar peptides that are obtained when electrophoretically purified mPSDp (Fig. 3, lane c) is subjected to the same peptide fingerprinting technique (compare panels a and b in Fig. 4). In fact, the fingerprint from a mixture of equal amounts (CPMs) of peptides from the mPSDp and the Triton-insoluble P~B, 52,000 Mr protein shows that the two-dimensional positions of their prominent, iodinated peptides (numbers 1-7) are indistinguishable (Fig. 4c). For comparative purposes, the tryptlc fingerprint of the 52,000 Mr protein front the Triton-insoluble mltochondrml fraction (the protein composition of this fraction is shown in Fig. 3, lane h) is also displa.~,ed m Fig. 4 (panel d). The peptide fingerprint of this mitochondrial pobpeptid¢ is markedly different from that of the mPSDp. With the exception of SPM, SJ, PSD and Triton-insoluble PzB fractions, the only other fraction that contained significant amounts of the mPSDp was the plasma membrane fraction harvested at the 0.8--1.0 M sucrose interface in grad;ents used to Isolate SPM fractions (see Methods). This fraction has a 'ighter buoyant density than the SPM fraction which sediments to the 1.0-1.3 M sucrose interface in the same gradient. Based on brain wet v,eight, the recovery of protein at the 0.8--1.0 M interface

274

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Fng. 4. Tryptnc fingerprints of radno~odmated peptxdes of 52,000 Mr proteins :n dufferent brain subcellular fractions: panel (a), major PSD protein; panel (b), 52,000 Mr protein of TX,-P~B fract,on (see Fig. 2); panel (c), equal mnxture (CPMs) of rad]o-iodmated p:ptides from samples tap and (b); panel (d), major stammg band of 52,000 Mr of the TX,-mltochondr=al fraction ('see Fig 2). Each sample contained approxmlately 5 - tOs CPM, autoraduoglaphic exposures ranged from 8 to 12 h. Electrophoresus was from left to right and chromatography was from bottom to top.

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Iqg. 5. a: electron mlcrograph of Triton X-IO0 m,,oluble malerml from the membrane Ira,.lure harvested at the 08-1.0 M interface of gradient,, u,,ed to 0urfl) SPM~, reset ,,hov~, a h~gh magmficatmn view of a post~ynapI~c membrane specmil/atmn IPSMs~ exh~bmng a po~,t,~ynaptt~. density IPSD}, and an ovcdymg Oostsynaptic membrane (PSM). b" an electron m~crograph ol the NLS insoluble malenal from tile Io0 M sucro e cu~hmn used m the fin,~l PSD gradient, and a h~gh magmficalmn m~et silowmg the bilammar structure of the NLS m~oluble, ~e~cle membrane~, c" one° d~men~ional SDS gel of: ~, postsyn~0t~c den,,~ty fractmn (20 !~g p r o l c m k u. the NLS m~oluble I 0 M cushmn material (45 /~g prolem); m, tile Iov, mmc ~trength ~ a s h of the NLS m~oluble 1.0 M¢m, hmn material m 0i); till,, material ~ not ~ed~mentable at 100,000 g I55 !~g prolem. ~ee text Ior detads/.

276 is one-third of the value for SPM fractions. We and others have observed that plasma membrane fractions which sediment to I.O M and 0.9 M sucrose interfaces contain considerably fewer synaptic junctional complexes than do SPM fract:ons that are harvested at 1.2 or 1.3 M sucrose interfaces30, 4e. Consistent with these electron microscopic results is the finding that when the 0.8-1.0 M sucrose interface fraction is treated with Triton X-100, a particulate fraction is generated which is highly enriched in synaptic junctional structures (arrows, Fig. 5a). In comparison to the yield of SJs from the standard SPM fraction, the recovery of total protein in the Triton-insoluble 0.8-1.0 sucrose interface fraction is approximately 4-fold lower. Based upon ultrastructural analyses, one qualitative difference was apparent between SJ and Tritoninsoluble 0.8-1.0 interface fiactions. The Triton-insoluble 0.8-1.0 interface fraction contains few intact synaptic junctions while postsynaptic membrane specialization~ (PSD with overlying postsynaptic membrane, no presynaptic membrane is present) were the primary structures in this fraction. The protein composition of the Tritoninsoluble 0.8-1.0 interface fraction is shown in Fig. 6a, lane (ii). The staining pattern of this fraction is strikingly similar to that of the standard SJ fraction [Fig. 6a, lane (i)].

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Fig. 6. One-dimensional SDS gels of different subcellular fractions prepared from brain, hver and lens, a: I, synaptlcjunction fraction; i~, Triton X-100-insoluble material from the 1.0 M interface of an SPM gradient; m, a purified myelin fraction, b: i, INT cross-linked, insoluble material of an S.3 fraction from lysed synaptosomes; .i, INT-cross-linked, insoluble material of $3 fraction from whole brain; id, Ss fraction from osmotically lyscd synaptosomes; iv, Sa fraction from whole brain, c: i, gap-junction fraction purified from chick lens 0 5 i~g protein); ii, synaptlc junctions; iii, Triton X-100-insoluble fraction from INT-cross-hnked hver plasma membranes; iv, INT-cross-linkcd, hver plasma membranes. (All wells contained 55 pg protein unless otherwise indicated.)

277 Most noteworthy is the observation that this SJ-like fraction, prepared from plasma membranes of light buoyant density, contains the mPSDp in relative amounts nearly identical to that of the standard SJ fraction. The unambiguous identtt of the m PSDp in this Triton-insoluble 0.~-1.0 interface fraction was verified by two-dimensional peptide fingerprinting (data not shown). In the preparation ofPSD i'ractions from synaptic plasma membranes, a modest amount of N-lauroyl sarcosinate (NLS)-insoluble material is recovered at the top of the !.0 M sucrose cushion in the final gradient used to separate PSDs fi..m NLSsoluble material. This NLS-insoluble material, when examined by electron microscopy, contains a rather homogeneous population of smooth, often vesicularized, membrane-hke material (Fig. 5b). At high magnification, these membranes display a classic bilaminar structure (insert to Fig. 5b). Examination of this material has ne~er revealed structures resembhng PSDs in morphology. Interestingly, this NLS-msoluble fraction contains large amounts of the mPSDp [compare Fig. 5c, lane I0 (PSDs) ~lth lane (ii) (N LS-msoluble 1.0 interface)]. The identity of this 52,000 M r protein as bona fide mPSDp was confirmed by peptide fingerprint analysis (data not sho~n). This finding appeared contradictory to our initial hypothesis that the mPSDp was localized solely to structures that morphologically resembled PSDs. We then discovered that diluting this N LS-insoluble, membrane-like fraction with 5 vols. of low ionic strength buffer (I-5 mM Tris-HCI or HEPES, pH 7.2)followed by centnfugatlon at 100.000 g for 60 rain, resulted in the quantitative solubilization of it~ total protein content [see Fig. 5C, lane (iii) for the protein composition of the supernatant fraction from the low ionic strength wash of the N LS-insoluble 1.0 M interface material shown m lane ¢Li)]. Concomitant with this washing and high speed centrift,gation step was the d,sappearance from this fraction of all morphologically identifiable plasma membrane structures {data not shown}. We think that these tindmgs are conststent with the interpretation that this mPSDp-containing. N LS-in~oluble membJ ane frattton ts made up of detergent-lipid-protein vesicles (micelles) that are artifiLmlly generated during the treatment of SPM fractions with high concentrations of NLS 13.9~,,. w,v} And. when these vesicles are diluted with low ionic strength buffer so that the concentration of NLS is no longer above its critical micelle concentration, they dissolve and their total complement of proteins becomes soluble. These results suggest that, to a limited extent, the mPSDp content of synaptic junctions and postsynaptlc densities is solubilized during the extraction of synaptic plasma membranes with NLS. Experiments designed to screen additional subcellular fractions for the mPSDp, employing similar strategies of INT-cross-linking and detergent extraction, were carried out on brain cytosol (S.~) fractions and plasma membranes prepared from rat liver. Myelin and a gap junction fraction prepared from lens membranes were also analyzed for the presence of the mPSDp. Brain cytosol fractions contain two prominent polypeptides of 51,000 and 52.000 Mr (Fig. 6b, lanes iii and iv). However, the peptide fingerprints of these two proteins (Fig. 7d and 7a. respectively) are very different from that of the mPSDp (compare to Fig. 4a). Moreover, when Sa fractions are treated with INT, both the 51,000 and 52,000 Mr proteins are nearly absent from the INT-cross-linked, insoluble material

278

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Fig. 7. Autoradmgrams of tryptfc pept=de fingerprints, a: the 52 K molecular weight polypcptJde from an S,~ fraction, b: the prox,ainent 53 K polypeptide of purified mitochondria, c: the 52 K polypept=de

from lens gap,junctions, d" the 51 K polypeptide from an S~ fraction, e: the major Wolfgram protein (52 K) from purified myehn, f: the Triton X-100 =nsoluble, 52 K protein from liver plasma membrane~ (Fig. 6b, lanes i and ii), a property uncharacteristic of the mPSDp. Plasma membranes purified from rat liver contain a few proteins in the 52,000 Mr region (Fig. 6c, lane iv). Based on peptide fingerprint analysis, none of these proteins resemble the mPSDp (data not shown). Moreover, when liver plasma membranes are treated with INT and extracted wnth Triton X-100, only faint Coomassie blue-staining components are present in the 51,000-54,000 Mr range (Fig. 6c, lane iii). The faint staining band from this Triton-insoluble, liver membrane fractmn that most closely resembles the mPSDp in Mr, was excnsed from the gel and subjected to peptide mapping (Fig. 7f). The peptlde fingerprint of this hver membrane 52,000 Mr protein(s) ns qmte distinct from that of the mPSDp (compare Figs. 7f and 4a). Gap junctions are present between neurons and represent a type of intercellular junction that is morphologically distinguishable from asymmetric synaptic june-

279 tions 4z. Although gap junctions have not yet been isolated from CNS hssues, they can be isolated from lens, a tissue rich in morphologically identifiable gap j,~nct~ons 23. Gap junctions purified from chick lens contain a 52,000 Mr protein m addluon to a major intrinsic protein of 26,000 daltons 48 (Fig. 6c, lane i). When analyzed by peptide mapping, however, the fingerpritit of the 52,000 dalton gap junction protein (F,g. 7c) ~s very different from that of the mPSDp (compare with F~g. 4a). Myelin is a non-neuronal plasma membrane that ~s easdy pun,led and v, hich contains a number of well characterized polypeptidesaL Purified m)elin contains a prominent proteio, the major Wolfgram protein 5z, whose molecular weight ~s vely similar to that of the mPSDp (see Fig. 6a, lane n,). Analysis of the major W o l f r a m protein by peptlde mapping shows that it is very different from the mPSDp (compare Figs. 7e and 4a). Nuclei purified from rat brain ~ere subjected to electrophores~s and examined for proteins simdar to the mPSDp on the basis of apparent molecular weight. Nuclc, purified by extraction with Triton X-100 °-7 contained only minor, faint Coomasstc blue-staining components in the 52.000 molecular weight range (these bands comprise less than 0.1 ~o of the total protein in nuclear fractions, data not shown) We have previously shown that the appearance of the mPSDp in both SPM and SJ fractions Is developmentally regulated: its amount in these fractions increases approxmmtely 2~fold during the third and fourth postnatal week~z-' Nucleal fractions isolated from rodent brains throughout this period of development dl,pla)ed no major agedependent ,ncreases in the amounts of protein compon,mts m the 50.000-54.000 molecular weight range {data not shown; see also ref. 27) These results ~hox~ that the mPSDp is undetectable in brain nuclei fractions.

Analysis o! Con A-binding glycoprowin~ The subcellular fractions discussed abo~c were analy/cd for their contcnt ol glycoproteins that contain mannosyl residues and can be ~dentdled and Iocah/cd in SDS-gels by their abihty to bind [;"~l]Con A 4G. We ha~e previously ¢haracterlTcd a distinct class of Con A-binding glycoproteins that are pre~cnt in SJ fractions isolated from mammalian CNS tissue2S,~L These Con A-binding glycoprotems (designated I. II, Ill and IV in Fig. 8b) are greatly enriched in SJ fractions when compared to synapt;c plasma membranes. These glycoproteins have been hlstcchemically localized to the external surface of the postsynaptic membrane that overhes the PSD at asymmetric synapses li,z'~. Shown in Fig. 8 is a composite autoradiogram of glycoproteins that bind [l"~i]Con A in different subceilular fractions. In Fig. 8a is displayed the pattern of Con A-binding glycoproteins in SJs and the Triton-insoluble 0.8-1 0 M sucrose interface fraction. The patterns of mannosyl containing glycoproteins in the~e two fractions are qualitatively very simdar with the major quant~tatlve difference being that the SJ fraction contains proportionately more of the 170,C00 Mr. component I The finding that these Con A-binding glycoproteins are enriched in the Tritoninsoluble 0.8-1.0 interface fraction is consistent with the electron mic~o~.~,ptc findmg~ presented earlier which show that this fraction 1, highly enriched in postsynaptlc membrane specializations (PSD plus overlying postsy~,',r~tic m .abrane).

2SO

Con A B,ndlng Glycoproteins in SubceJlular Fractions (c) (b} (a) i

170 120 105

~

=~

- -

I

IV

52 46

25 f

~

SJ

TX, 1.0 ~nt'f.

f

f

• t

.

f

,

t

P3B INT- TX I SJ p3B INT-P3B

t

t

MITO. TX, MITO.

Fig. 8. Autorad'ogram of [l~'~l]Con A.binding glycoproleins of subcellular fractions as resolved in one-dimensional $D$ gels. a: synaptic junction fraction (SJ) and the Triton X-100-insoluble 1.0 M interface material (TXj 1.0 snt'f.), b: P:;B, INT-treated P:sBt I N T P:3B), Triton X-100-insoluble, INTtrealed P:jB (TXt I N T P.~Bhand an $.1 fraction with glycoproteins !, II, III a and b, and IV marked by arrows, c: INT-treated mitochondrial fraction (MITO}, and the Triton X.100 insoluble, INT-treated mltochondrial fraction (TXj MITO). Autoradiographzc exposures ranged from 24 to 36 h. (All samples contained 55/tg protein.)

Fig. 8 displays the Con A-binding glycoproteins present in PsB and INT-PsB fractions (panel b). The glycoprotein pattern of this fraction is unaltered by the INT treatment and is very similar to that of SPM fractions (data not shown; see ref. 47). However, unlike SPM fractions, treatment of the INT-PsB fraction with Triton X-100 solubilizes almost all of the Con A-binding glycoproteins in this fraction (Fig. 8b). This agrees well with the results described earlier which showed that although the Tritoninsoluble INT-PsB fraction contained predominantly PSD-like structures, these PSDs displayed no overlying postsynaptic membrane (see Fig. 2b). These results are consistent with the notion that the Con A-binding glycoprotcins originally described in SJs, may be a marker specific to the postsynaptic membranes of asymmetric synapses. Also shown in Fig. 8 are the Con A-binding glycoproteins present in the Tritoninsoluble mitochondrial fractions. The amounts of mannosyl containing glycoproteins in these two fractions (panel c) were barely detectable under conditions of equivalent protein loading and autoradiographic exposure compared to those used for the other fractions in this figure.

281 DISCUS,'~ION These resuRs show that the mPSDp in the rat forebrain is confined to subcellular fractions that are enriched in structures that morphologically resemble postsynaptlc densities (PSDs). It is generally accepted that PSDs isolated from the mammalian forebrain originate from the submembranous surface of the postsynaptic membrane at asymmetric synapses (type I synapse; see refs. 9, 21). PSDs possess a rather distinct type of subunit structure. A number of laboratories have verified that the PSD contains 20-30 nm dense staining bodies (particle-like aggregates)7,8,1°. Carlin et al. 7 have shown that these 20-30 nm aggregates appear to be interconnected by 6-9 nm diameter filaments. These filaments are probably analogous to the branching network of short 5 nm fibres that Matus and co-workers have described for a posts:ynapuc junctional lattice fraction that is prepared by extracting synaptic plasma membranes with sodmm deoxycholate40, ~1. Studies by Landis and co-workers "~4.a~ have shown that the postsynaptic membrane at presumptive asymmetric synapses contains clusters of 8-13 nm intramembranous particles (IMPs). Landis et al. 34 observed that these segregated patches of IMPs are characteristic of type i, excitatory synapses and not of the type !!, inhibitory synapse. Whether or not there exists a structural relationship or fun:tional interaction between IMPs and dense staining l:odies remains unknown Minus et al. ~9 have suggested that dense bodies may actually penetrate the postsynaptic membrane and may, therefore, comprise these IMPs. In this context, early studies by Peters and Kaiserman-Abramof 4~ showed that the 30 nm part;clcs observed in PSDs in situ appeared to traverse the postsynapuc membrane and extend into the deN. Although much is known about the ultrastructure of the asymmetric synapse. and especially the PSD, very little is known about the identity of spccllic molecules that make up its different morphological components. Carlin et al. 7 have recently isolated PSDs from the canine cerebellum. They observed that cerebellar PSD~ contained considerably less mPSDp than is present in PSDs isolated from cerebral cortex. The prominent morphological difference observed between cerebral and cerebellar PSDs was that the former displayed easily recogmzable 20-30 nm dense particles and the latter did not. They concluded that the mPSDp is somehow related to the presence and/or formation of these dense particles, in addmon. Carlin et al. 7 suggested that the PSDs isolated from the cerebellum are primarily type I!. inhibitory synapses, while those from the cerebral cortex are type I synapses and thus, the mPSDp might be preferentially associated with excitatory synapses. We have isolated SJ fractions from rat cerebellum by the INT procedure and observed a 20-fold reduction in their mPSDp content. We have also isolated SJs from rat forebrain minus cerebral cortices and observed that they contain approximately 50",, lower amounts of the mPSDp when compared to SJs prepared from the cerebral cortex. This difference in mPSDp content between cerebrum and underlying forebrain and cerebrum ~,s cerebellum may reflect the high proportions of asymmetric relative to symmetric synapses that are present in the cerebral cortex 9a3. In this context, it is difficult to conclude whether symmetric synapses from all brain regions contain very httle mPSDp or

282 that more than one type of asymmetric synapses may exist. In c~ntrast, the amounts of the four prominent Con A-binding glycoproteins (these components presumably reside on the external surface of the postsynaptic membrane2s,s2) are quantitatively indistinguishable between SJs isolated from cerebral cortex and forebrain minus cerebral cortex. These results show that microsomes (P3B), when treated under the same conditions used to prepare SJ from $PM fractions, will yield a detergent-inso:uble fraction which is highly enriched in structures that resemble l~Ds. This Triton X-100insoluble microsome fraction contains enriched amounts of the mPSDp when compared to the fraction from which it is derived. Moreover, the overall protein composition of SJ and Triton-insoluble microsome fractions are very similar (Fig. 3). In contrast to SJ fractions, however, the microsome-derived PSD fraction is devoid of postsynaptic junctional membranes and the Con A-binding glycoproteins that reside in these membranes*. We are uncertain as to the correct interpretation of this finding. Perhaps the microsomal fraction contains a population of synaptic junctional complexes whose postsynaptic membrane is more sensitive to detergent solubilization compared to SJs that are derived from the SPM fraction. On the other hand, these microsome-derived structures may represent PSDs that originate from symmetric synapses in the forebrain, whose junctional membranes are soluble in Triton X-100. In addition to containing large amounts of the mPSDp, the microsome-derived PSD fraction contains approximately twice as much tubulin when compared to SJ fractions (compare lanes b and d in Fig. 3). This observation is consistent with earlier studies in which tubulin was shown to be a significant component of rodent SJ and PSD fractions~,2g,sJ. A number of proteins such as actin .~,.~.~0,tubulintg,'ze,sl0 myosin s, caimodulin 'zo, cyclic AMP-dependent protein kinases and substrate phosphoproteins~O,50 have been identified and characterized in subcellular fractions enriched in SJs or PSDs. Without exception, these proteins are also present in brain subcellular fractions other than SJs and PSDs, and in cell types that do not possesssynapses. The studies presented herein have explored whether or not the mPSDp constitutes a molecular marker specific to the asymmetric, type I synapse. Our approach, based on the technique of subcellular fractionation, represents an indirect analysis of the cellular distribution of the mPSDp in situ. Thus, conclusions based on these results must be discussed in view of certain limitations. For example, we are presently unable to carry out a non-specific adsorption study in which an exogenous, purified protein (usually in a radiolabeled form) or purified organdie is added to tissues during homogenization to examine whether or not the component of interest truly co-purifies with the appropriate subceUular fraction(s). These experiments cannot be done because isolated PSDs are unusually adhesive and will stick to almost anythinge,la. Experiments based on the use of purified mPSDp are unrealistic since the only method presently available to solubilize and purify this protein is by SDS-gel electrophoresis. This would produce * in SJ fracttons isolated from adult brain, two-thirdsof all intact SJs and postsynapticmembrane specializations contain easily identifiablesegmentsof postsynapticmembraneaz.

283 the mPSDp in a denatured, non-physiological state. Secondly, our ability to detect the mPSDp in the different subcellular fractions relies on the sensitivity of staining with Coomassie blue and being able to resolve and identify mPSDp specific [1251]peptides in the various peptide fingerprints. We have estimated by dei~sitometry that protein quantities approaching 80-100 ng are detected by these staining procedures. With regard to the peptide mapping technique, we have shown that distinct, protein-specific [1251]peptldes can be identified in protein mixtures even when the protein of interest represents as lit,!e as one-fifth of the total protein in a single gel slice 3~. When these two limits of resolution are combined, the mPSDp will only be detected in a given fraction when it constitutes at least 0.04~ by weight of that fraction's total protein content (assuming the fraction contains 20-40 prominent polypeptldes). Finally, the mPSDp may be a prominent molecular component of a subcellular organelle that ~s not appreciably enriched in the fractions examined throughout these studies. For example, we did not examine coated vesicles for the presence of the mPSDp. Howevel. studies by others have shown that clathrin (Mr :- 175,000}, the major coated vesicle protein, and other presumptive vesicle-associated proteins are clearly d~fferent from the mPSDp on the basis of molecular weight 24,4~. Furthermore, we have made the assumption that the mPSDp, wherever present in the brain or non-neural tissues. ~s responsive to INT-dependent cross-linking and thereafter is insoluble m Triton X-l130 Although we have u:ed this strategy in an attempt to enrich for the mPSDp in various subeellular fractions, this property may not pertain to the mPSDp -zhat could exist at non-synaptic locations. Our results ~,how that there is a very stron~ correlation between the pre, cnce in any subcellular fraction of asymmetric synaptic junctional structures and the existence in these fractions of substantial amounts of the mPSDp. The mPSDp ts not detectable in brain fractions enriched in myelin, mitochondria, nuc[e~ and cytosol, nor ~s ii detectable in membranes purified from liver and lens, The,~e results also show a strong interdependence between the presence of morphologically idenufiable posts~napt~c membranes (presumably those that overlie PSDs originating from type I synap~,c~) and the co-existence of 4 major Con A-binding glycoproteins that have been characterized in isolated SJs22,2s,47. On the basis of these observations and prewous papers, ~c believe that the mPSDp is a marker ~pecifie to the postsynaptic density of asymmetric. type ! synapses. Likewise, the Con A-binding glycoprotems appear to be a suitable marker for the postsynaptic membranes of this same class of synapses. We and others have shown that these glycoproteins are not found in appreciable amounts m microsomes, myelin, mitochondria, synaptic vesicles, extrajunctional synapuc membranes and axolemma14.~z,'zs. The unequivocal locahzation of these presumptive, synapse specific macromolecules in neural t~ssues ~ill likely result from immunohistochemical studies that u~e antibodies specific to these d~fferent synaptic macromolecules. Previous reports have shown that the mPSDp ~s extremely m,~olublc",~,z'j Our finding that the mPSDp cannot be detected in two separate cytosolic (S:~) fractions ~s consistent with earlier observations. Although little ~s presently known about the turnover rates of specific synaptic polypeptides, it will be important to understand how

284 the neuron regulates the synthesis, transport to and the assembly of the mPSDp into postsynaptic densities. If the mPSDp is insoluble at the site of synthesis one might speculate that it must be synthesized by ribosomes associated with the rough ¢ndoplasmic reticulum via the signal sequence model of membrane assemblyaS,a¢, and then shuttled to the synapse by a transport/assembly vesicle. In isolated SJ fractions, the levels of the mPSDp increase approximately 20-fold during the third and fourth postnatal weeks3z. The synthesis and assembly of the mPSDp may be most easily studied within a developmental setting, when the levels of this major synaptic protein increase dramaticallyt4,~2. At present : -- can only speculate as to the functional role(s) that the mPSDp may fulfill at asymmetric synapses. We have previously proposed that this protein's unusual solubility properties14.2g, and its inherent propensity to form intermolecular disulfide bonds w~!h itself and other proteins z~, make it a suitable candidate for the primary structurai component which forms the supramolecular scaffolding of the type 1 synaptic junction. In this manner, the mPSDp would impart structural stability to the asymmetric synapse. In addition, the mPSDp may constitute a superstructure to which an actomyosin contractile system.~.i4 could anchor and exert far-reaching, regulatory actions on the lateral mobility of components (e.g. transmitter receptors) embedded in the overlying postsynaptic membrane. Recent reports have shown that the mPSDp can be phosphorylated in isolated SJ and PSD fractions¢,3°. Although the phosphorylation of this protein is refractory to the addition of cyclic AMP 7,.';", recent findings by Carlin et al. ¢ have shown that the phosphorylation of the mPSDp may be linked to calcium through the action of calmodulin. We have previously shown that the mPSDp is a distinct, l',omogenous polypeptide and Js clearly different from all other filamentous polypeptides anc~ proteins to which it has been comparedl4,29. We have shown that the developmental appearance of the mPSDp in SJ fractions isolated from immature brains is temporally coordinated with the morphological emergence of synaptic contacts~'-'. These observations, together with findings concerning the synapse-specific localization of the mPSDp, strongly support the notion that this protein is an important structural/functiona~ element of asymmetric synapses. ACKNOWLEDGEMENTS

We thank Ms Christine Neibling for her help in preparing this manuscript and Steve Kimura and Ellen Lewis for their technical help. We also extend a special thanks to Carl Cotman, whose interactions stimulated the initiation of certain aspects of this work. This research was supported in part by NIH Grant NS 15554 (to P.T.K.). PTK is a recipient of RCDA NS 00605. This is Contribution 81-450-j from the Kansas Agriculture Experiment Station, Kansas State University, Manhattan, KS. ABBREVIATIONS SJ PSD SDS INT

synaptic junctton postsynaptic density sodmm dodecyl sulfate iodonilrotetrazohum violet

SPM synaptic plasma membrane mPSDp major PSD protein Con A Concanavalin agglutinin

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