©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Neurofilament Protein Heterotetramers as Assembly Intermediates (*)

Jeffrey A. Cohlberg (§) , Hamid Hajarian , Tan Tran , Parvaneh Alipourjeddi , Alexander Noveen

From the (1) Department of Chemistry and Biochemistry, California State University, Long Beach, California 90840

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Evidence is presented for the existence of a soluble heterotetramer containing the low and middle molecular weight neurofilament (NF) proteins, NF-L and NF-M, and one containing the low and high molecular weight proteins, NF-L and NF-H, and for their role in filament assembly. When a mixture of either pair of proteins was renatured in 2 M urea, 20 m M Tris, pH 7.2, a new band representing a complex was observed in native gel electrophoresis. No new band was observed with a mixture of NF-M and NF-H. Two-dimensional gel electrophoresis showed that treatment of the complexes with SDS caused them to dissociate into their constituent polypeptide chains. Native neurofilaments dissociated in 2 M urea into a mixture of LM and LH complexes. Titration of NF-L with NF-M indicated that complex formation was complete at an approximately equimolar ratio of the two proteins. The LM complex had a sedimentation coefficient, s, of 4.4 S, consistent with a tetrameric structure. Dialysis of a solution of the LM complex against 50 m M 4-morpholineethanesulfonic acid, 0.17 M NaCl, pH 6.25, led to the formation of 10-nm filaments in good yield. These results suggest that NF protein heterooligomers are intermediates in NF assembly and disassembly.


INTRODUCTION

The neurofilaments (NF)() of adult central nervous system neurons are comprised principally of three polypeptide chains, termed NF-L (low), NF-M (middle), and NF-H (high) according to their molecular masses, of 62, 98, and 113 kilodaltons, respectively (1, 2) . Although the biochemistry and cell biology of NF have been extensively studied (1, 2, 3, 4, 5) , understanding of their structure and assembly is limited.

Like other members of the intermediate filament family (6, 7, 8) , NF proteins have a central helical rod domain about 310 amino acids long which participates in the formation of coiled coil dimers, flanked by nonhelical head and tail domains. The large size of NF-M and NF-H is due to extra mass in their tail domains, which have been shown to project out from the core of the filament (9) . The polypeptide composition of NF appears to be variable rather than fixed, with different ratios of the three proteins found in different tissues and with changes observed during growth and differentiation (10) .

NF dissociate into a mixture of the three proteins when dissolved in concentrated urea and reassemble into filaments when incubated at moderate salt concentrations and slightly acidic pH (11, 12, 13) . Purified NF-L readily forms filaments by itself in vitro, and studies of the pathway of NF assembly have thus far concentrated on reconstitution of homopolymer filaments from NF-L. Several intermediates in this process can be stabilized in soluble form under appropriate conditions. Thus NF-L assembly proceeds through dimers (14) , tetramers (15, 16) , and octamers (17) , with further longitudinal and lateral aggregation finally leading to filaments (17, 18) .

However, the NF-L homopolymer filaments whose in vitro assembly has been intensively studied never occur in vivo; rather, NF-L is always expressed in combination with at least one other neuronal IF protein (4) . In fact, when expressed by transfection in cultured cells, none of the three proteins, including NF-L, can polymerize by itself, but NF-L copolymerizes with NF-M or NF-H in appropriately cotransfected cells (19, 20) . In vitro studies on NF-M and NF-H and on mixtures of proteins are generally consistent with the results of the transfection studies; NF-M, NF-H, or a combination of these two proteins form shorter and more irregular filaments and do so with relatively low efficiency, whereas each protein can coassemble efficiently with NF-L ( e.g. Refs. 9, 21, and 22).

None of the studies on filament formation from mixtures of NF proteins has examined the details of the process or the nature of intermediates. Therefore, although NF-L homopolymer assembly in vitro has been extensively characterized, little is known about the assembly pathway of the heteropolymer filaments actually found in cells.

In this study we demonstrate by nondenaturing polyacrylamide gel electrophoresis (``native gels'') the formation of two soluble heterotetramers, one containing NF-L and NF-M and the other containing NF-L and NF-H. Upon treatment with 2 M urea, intact NF dissociate into a mixture of the two complexes. The NF-LNF-M complex is able to serve as a starting point for filament assembly. The implications of these results for the pathway of NF assembly and disassembly in vivo are discussed. Preliminary accounts of some of these results have been presented (23, 24) .


EXPERIMENTAL PROCEDURES

Purification of NF Proteins

NF were isolated from bovine spinal cord, and NF proteins were purified by chromatography on DEAE-cellulose in the presence of 6 M urea as described previously (25) , except that Toyopearl DE-650 Superfine (Supelco) was used. This gave much better resolution than Whatman DE52. For experiments on whole neurofilaments, crude spinal cord extract clarified by low-speed centrifugation was chromatographed on Sepharose 4B in 20 m M PIPES, 1 m M EGTA, 1 m M MgCl, 10 µg/ml leupeptin, 0.4 m M PMSF, and 1 m M DTT, pH 6.8, and the void volume fractions, found to contain only the three NF proteins, were used in further experiments (9, 26) . Chicken gizzard desmin was purified by the method of Geisler and Weber (27) , with an additional step of single-stranded DNA-cellulose chromatography (28) to remove small amounts of degraded desmin. Porcine lens vimentin was purified by the method of Geisler and Weber (29) .

Sedimentation Velocity

Sedimentation velocity experiments were performed in a Beckman model E centrifuge equipped with a photoelectric scanner. The recorder voltages were input to a Macintosh SE computer through a MacAdios 8ain interface (GW Instruments). Solvent viscosities were measured with a capillary viscometer and densities determined pycnometrically for correction of observed sedimentation coefficients to s.

Urea Native Gel Electrophoresis

Protein stock solutions were dialyzed versus 4 M guanidine hydrochloride containing 1 m M DTT and 0.4 m M PMSF. Protein concentrations were determined by microbiuret assay (30) . Molar concentrations were calculated using molecular weights of 6.2 10for NF-L, 9.8 10for NF-M, and 1.13 10for NF-H, averages of molecular weights determined from amino acid sequence data (31) . Individual proteins were mixed and dialyzed overnight versus two changes of 2 M urea, 20 m M Tris, 6 m M mercaptoethanol, and 0.4 m M PMSF, pH 7.2, in Sartorius microcollodion bags.

Since 2 M urea electrophoresis with a continuous Tris chloride buffer system of uniform pH gave poor resolution and reproducibility, a discontinuous buffer system was used. The Tris-glycine system used by Laemmli (32) was modified by lowering the ionic strength, raising the pH of the stacking gel and lowering the pH of the running gel, since high salt concentrations and extremes of pH might cause the proteins to precipitate or dissociate. The running gel, 16 cm 14 cm 1.5 mm, contained 4.5 ml of acrylamide solution (30% acrylamide, 0.8% N,N`-methylenebisacrylamide), 7.5 ml of 8 M urea, 0.25 M Tris, pH 8.4, and 17.74 ml of HO (final pH 8.0). Polymerization was initiated by addition of 10 µl of TEMED and 250 µl of 10% ammonium persulfate. The stacking gel contained 1.4 ml of acrylamide solution, 2.5 ml of 8 M urea, 80 m M Tris, pH 7.6, and 5.99 ml of HO (final pH 7.2). Polymerization was initiated by adding 10 µl of TEMED and 100 µl of 10% ammonium persulfate. Samples of approximately 40-µl volume were layered onto the bottoms of 6-mm-wide wells. The electrode buffer contained 0.47 g/liter Tris and 2.38 g/liter glycine (pH 8.6). Because a dilute electrode buffer was used, electrophoresis was conducted in an apparatus containing upper and lower buffer chambers with volumes of 3 liters each in order to provide the required buffer capacity. Electrophoresis was conducted at 100 V (6 mA) for 15 h until the tracking dye reached the bottom of the gel. The pH of the lower buffer dropped to about 8.0 during the run.

For two-dimensional gel electrophoresis, a lane from a 2 M urea gel was excised with a razor blade and incubated for 60 min in SDS sample buffer (0.0625 M Tris-HCl, pH 6.8, containing 10% glycerol, 2% SDS, and 5% 2-mercaptoethanol) containing 0.001% bromphenol blue at room temperature. The second dimension SDS gels contained 6-7.5% acrylamide and were prepared according to Laemmli (32) . The first dimension gel was embedded on top of the stacking gel of the second dimension slab using 1% agarose dissolved in SDS sample buffer lacking glycerol.

Filament Reconstitution

Protein samples in 2 M urea, 20 m M Tris, 1 m M DTT, 0.4 m M PMSF, pH 7.2, were dialyzed at 37 °C versus 50 m M MES, 0.175 M NaCl, 0.5 m M EGTA, 1 m M DTT, 0.4 m M PMSF, pH 6.25 (33) . An aliquot of the suspension was applied to a Formvar-coated copper grid, stained with 2% uranyl acetate, and examined at 80 kV in a Jeol 1200EX-II electron microscope.


RESULTS

NF Proteins Retain Their ``Native'' States in 2 M Urea

Electrophoresis of NF proteins was initially performed in the presence of 4 M urea, in which keratins readily form complexes (34) . In these experiments the three NF proteins migrated independently and showed no evidence of complex formation (data not shown). Attempts at performing native gel electrophoresis in aqueous buffers lacking denaturants were unsuccessful; very little protein entered the gel, and heavy staining was observed at the bases of the sample wells. It was then found that inclusion of 2 M urea in the gel was sufficient to render the proteins soluble, and such gels provided evidence of heterooligomer formation (see below). We therefore performed sedimentation velocity experiments to verify that NF proteins retain their ``native'' states in 2 M urea ( i.e. exhibit the same states in 2 M urea as they do in dilute pH 8 buffers), as has been reported for desmin (35) and keratins (36) .

Fig. 1 shows the sedimentation coefficient as a function of urea concentration for different intermediate filament proteins. The s value of desmin remains almost constant up to a urea concentration of 6 M and begins to decrease at 7 M urea, in agreement with the findings of Kaufmann et al. (35) that desmin tetramers dissociated to dimers in 7 M urea. Desmin existed as dimers in 2-3 M guanidine hydrochloride and then dissociated to monomers at 4 M guanidine. The svalues in 4 and 6 M guanidine, 2.1 and 1.9 S, are close to the value of 2.0 S expected for a randomly coiled polypeptide chain of 463 amino acid residues (calculated from the data in Ref. 37). Similar behavior for desmin in guanidine solutions was reported by Quinlan et al. (14) . For both NF-L and vimentin, a concentration of 5 M urea is required before any decrease in the s value is observed, suggesting that tetramers are stable up to 4 M urea. The sedimentation coefficient of NF-M is virtually independent of urea concentration, indicating that it undergoes no change in aggregation state within this range of urea concentrations. Thus, gel electrophoresis in the presence of 2 M urea should reflect the behavior of individual NF proteins and protein complexes in their ``native'' states.


Figure 1: NF proteins retain their native states in 2 M urea. Sedimentation coefficients of desmin (), vimentin (), NF-L (), and NF-M () in 10 m M Tris, pH 8.0, as a function of urea concentration, and desmin as a function of guanidine hydrochloride concentration ().



NF-L Forms a Heterooligomer with NF-M or NF-H

The mobilities of the three individual NF proteins in the 2 M urea gels are shown in Fig. 2, lanes 1-3. It should be noted that electrophoretic mobilities in native gels are functions of both size and charge, and no direct conclusions can be drawn about the relative sizes of the proteins under these conditions from their mobilities in this gel system. Presumably NF-L migrates as a tetramer and NF-M and NF-H as monomers or monomers in equilibrium with dimers (25) . At higher acrylamide concentrations, the order of the bands changes, with NF-M migrating faster than NF-L (data not shown), consistent with a larger size for NF-L than NF-M under these conditions (38) .


Figure 2: 2 M urea gel electrophoresis of NF protein mixtures shows complex formation. Samples (except lane 6) were dialyzed overnight versus 4 M guanidine hydrochloride, 0.1 m M PMSF, 1 m M DTT, and then overnight versus 2 M urea, 20 m M Tris, pH 7.2, 0.1 m M PMSF, 1 m M DTT. Electrophoresis was performed as described under ``Experimental Procedures.'' Lane 1, NF-L; lane 2, NF-M; lane 3, NF-H; lane 4, NF-L + NF-M; lane 5, NF-L + NF-H; lane 6, whole neurofilaments obtained by gel filtration of crude spinal cord extracts were dialyzed overnight versus the 2 M urea buffer; lane 7, NF-M + NF-H; lane 8, NF-L + NF-M + NF-H.



For NF-H, in addition to the principal band migrating near the middle of the gel, a second band near the top of the gel was observed. The relative intensities of the two bands are variable; probably the band near the top of the gel represents an aggregated form of NF-H. In some experiments a minor second band for NF-M was observed which approximately comigrated with NF-L; the origin of this band is unknown.

Lane 4 in Fig. 2shows a sample in which NF-M and NF-L were mixed, dialyzed overnight against 4 M guanidine hydrochloride, then dialyzed overnight against 2 M urea, 20 m M Tris-HCl, pH 7.2. The band for NF-M is missing, the band for NF-L is greatly reduced in intensity, and a new band with a lower mobility appears. This new band presumably represents a heterooligomer containing both polypeptides (``LM complex''). A mixture of NF-L and NF-H treated in the same fashion ( lane 5) showed reduced intensities for the NF-L and NF-H bands and the appearance of a new lower mobility band presumably representing an NF-LNF-H heterooligomer (LH complex) which migrated more slowly than the LM complex. Mixtures of NF-H and NF-M ( lane 7) showed only bands corresponding to the individual proteins, indicating that no MH complexes were formed. When a mixture of all three proteins was examined ( lane 8), both LM and LH complexes were observed, with some residual bands of reduced intensity corresponding to uncomplexed NF-L and NF-H.

For all mixtures, results similar to those shown were obtained when the guanidine dialysis step was omitted, or when proteins were dialyzed directly from the 6 M urea buffer used for ion exchange chromatography into 2 M urea, 10 m M Tris, pH 7.2, and then mixed just before electrophoresis. However the best yields of complex were most consistently obtained when the guanidine dialysis step was included.

Two-dimensional Gels Confirm the Identity of the Complexes

The identity of the putative complexes was confirmed by the use of two-dimensional gel analysis. Native gel electrophoresis in the presence of 2 M urea was performed in the first dimension; then the lanes were excised and incubated in SDS in order to dissociate the oligomers into their constituent polypeptides, which were resolved in a second dimension in the presence of SDS. Fig. 3 A shows a pattern obtained from a mixture of NF-L and NF-M at a molar ratio (M:L) of 0.47; a band corresponding to a complex dissociated in the presence of SDS into NF-M and NF-L, whereas a second band with greater mobility contained only NF-L. Fig. 3 B shows a pattern obtained with a molar excess of NF-M, indicating an LM complex, an NF-M band, and a small amount of uncomplexed NF-L. A mixture of NF-L and NF-H is shown in Fig. 3 C, with a band for LH complex dissociating into NF-H and NF-L in the presence of SDS, along with separate bands for free NF-H and NF-L. A mixture of all three proteins (Fig. 3 D) shows LH and LM complexes dissociating into the expected components in the second dimension, along with spots for each of the three uncomplexed proteins.


Figure 3: Two-dimensional gel electrophoresis of NF protein complexes. First dimension was 2 M urea electrophoresis from left to right and second dimension was SDS gel. A, NF-L + NF-M, molar ratio (M:L) 0.47; B, NF-L + NF-M, molar ratio (M:L) 1.73; C, NF-L + NF-H; D, NF-L + NF-M + NF-H; E, whole NF (see Fig. 2, lane 6).



Dissociation of Whole Neurofilaments into LM and LH Complexes

For this experiment, a pure neurofilament preparation containing only the NF triplet proteins was obtained by gel filtration of a crude spinal cord extract as described under ``Experimental Procedures.'' Fig. 2, lane 6, reveals that dialysis of these filaments against 2 M urea, 20 m M Tris, pH 7.2, caused them to dissociate into a mixture of LM and LH complexes. The corresponding two-dimensional gel (Fig. 3 E) confirmed that the spots corresponding to the LH and LM complexes accounted for virtually all the protein in the solution. This experiment lends support to the idea that the LM and LH complexes represent structural entities present within intact filaments.

Titration Experiments Indicate a 1:1 Stoichiometry

A complexation titration was performed, in which the amount of NF-L was held constant while the amount of NF-M was increased, as shown in Fig. 4. In lanes 2-5, addition of increasing amounts of NF-M led to the gradual disappearance of the NF-L band, whereas all the NF-M was incorporated into the complex. (Only faint NF-M bands are seen in lanes 4 and 5.) By lane 5 only a faint NF-L band remained, corresponding to a small amount of NF-L which was not able to associate with NF-M. Addition of further NF-M led to the appearance of a strong NF-M band, indicating that after the equivalence point was reached, excess free NF-M was present. The mixture of lane 5, at a molar ratio M:L of 1.17, is close to the equivalence point at which complex formation was complete. This suggests that the LM complex contains equimolar amounts of the two proteins. A similar equivalence point was obtained in an experiment in which the amount of NF-M was held constant and the amount of NF-L was varied (data not shown). Also, a similar equivalence point was obtained in experiments where the protein concentration was determined by the method of Bradford (39) instead of the microbiuret assay. It should also be noted that a molar ratio (M:L) of 1 corresponds to a mass ratio of 1.58; this is consistent with the two-dimensional gel patterns (Fig. 3), which always showed a greater intensity of the NF-M spot than the NF-L spot derived from the dissociation of a band for ML complex.

One example of a titration of NF-L with NF-H is shown in lanes 8-13. In this experiment, addition of increasing amounts of NF-H led gradually to near complete disappearance of the NF-L band. Initially ( lane 8) all of the NF-H was incorporated into the complex, but with increasing amounts of NF-H added a significant portion of the NF-H remained uncomplexed. The proportion of NF-L and NF-H which appeared competent in complex formation differed among protein preparations, but complete complex formation was never observed. Therefore, we did not attempt to interpret these experiments in terms of an equivalence point.

LM Complex Can Serve as an Intermediate in Filament Assembly

A preparation of pure LM complex (Fig. 5, inset, fourth lane) in 2 M urea, 20 m M Tris, pH 7.2, was dialyzed against a buffer favoring filament reconstitution at 37 °C (see ``Experimental Procedures'') and examined by electron microscopy. Filaments with a diameter of 10-12 nm were observed, with virtually no unpolymerized protein appearing on the grid. This suggests that the LM complex can serve as an intermediate in filament assembly (see ``Discussion'').


Figure 5: Reconstitution of filaments from LM complex. An equimolar mixture of NF-L and NF-M in 4 M guanidine hydrochloride was dialyzed versus 2 M urea, 20 m M Tris, pH 7.2, and an aliquot was taken for native gel electrophoresis. The solution was further dialyzed for 3 h at 37 °C versus 50 m M MES, 0.175 M NaCl, 0.5 m M EGTA, 0.4 m M PMSF, 1 m M DTT. An aliquot was stained with 2% uranyl acetate and examined in a Jeol 1200-EXII electron microscope operated at 80 kV. The bar represents 100 nm. Inset, 2 M urea electrophoresis of LM complex used for reconstitution (fourth lane) along with NF-L, NF-M, and NF-H standards.



Sedimentation Velocity of LM Complex Suggests a Tetrameric Structure

A series of absorbance traces from a sedimentation velocity experiment on a solution of LM complex in 2 M urea, 20 m M Tris, pH 7.2, is shown in Fig. 6. A 2 M urea gel of this solution showed virtually 100% complex, similar to Fig. 5. A well defined boundary indicative of a single sedimenting species was observed, with initial absorbance traces (not shown), indicating that about 29% of the material formed rapidly sedimenting aggregates. (Aggregation was a more severe problem when renaturation was performed at higher protein concentrations.) A sedimentation coefficient, s, of 4.4 S was observed, compared with values of 3.7 S for NF-L tetramers and 5.5 S for NF-L octamers (17) . This value was confirmed by sucrose gradient velocity sedimentation of a preparation of the complex dialyzed against 10 m M triethanolamine, pH 8.0, which showed NF-L and NF-M cosedimenting at the same rate as bovine serum albumin, whose sis 4.31 S (40) (data not shown). This sedimentation coefficient is consistent with a tetrameric structure for the LM complex (see ``Discussion'').


DISCUSSION

LM and LH Complexes as Structural Entities of Filaments and Intermediates in Filament Assembly

This study has demonstrated that LM and LH complexes form when appropriate mixtures of NF proteins are renatured together in a low ionic strength buffer containing 2 M urea. The sedimentation coefficient observed for the LM complex, intermediate between the values for the NF-L tetramer and NF-L octamer, is consistent with a tetrameric structure for this oligomer. An increase in the s value of the tetramer from 3.7 S for Lto 4.4 S for LMis exactly what would be predicted from the dependence of sedimentation coefficient on the 2/3 power of the molecular weight (41) . A tetrameric structure is also supported by the results of preliminary cross-linking studies employing dithiobis(succinimidyl propionate) (24, 42) .

The relevance of the heterotetramers to neurofilament structure and assembly is indicated by two key findings. First, when filaments isolated by gel filtration of crude spinal cord extract, which had not previously been subjected to conditions favoring filament disassembly, were exposed to 2 M urea, they dissociated into a mixture of LM and LH complexes. Second, when a solution containing virtually 100% LM complex was dialyzed against a standard filament reassembly buffer, filaments were obtained in good yield.

It could be argued that neither of these findings proves the relevance of the complexes to NF assembly. In regard to the first observation, it is conceivable that the 2 M urea treatment initially gave different dissociation products which subsequently rearranged to form a mixture of LM and LH complexes with virtually no free protein remaining, but this seems unlikely. In regard to the filament reassembly, it is possible that the LM heterotetramers are not productive assembly intermediates, that the LM complexes dissociated into NF-L and NF-M which then reassociated via completely different intermediates to form filaments. For example, although tropomyosin is composed of coiled coil heterodimers, renaturation under certain conditions favors formation of and homodimers which can then reequilibrate to heterodimers (43, 44, 45) . One should, however, note for comparison the behavior of a dead-end complex of vimentin and keratin, which when added to a keratin filament reconstitution mixture not only was incapable of forming filaments but actually inhibited further filament formation (46) . Rigorous proof that LM complex can serve as a true assembly intermediate rather than a dead-end complex would require demonstration that a covalently cross-linked LM complex is capable of forming filaments ( cf. Ref. 46), and such studies are now in progress.

The specificity observed in heterooligomer formation is the same as that observed in NF assembly; thus, the specific requirement for NF-L and one of the other NF proteins seen in assembly experiments (see the Introduction) can be explained by the behavior of the proteins at the level of tetramer formation. Thus the failure of NF-M and NF-H to coassemble in the absence of other IF proteins can be explained by their failure to form a heterotetrameric assembly intermediate.

While Carden and Eagles (47) did observe the formation of disulfide cross-links between NF-M and NF-H upon oxidation of the soluble material formed from dissociating NF at low ionic strength, it should be noted that the NF preparations used in these studies contained considerable non-NF protein contamination, and the soluble material was heterogeneous in size (48) .

Detailed Structure of the Complexes

The detailed structure of the LM and LH complexes is not yet certain. While the titration data suggest that LM tetramers contain two copies each of NF-L and NF-M, other models cannot be ruled out. For example, electron microscopic observations of the frequency of NF-H tail domain projections in NF reconstituted from a mixture of NF-L and NF-M led Hisanaga and Hirokawa (9) to conclude that there was one such projection per tetrameric unit, not two; this leads to the expectation that a tetramer containing three NF-L polypeptide chains associated with one chain of either NF-M or NF-H (LM or LH) would constitute a structural entity within filaments. However, these authors mentioned the possibility that each projection could represent more than one tail domain, an idea consistent with the titration data of Fig. 4. Clearly further work is required to settle these questions. Experience with the incorrect three-chain model for keratins (49) , which was also based largely on evidence from titration studies, argues for caution in this regard.


Figure 4: Titration studies of NF complex formation. Each lane of the 2 M urea gel contained a constant amount of NF-L. Lanes 2-7 also contained NF-M at molar ratio (M:L) of 0.29, 0.58, 0.88, 1.17, 1.46, and 1.75. (Note the gradual disappearance of the NF-L band, which is completed by lane 4.) Lanes 8-13 also contained NF-H at molar ratios (H:L) of 0.20, 0.39, 0.59, 0.79, 0.99, and 1.18. The incompleteness of HL complex formation makes it impossible to determine an equivalence point.



In an electron microscopic study (50) , antigenic epitopes on the NF-L tail domain which were accessible in filaments reconstituted from NF-L alone were found to be relatively inaccessible in native NF; this was taken to suggest that NF-L/NF-M and NF-L/NF-H heterodimers occur within NF. The results presented here do not distinguish between a tetramer consisting of a dimer of homodimers, e.g. (L)(M), or a dimer of heterodimers, e.g. (LM).

Finally, the present results do not identify the molecular interactions responsible for the observed specificity in heterotetramer formation. Initial results with truncated mouse NF-L constructs missing head or tail domains suggest that this specificity is due to specific interactions among the central rod domains of the three proteins.() These studies will be reported elsewhere.

Significance for Assembly in Vivo

These studies suggest that NF protein heterooligomers, rather than individual NF proteins, are the intermediates for filament assembly and disassembly in vivo. According to this model, in de novo filament assembly, newly synthesized NF proteins would form LM and LH tetramers, which would then associate to form 10-nm filaments. During filament disassembly, e.g. that triggered by head domain phosphorylation (17, 51, 52) , LM and LH tetramers would dissociate from the filaments and be released into the soluble phase. The incorporation of axonally transported protein into the stationary filamentous network (51, 53) would occur through exchange of heterotetramers released from the filaments with heterotetramers present in the moving phase (54, 55) ; replacement of one type of heterotetramer by another with a different protein composition would then be responsible for changes in the protein composition of NF observed during development (10) .

While the NF triplet proteins are the principal constituents of NF in mature neurons of the central nervous system, in some neuronal cells and neuronal precursors they are present with other ``neuronal IF proteins,'' peripherin, -internexin, vimentin, and nestin (2, 5) . In these neurons, as yet uncharacterized heterotetramers or other heterooligomers containing NF triplet proteins in combination with vimentin, peripherin, or -internexin would serve as assembly intermediates.


FOOTNOTES

*
This work was supported by National Science Foundation Grants DCB-8902594 and DCB-8904460, by a summer fellowship (to T. Tran) from the Howard Hughes Medical Institute Biological Sciences Initiative, and by the California State University, Long Beach, CA. The purchase of the electron microscope was funded in part by National Science Foundation Grant BBS 88-20774. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, California State University, Long Beach, 1250 Bellflower Blvd., Long Beach, CA 90840. Tel.: 310-985-4944; Fax: 310-985-2315.

The abbreviations used are: NF, neurofilament; NF-L, NF-M and NF-H, low, middle, and high molecular weight neurofilament proteins; DTT, dithiothreitol; IF, intermediate filament; PMSF, phenylmethylsulfonyl fluoride; PIPES, 1,4-piperazinediethanesulfonic acid; TEMED, N,N,N`, N`-tetramethylethylenediamine; MES, 4-morpholineethanesulfonic acid.

T. Tran, A. Ansari, J. Cohlberg, M. Lee, and D. Cleveland, unpublished experiments.


ACKNOWLEDGEMENTS

We thank Dr. Tom Douglass for assistance in the preparation of Fig. 5.


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