From the
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
The neurofilaments (NF)
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-L
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
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.
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 s
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-L
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.
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.
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
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) .
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.
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,
We thank Dr. Tom Douglass for assistance in the
preparation of Fig. 5.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, 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.
(
)
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.
NF-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) .
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 10
for NF-L, 9.8
10
for NF-M, and 1.13
10
for 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.
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 H
O (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 H
O (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.
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.
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) .
values 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.
NF-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.
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.
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
s
is 4.31 S
(40) (data not shown). This sedimentation coefficient is
consistent with a tetrameric structure for the LM complex (see
``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 L
M
is 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) .
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.
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
L
H) 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)
.
(
)
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) .
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.