(Received for publication, March 14, 1995; and in revised form, August 11, 1995)
From the
The multimeric clathrin assembly proteins AP-1 and AP-2 with
molecular masses of 270 kDa and the monomeric clathrin assembly
proteins AP
and auxilin with molecular masses of
90
kDa catalyze the assembly of clathrin into artificial clathrin baskets
under physiological conditions. We have now identified a much smaller
20-kDa clathrin assembly protein in 0.5 M Tris, pH 7.0,
extracts of bovine brain coated vesicles and purified it to near
homogeneity. A polyclonal antibody against this protein did not
cross-react with any of the other assembly proteins, and sequencing
data suggest that this new protein is similar or identical to myelin
basic protein (MBP). At a molar ratio of 3 molecules per clathrin
triskelion, MBP catalyzes polymerization of clathrin into artificial
baskets that appear structurally similar to the baskets assembled by
the other assembly proteins. In addition, like the other baskets, the
clathrin-MBP baskets are uncoated by hsp70. MBP represents a
significant fraction of the total assembly protein activity present in
0.5 M Tris, pH 7.0, extracts of coated vesicles. It is not
clear if it acts as an assembly protein in vivo, but because
it is well characterized and easily available, MBP will be a useful
protein to investigate the mechanism of clathrin assembly and
disassembly in vitro.
Clathrin-coated vesicles are organelles involved in a number of
cellular transport processes including receptor-mediated endocytosis,
transfer of proteins from the trans-Golgi network to a pre-lysosomal
compartment (reviewed in (1) and 2), and the recycling of
synaptic vesicles at nerve terminals(3, 4) . The main
constituent of coated vesicles is clathrin, a triskelion composed of
three 190-kDa subunits and three
23-27-kDa light
chains(5, 6) . In addition to clathrin, a number of
proteins referred to as assembly proteins or adaptins copurify with
coated vesicles (reviewed in (7) ). Under physiological
conditions, these proteins induce polymerization of clathrin into
artificial clathrin baskets that resemble coated vesicles (8) .
In addition, several of these proteins have been shown to bind to the
cytoplasmic tails of receptors that are concentrated in clathrin-coated
pits(9) . To date, four different assembly proteins have been
discovered. AP-1 (
)and AP-2 are multimeric subunit complexes (10, 11) of M
270,000; the
former is localized to coated vesicles derived from the trans-Golgi
apparatus and the latter to coated vesicles derived from the plasma
membrane(12, 13) . Two other assembly proteins,
AP
(also referred as AP-3) (14, 15) and
auxilin (16) are monomeric, with M
values
of
90,000. These proteins have been isolated only from neuronal
cells and may be involved in the recycling of synaptic
vesicles(16) .
In vitro, both coated vesicles and
clathrin baskets assembled from purified clathrin and assembly proteins
are uncoated by hsp70 in an ATP dependent
reaction(17, 18, 19) . We recently found
that, in addition to hsp70, a 100-kDa protein cofactor which appears to
be auxilin is required for
uncoating(20, 21, 36) . Auxilin is heat
sensitive, but while purifying auxilin we obtained evidence for a
50-kDa heat-stable protein cofactor which, like auxilin, supported
uncoating by hsp70. (
)While attempting to purify this latter
cofactor, we found that a protein fraction with a M
of less than 50,000 was present in the 0.5 M Tris, pH
7.0, extracts of bovine brain coated vesicles which, rather than
supporting uncoating by hsp70, actually suppressed it. Further
examination of this fraction revealed it to contain a protein with
strong clathrin polymerizing activity. We now report the purification
of this 20-kDa protein and identify it as myelin basic protein (MBP).
Hsp70 was prepared as described previously (17) employing a method which is similar to the method of Schlossman et al.(18) .
Superose-6 column chromatography of a 0.5 M Tris, pH
7.0, extract of coated vesicles yielded the characteristic elution
profile reported previously (7) with a major peak of clathrin
followed by a broad peak containing assembly proteins (Fig. 1A). A small peak prior to clathrin contained
aggregated clathrin and a small peak following the assembly protein
peak contained several uncharacterized proteins that were previously
ignored (Fig. 1B). The 100-kDa protein cofactor
subsequently identified as auxilin, which was found to be essential for
the uncoating of clathrin baskets by hsp70, was purified from the
descending portion of the assembly protein peak. At the end of the
assembly protein peak we observed the presence of a heat-stable protein
cofactor with M
of
55,000-60,000 which
could substitute for auxilin in supporting the uncoating reaction. In
the course of characterizing this protein, we found that some fractions
in the minor peak that followed the assembly protein peak strongly
suppressed the uncoating activity of hsp70, suggesting that they might
be inducing the polymerization of clathrin. Preliminary work suggested
that they were causing clathrin polymerization under the same
conditions as the other assembly proteins which induce the formation of
clathrin baskets.
Figure 1:
A, Superose-6 column chromatography of
total coated vesicle extract. Coated vesicle proteins were extracted
from coated vesicles after treatment with 0.5 M Tris, pH 7.0,
and chromatographed on a Superose-6 column according to published
procedures. All the high molecular weight assembly proteins (fractions
56-70) elute behind clathrin (fractions 43-55) as a broad
peak followed by a small peak containing uncharacterized proteins.
Fractions 75-81 were pooled for purification of the 20-kDa
protein as indicated by the bar. B, SDS-PAGE on
4-20% gradient gel, of Superose-6 column fractions. Numbers
above lanes correspond to the column fractions in A. The
molecular weight standards are on the extreme left lane. T refers to the total protein loaded on the column. In the left gel,
10 µl of each protein was loaded and in the right gel 20 µl.
Note the presence of AP, AP-2, AP-1, and auxilin in
fractions 58-70 and the presence of a band corresponding to M
20,000 in fractions
76-81.
Figure 2:
A, The M
20,000
containing fraction induces clathrin to form sedimentable baskets.
Fractions containing clathrin and M
20,000
in Fig. 1(75-81) were pooled and dialyzed against coated
vesicle isolation buffer (Buffer A). Baskets formed were pelleted by
centrifugation at 40,000 rpm at 4 °C for 2 h in a Ti 45 rotor and
dissociated in 0.5 M Tris, pH 7.0; concentrated by ammonium
sulfate precipitation and gel filtered on a Superose-6 column. B, Superose-6 column fractions of the dissociated baskets were
analyzed by SDS-PAGE. Numbers above the lanes correspond to the column fractions shown above. The molecular
weight standards are in the extreme left lane. T represents
the total protein loaded on the column. Note the complete absence of
known assembly proteins and enrichment of M
20,000 in the assembly protein peak from the dissociated clathrin
baskets.
Each fraction of the redissolved
polymerized clathrin eluted from the Superose-6 column chromatography
was tested for its ability to polymerize fresh clathrin. The fractions
that had the highest activity (fractions 74-81) were then pooled
and fractionated further on a hydroxylapatite column, equilibrated in
0.5 M Tris, pH 7.0. Fig. 3shows the SDS-gel
electrophoretic pattern of both the flow-through fractions and the
proteins that are eluted with 0.5 M sodium phosphate, pH 7.0.
The flow-through fractions contained predominantly a protein of M
20,000 and examination of the assembly activity
of these fractions suggested that they accounted for
80% of the
total clathrin assembly activity. The lower molecular weight bands are
probably digestion products of the 20-kDa protein since their amount
increases during the hydroxylapatite chromatography. Therefore, it
appears that this 20-kDa protein was able to strongly induce clathrin
polymerization. Table 1summarizes the purification of this
protein; approximately 40% of the protein was recovered with a 6-fold
increase in specific activity.
Figure 3:
Purification of M
20,000 by hydroxylapatite column chromatography. Fractions
containing
20-kDa protein from the Superose-6 column in Fig. 2(74-81), were pooled and loaded on a hydroxylapatite
column equilibrated in 0.5 M Tris, pH 7.0. The 20-kDa protein
eluted as a flow-through fraction. The molecular weight standards are
in the extreme left lane. T refers to the total protein loaded
on the column. Flow-through, refers to purified 20-kDa
protein. High salt, refers to the protein bound to the column
and eluted using 0.5 M phosphate, pH
7.0.
Figure 4:
Immunoblot analysis of 20-kDa protein
in assembly protein fractions. Known classes of assembly protein
fractions were probed with a polyclonal antibody against
20-kDa
protein to ensure that it was not derived from them. Lanes 1 and 5, total pool of all known assembly proteins; lanes 2 and 6, AP
and a recently
identified p140 containing fraction(25) ; lanes 3 and 7, purified AP-2; lanes 4 and 8, purified
20-kDa protein. Lanes 1-4, stained by Coomassie Blue and lanes 5-8, the corresponding
immunoblot.
Figure 5:
Sequence analysis of the 20-kDa
protein. The protein was digested with endoproteinase Lys-C and four
peptides P1, P2, P3, and P4 were sequenced after fractionation on high
performance liquid chromatography. The peptide sequences were aligned
with the sequence of bovine myelin basic
protein(25, 26) .
Figure 6: A, assembly of clathrin baskets by MBP. Clathrin (0.5 µM) was combined with varying concentrations of MBP (0-1.2 µM) and dialyzed against coated vesicle isolation buffer. The solutions were spun in a TL-100 rotor at 100,000 rpm for 6 min, at 4 °C and the concentration of clathrin in the supernatant in each case determined by SDS-PAGE. Lane 1, 0.5 µM clathrin; lane 2, 1 µM MBP; lane 3, 3 µM MBP; lanes 4-11 contain 0.5 µM clathrin and 0, 0.17, 0.34, 0.51, 0.68, 0.85, 1.0, 1.2 µM, MBP, respectively. The reduction in clathrin upon the addition of MBP represents the clathrin assembled into baskets. The composition of the baskets formed at 0.68 µM MBP and 0.5 µM clathrin is shown in lane 12. B, plot of the percentage of clathrin assembled as a function of the concentration of MBP. The data in A were replotted to give the percentage clathrin assembled at varying MBP concentrations.
Figure 7:
Electron micrographs of reassembled
clathrin baskets. Clathrin baskets were assembled from clathrin and
MBP. Bar represents 100 nm. A, magnification is
84,750 . B, magnification is 115,260
.
We next tested the ability of the MBP-clathrin baskets to be
uncoated by hsp70. As noted above, we recently determined that a
100-kDa protein cofactor, probably auxilin, is required for the
uncoating of clathrin baskets assembled by AP-2 (20) or
AP(21, 36) . Fig. 8shows that a
similar effect occurs with clathrin baskets assembled by MBP; like the
AP-2-clathrin and the AP
-clathrin baskets, the
MBP-clathrin baskets were uncoated by hsp70 in a cofactor dependent
manner. Furthermore, the time course of uncoating was similar to that
observed with coated vesicles, AP-2-clathrin baskets, and
AP
-clathrin baskets (data not shown). Therefore, the
MBP-clathrin baskets appear to be similar to the other clathrin
substrates in their ability to be uncoated by hsp70.
Figure 8:
Cofactor dependence of the uncoating of
clathrin-MBP baskets by hsp70. Clathrin-MBP baskets (0.2 µM clathrin) were incubated with hsp-70 (0.3 µM) in
presence of 20 µM ATP and varying concentrations of
100-kDa cofactor for 15 min and the uncoating activity was determined.
Note that in the absence of cofactor very little uncoating occurs as
observed previously with clathrin-AP-2 baskets (20) or
clathrin-AP baskets(21) .
Since MBP is a basic protein, we were interested in determining whether other basic proteins could also induce clathrin basket formation. Table 2shows that, of the five proteins tested, three had no effect on the clathrin. The other two proteins, histone I and the polylysines, did affect the clathrin. However, with these proteins we could not detect polymerization of the clathrin into baskets. Rather, even at very low ratios of these proteins to clathrin, the clathrin precipitated in proportion to the amount of basic protein added and electron microscopy of the precipitates revealed no basket-like structures (data not shown).
Like the previously discovered higher molecular weight
clathrin assembly proteins, AP-1, AP-2, AP, and auxilin,
MBP copurifies with coated vesicles and interacts with clathrin in
vitro under physiological conditions inducing it to polymerize
into baskets. Furthermore, like the plasma membrane-associated assembly
protein, AP-2 (and perhaps the trans-Golgi membrane associated assembly
protein, AP-1), MBP catalyzes the polymerization of clathrin with a
defined stoichiometry of 3 MBP molecules per clathrin triskelion.
Therefore, MBP is acting like a clathrin assembly protein.
MBP accounts for a little less than half of the total clathrin-assembly promoting activity in bovine brain coated vesicles isolated by conventional differential gradient centrifugation procedures. However, this does not mean that, in vivo, MBP makes up half of the assembly proteins present in coated vesicles. Since MBP is associated with myelin(27) , the percentage of MBP present in our preparation of clathrin-coated vesicles may reflect the amount of white matter present in the bovine brain starting material from which we isolate the vesicles rather than the amount of MBP directly involved as a clathrin assembly protein.
Since MBP is a strongly basic protein, the question arises as to whether its ability to polymerize clathrin is a nonspecific effect. It has previously been reported that several basic compounds enhance the rate of clathrin polymerization(28) . However, these studies were carried out before assembly proteins were discovered (8) and therefore the clathrin used may have been contaminated with assembly proteins. In addition, turbidity was used to follow polymerization and therefore it was not clear if the clathrin was actually forming baskets or was simply being precipitated by the basic proteins tested. In our experiments on the effect of basic proteins on clathrin polymerization, we found that 3 of the basic proteins we tested did not assemble clathrin even at 5-10-fold higher concentrations than the concentration where MBP is effective. Two proteins, histones and polylysines, did precipitate the clathrin but the precipitate did not exhibit any structure when viewed by electron microscopy nor did hsp70 depolymerize the precipitate. This is in contrast to the effect of MBP where the polymerized baskets appear normal by electron microscopy and are normally uncoated by hsp70 both in regard to the time course of uncoating and the requirement for 100-kDa cofactor. Finally, in contrast to the action of the histone and polylysines, where clathrin precipitated even at low ratios of basic protein to clathrin, with MBP the polymerization of clathrin had a simple stoichiometry of 3 MBP molecules per clathrin. Therefore, we were not able to duplicate the effect of MBP by using other basic proteins, which suggests that the effect of MBP may be specific.
MBP is a major component of the myelin membrane and is present in the oligodendrocytes which form myelin(29) . Although it is clear that changes in MBP cause pathological changes in the function of myelin, the exact function of MBP in the formation and properties of myelin are not known(30) . Recently it has been reported that, like other proteins, e.g. STOP 220, MBP stabilizes microtubules derived from bovine brain (31) which may be related to the observation that MBP is associated with microtubules in oligodendrocyte cell cultures(32, 33) . It is therefore possible that MBP forms a bridge between clathrin-coated vesicles and microtubles. However, as yet, there is no direct evidence that MBP is associated with clathrin in vivo.
Clathrin-coated vesicles have been observed in specialized regions of the axolemma, at the site of adhesion between the axon and its myelinating process; it has been suggested that these coated vesicles may be involved in the insertion or deletion of the junctional membrane (34) . In addition, a large amount of endocytosis occurs in oligodendrocytes because of their uptake of iron by the transferrin pathway(35) . It is possible that MBP may be involved in the formation of clathrin-coated vesicles in one or both of these processes. In any case, whether or not MBP interacts with clathrin in vivo, its easy availability and small size should make it very useful in studying the mechanism of the assembly of clathrin into clathrin baskets and the disassembly of these baskets by hsp70.