(Received for publication, September 20, 1994)
From the
We have isolated an inositol hexakisphosphate binding protein
from rat brain by affinity elution chromatography from Mono S cation
exchange resin using 0.1 mM inositol hexakisphosphate
(InsP). The amino acid sequences of six tryptic peptides
from the protein were identical to the sequences predicted from the
cDNA encoding a previously isolated protein designated as AP-3 or
AP180. This protein is localized in nerve endings and promotes assembly
of clathrin into coated vesicles. The isolated protein-bound InsP
with a dissociation constant of 1.2 µM and a
stoichiometry of 0.9 mol of InsP
bound/mol of AP-3.
Recombinant AP-3 expressed in Escherichia coli also bound
InsP
with a similar affinity. InsP
inhibited
clathrin cage assembly mediated by AP-3, in an in vitro assay,
but had little effect AP-3 binding to preformed cages. We speculate
that InsP
and perhaps highly phosphorylated inositol lipids
may play a role in coated vesicle formation.
Inositol polyphosphates containing five- and six-phosphate
groups (inositol pentakisphosphate and inositol hexakisphosphate) are
ubiquitous in eukaryotic cells, yet their functions remain unknown. In
an attempt to define proteins that interact with these moieties
Theibert et al.(1) and Chadwick et al.(2) isolated inositol polyphosphate binding proteins from rat
and bovine brain, respectively. Both groups showed that the protein
isolated was identical to clathrin assembly protein 2
(AP-2)()(3, 4) . This conclusion was based
on the finding that the amino acid sequence of the inositol
polyphosphate binding proteins was identical to that of the
subunit of AP-2. AP-2 is thought to function in the formation of coated
vesicles at the plasma membrane by recruiting receptors and clathrin
into growing coated pits. Beck and Keen (5) have shown that
inositol polyphosphates inhibit AP-2-mediated clathrin assembly in an in vitro assay.
We recently devised a scheme for the
isolation of inositol polyphosphate 4-phosphatase, an enzyme of the
inositol phosphate signaling pathway, that utilized an affinity elution
protocol(6) . In the course of this work, we noted a major
contaminant protein of approximately 160 kDa that was eluted from a
cation-exchange resin (Mono S) by low concentrations of inositol
hexakisphosphate (InsP). We now report that this protein is
identical to a protein that was previously isolated as clathrin
assembly protein 3 (AP-3), another protein involved in assembly of
clathrin-coated vesicles(7, 8, 9) . We also
report that both native and recombinant AP-3 bind inositol
polyphosphates with high affinity and that InsP
inhibits
AP-3-mediated clathrin assembly.
Clathrin (200 µg/ml) and AP-3
(50 µg/ml) were incubated with InsP for 10 min on ice.
Assembly was induced by addition of 10
concentrated buffer C to
give a final concentration of 1
. The final composition of buffer
C was 0.1 mM MES, pH 6.5, 0.5 mM MgCl
, 1
mM EGTA, and 0.02% NaN
. After 1 h on ice, assembly
was analyzed by ultracentrifugation for 20 min at 100,000
g in a Beckman TL-100 centrifuge followed by SDS-PAGE of supernatant
and pellet fractions as described previously(12) . The
estimation of cage content was made by densitometry of
Coomassie-stained gels using a molecular dynamics laser
densitometer(13) .
A purification scheme for the rat brain
inositol-polyphosphate 4-phosphatase was recently described that
employed affinity elution from a Mono S cation exchange column with
InsP and inositol hexasulfate(6) . Prior to the
elution of the 4-phosphatase, a major contaminating protein of 160 kDa
was eluted with 0.1 mM InsP
. This protein was
nearly homogenous as determined by SDS-PAGE as shown in Fig. 1.
We carried out tryptic digestion of the protein in order to determine
whether it represented a previously identified protein. We separated
the tryptic peptides by HPLC as described under ``Experimental
Procedures'' and amino acid sequence was obtained from six
peptides as shown in Table 1. A search of the GenBank data base
using tblastn (14) indicated that the peptide sequences
obtained were identical to the predicted amino acid sequence of a
protein that has been previously described by several names including
clathrin assembly protein 3/phosphoprotein F1-20/AP
180(10, 15, 16) . The only exception to the
sequence identity was at position 854, where the cDNA sequence predicts
an arginine residue. There was no arginine signal at this cycle in the
sequence, rather we saw a peak that co-migrated with a standard of N-monomethylarginine(17) . Thus it is likely that this
residue is methylated in the rat brain protein. The significance of
this putative modification is unknown.
Figure 1:
SDS-PAGE analysis
of the AP-3 protein eluted from Mono S with InsP. Protein
eluted with 0.1 mM InsP
was concentrated and run
on a 6% polyacrylamide gel and stained with Coomassie Blue. Left
lane, molecular weight markers; right lane,
AP-3.
We will refer to the protein as AP-3 in this report. It is a monomeric protein of 897 amino acids that is found exclusively in brain(10, 16, 18) . It has also been isolated from synaptosomes where it was designated as Fl-20(16) . In vivo and in vitro studies have indicated that AP-3 is a phosphoprotein (18) that is abundant in both soluble and vesicle fractions. AP-3 migrates anomalously upon SDS-PAGE in that its apparent molecular mass ranges from 155 to 185 kDa depending on the conditions, while its molecular mass predicted from the cDNA is 91.4 kDa. This retarded gel mobility may result from the fact that AP-3 is a phosphoprotein and has a highly acidic domain of 50 kDa.
AP-3
induces the assembly of clathrin cages in vitro and is about 4
times more effective than AP-1 or AP-2 in the in vitro assembly assays(12) . There is slight homology (10% amino
acid identified over 100 amino acids) between the chain of AP-2,
which contains the inositol polyphosphate binding site and AP-3 (4.5
standard deviations using the NBRF/PIR relate program)(19) .
Since AP-2 and AP-3 are both involved in clathrin assembly and both
were serendipitously isolated as inositol polyphosphate binding
proteins, we investigated whether AP-3 binds inositol polyphosphates
using an assay similar to that used in the study of AP-2(1) .
We measured [H]InsP
binding in the
presence of increasing concentrations of unlabeled inositol
polyphosphates as shown in Fig. 2. InsP
(IC
= 1 µM) was the most effective inositol
polyphosphate in displacing [
H]InsP
followed by Ins 1,3,4,5,6-P
(IC
=
3 µM), Ins 1,3,4,5-P
(IC
=
10 µM), and Ins 1,4,5-P
, respectively. This
order of specificity is the same as that found in the study of
AP-2(1) . Scatchard analysis of the
[
H]InsP
displacement curve yielded a K
of 1.2 µM and an apparent binding
capacity of 0.9 mol of InsP
bound per mol of AP-3 as shown
in the inset of Fig. 2. The K
of
InsP
for AP-2 has been reported as 12 nM(1) and 0.12 µM(2) , which is
significantly lower than the K
for AP-3. The
relationship between these binding parameters and intracellular events
is difficult to fathom since the intracellular concentration of
InsP
has been estimated to be very high at 50 µM in HL60 cells (20) and 0.7 mM in Dictyostelium discoideum(21) .
Figure 2:
The
displacement of [H]InsP
from rat
brain AP-3 by increasing concentrations of various inositol phosphates.
The amount of [
H]InsP
(23,000 cpm
added) bound to rat brain AP-3 (1.6 µg) was determined using the
polyethylene glycol precipitation method described under
``Experimental Procedures'' in the presence of unlabeled Ins
1,4,5-P
(
), Ins 1,3,4,5-P
(
), Ins
1,3,4,5,6-P
(
), and P
(
) Values
shown are the average of duplicates. Scatchard analysis of InsP
binding is also shown (inset, p = protein
concentration (M)).
We also measured the
binding of recombinant AP-3 to InsP to exclude the
possibility that a contaminating protein accounted for the binding
shown in Fig. 2. The recombinant protein bound InsP
with an affinity similar to that of purified rat brain AP-3 as
shown in Fig. 3. The amount of InsP
bound to the
recombinant protein is only one-fourth of that bound to the isolated
rat brain protein. This may result from proteolysis of the protein as
isolated from E. coli, and during subsequent storage since we
found that recombinant AP-3 was unstable with respect to InsP
binding upon repeated freezing and thawing and storage.
Figure 3:
The displacement of
[H] from recombinant AP-3 by increasing
concentration of unlabeled InsP
. The amount of
[
H]InsP
bound (23,000 cpm added) to
recombinant AP-3 (2.8 µg) was measured by polyethylene glycol
precipitation in the presence of the indicated concentrations of
unlabeled InsP
.
We
also investigated whether InsP had an affect on clathrin
cage assembly mediated by AP-3 as shown in Fig. 4a. In
this experiment, cage assembly was inhibited by InsP
with
50% inhibition at 0.1 mM. This concentration is about 100
times the concentration of InsP
required for binding to
AP-3, but the assays were not performed under the same conditions. The
assembly assay was done at pH 6.5, and the binding experiments were
done at pH 7.5. We could not do binding assays at the lower pH due to
binding of InsP
to carrier
globulin under this
condition. It was also not possible to perform the assembly experiments
at pH 7.5 because assembly is very inefficient at elevated pH. The
study on the effect of InsP
on assembly of clathrin cages
promoted by AP-2 also required these high concentrations to demonstrate
inhibition(5) . In fact the curve shown in Fig. 4a obtained using AP-3 is almost identical to that found using
AP-2(5) . Moreover, Keen and co-workers did not use isolated
AP-2 in their experiment, and it is therefore possible that they
observed an effect of InsP
on assembly mediated in part by
contaminating AP-3 in their preparation. Recall that AP-3 is more
potent in assembly assays than AP-2(12) . We also determined
whether InsP
had an effect on clathrin cages preformed in
the presence of calcium ions without any AP-3. Upon warming of the
cages to 37 °C, there is rapid exchange between free and cage-bound
clathrin. There was a modest effect of InsP
in this
experiment as shown in Fig. 4b. This suggests that the
effect of InsP
on cage assembly depends on AP-3 and not
upon an interaction directly with clathrin. We also found that
InsP
does not bind to clathrin-Sepharose, further
supporting this idea. The stoichiometry of InsP
binding to
AP-3 is 1:1, while we presume that there are two sites for interaction
of AP-3 with clathrin. This notion is further supported by the
experiment shown in Fig. 4c where the effect of
InsP
on the binding of AP-3 to preformed clathrin cages was
measured. In this case AP-3 bound to the cages despite InsP
implying that it is able to bind by a site distinct from that
occupied by InsP
.
Figure 4:
The effect of InsP on clathrin
cages. a, clathrin and AP-3 were incubated with or without
InsP
at the indicated concentrations as described under
``Experimental Procedures.'' In this experiment 100% is the
concentration of clathrin cages in the absence of InsP
. b, the stability of clathrin cages at 37 °C was determined
in the presence and absence of InsP
as described under
``Experimental Procedures.'' c, binding of AP-3 to
preformed clathrin cages was carried out in the presence or absence of
InsP
as described under ``Experimental
Procedures.'' 100% binding corresponds to the amount of AP-3 bound
in the absence of InsP
. In this experiment there was 0.6
mol of AP-3 bound/clathrin triskelion, which is about half of the
saturating value.
One difficulty in developing a model
for a role of InsP in coated vesicle assembly is that the
concentration of InsP
in cells is at least an order of
magnitude higher than would be required to fully saturate both AP-2 and
AP-3. Furthermore, InsP
levels do not appear to vary
rapidly in cells(20, 21) . Despite these
considerations it is tempting to speculate that inositol phosphates
participate in this process given that both assembly proteins bind
inositol polyphosphates with high affinity. AP-3 is present in neuronal
cells in concentrations comparable with those of clathrin, and most of
the AP-3 is cytosolic. In differentiated PC12 cells, AP-3 levels exceed
those of clathrin by 5-10 times(22) . Perhaps AP-3 is
sequestered by its association with cellular InsP
, thereby
preventing coated vesicle assembly. According to this idea, something
would be required to displace InsP
in order to initiate
cage assembly. One possibility is the formation of highly
phosphorylated 3-phosphate containing phosphatidylinositols in
membranes destined for coated vesicle formation ex.
phosphatidylinositol (3, 4, 5) tetrakisphosphate. If AP-3 has a
higher affinity for the lipid than for InsP
then AP-3 could
be recruited to the membranes and initiate the process of
clathrin-coated vesicle assembly.
It was recently shown that the yeast gene VPS34 required for vesicular transport to the yeast vacuole encodes for a protein homologous to mammalian phosphatidylinositol 3-kinase(23) . This enzyme is required for formation of the highly phosphorylated phosphatidylinositols. Additionally the phosphatidylinositol 3-kinase binding sites on the PDGF receptor have been shown to be required for PDGF-dependent receptor endocytosis(24) . Further experiments will be required to determine the possible role of soluble and lipid inositol phosphates in the regulation of vesicular transport. In particular it would be interesting to determine if the levels of the highly phosphorylated phosphatidylinositols vary during coated vesicle assembly in synaptosomes.