From the Department of Internal Medicine, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
§ Department of Pharmacology, University of Wisconsin
Medical School, Madison, Wisconsin 53706
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ABSTRACT |
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Adaptors appear to control clathrin-coat
assembly by determining the site of lattice polymerization but the
nucleating events that target soluble adaptors to an appropriate
membrane are poorly understood. Using an in vitro model
system that allows AP-2-containing clathrin coats to assemble on
lysosomes, we show that adaptor recruitment and coat initiation
requires phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2) synthesis. PtdIns(4,5)P2 is
generated on lysosomes by the sequential action of a
lysosome-associated type II phosphatidylinositol 4-kinase and a soluble
type I phosphatidylinositol 4-phosphate 5-kinase. Phosphatidic acid,
which potently stimulates type I phosphatidylinositol 4-phosphate
5-kinase activity, is generated on the bilayer by a phospholipase
D1-like enzyme located on the lysosomal surface. Quenching phosphatidic
acid function with primary alcohols prevents the synthesis of
PtdIns(4,5)P2 and blocks coat assembly. Generating
phosphatidic acid directly on lysosomes with exogenous bacterial
phospholipase D in the absence of ATP still drives adaptor recruitment
and limited coat assembly, indicating that PtdIns(4,5)P2
functions, at least in part, to activate the
PtdIns(4,5)P2-dependent phospholipase D1. These
results provide the first direct evidence for the involvement of
anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators.
An area of membrane that will give rise to a clathrin-coated bud
is demarcated by the placement of adaptors at that site. This
necessitates that adaptor recruitment onto membranes be controlled with
good precision. The first glimpse of the real complexity of the adaptor
recruitment process came from studies using the fungal metabolite
brefeldin A (1, 2). When added to cells, this compound causes an
extremely rapid disappearance of AP-1 adaptors and clathrin, from the
trans-Golgi network
(TGN)1 (1, 2), the site where
this heterotetrameric adaptor complex is usually massed to initiate the
formation of clathrin-coated buds (3). The effect of brefeldin A led to
the demonstration that the binding of AP-1 to the TGN is regulated by
ADP-ribosylation factor (ARF) in a cycle of GTP binding and hydrolysis
(4, 5).
We proposed a model in which an AP-1-specific, ARF-activated docking
site initiates clathrin-coat assembly at the TGN (5). Bound to an
ARF·GTP-activated docking site, AP-1 would begin to recruit clathrin
(6, 7). Sustained recruitment of both AP-1 and clathrin would result in
the formation of an extensive polyhedral lattice. The association
between AP-1 and the presumptive docking molecule is terminated on
hydrolysis of ARF-GTP to GDP (8). We envisioned that the laterally
expanding lattice would be tethered to the underlying membrane by
AP-1-docking protein associations, primarily occurring at the perimeter
of the growing coat. The density of AP-1 within the developing bud
would be sufficiently high so that even low-affinity interactions
between protein-sorting signals and the µ1 subunit of the adaptor
heterotetramer (9, 10) would result in preferential retention of select
transmembrane proteins within the structure. If a sorting signal on a
protein to be included within the clathrin coat disengaged from one
µ1 subunit, it would immediately encounter another AP-1 molecule so
the mobility of sorted proteins would be severely restricted over the
period in which the bud grows. Brefeldin A subverts coat assembly at
the TGN by interfering with earliest known step of the process,
catalyzed nucleotide exchange (11, 12), blocking ARF·GTP delivery
onto the membrane and all downstream events.
Unlike the clathrin-coated structures on the TGN, clathrin-mediated
endocytosis is not perturbed by brefeldin A (1, 2). This clearly
establishes that the recruitment of AP-1 and AP-2 adaptors is regulated
differently but, currently, very little is known about the nucleating
events that precede AP-2 translocation onto membranes (9, 13).
Overexpression of several plasma-membrane receptors normally
internalized in clathrin-coated vesicles does not alter the
steady-state distribution of AP-2 (14-16), arguing that unregulated
association between protein-sorting signals and AP-2 adaptors is
unlikely to begin the clathrin-coat assembly process. Synaptotagmin, a
calcium- and phospholipid-binding protein first identified in synaptic
vesicles, has been suggested to be a high-affinity AP-2-docking
molecule (17). The mild phenotype of synaptotagmin I-null animals
argues against this idea, but there are at least 7 other synaptotagmin
isoforms, each able to bind AP-2 (18). If synaptotagmin is an
AP-2-docking protein, the association with AP-2 must be tightly
regulated because synaptotagmin I is predominantly a synaptic vesicle
protein and synaptic vesicles are not clathrin coated until they are
rapidly retrieved following their fusion with the presynaptic plasma membrane.
Stage-specific assays for endocytosis show that early, but as yet
uncharacterized events in clathrin-coat assembly at the cell surface
require ATP hydrolysis (19). Polymerization of AP-2 and clathrin into
coats on lysed synaptosomes also requires ATP (20). Recently, we showed
that AP-2-containing clathrin coats can also assemble and invaginate on
lysosomes in a strictly ATP-dependent fashion (21).
Three-dimensional EM images reveal that the polyhedral lattice formed
on the lysosome surface is identical to clathrin coats formed at the
cell surface. Using this model system, we have now carefully dissected
the role of ATP in initiating clathrin-coat assembly. Our results show
that phosphoinositides, in particular phosphatidylinositol
4,5-bisphosphate (PtdIns(4,5)P2), are critical regulators
of AP-2 adaptor binding. Direct evidence for a positive feedback loop
between type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and
phospholipase D (PLD) is presented. Both of the lipids generated by
this regulatory loop, PtdIns(4,5)P2 and phosphatidic acid
(PtdOH), appear to play important roles in initiating clathrin-coat
assembly on lysosomes.
Materials--
[ Antibodies--
mAb 100/2, directed against the Subcellular Fractionation and Protein Purification--
Rat
brain cytosol and rat liver Golgi membranes and lysosomes were prepared
as described previously (5, 21). Lysosomes were resuspended in 20 mM Tris-HCl, pH 7.4, 250 mM sucrose with 25 µg/ml each of antipain, aprotinin, chymostatin, leupeptin, and
pepstatin A (protease inhibitors). A plasmid bearing the SH3 domain of
amphiphysin I fused to glutathione S-transferase (GST) was
provided by H. McMahon (29). Bovine ADP-ribosylation factor 1 (ARF1),
with amino acids 3-7 replaced with the corresponding residues from
yeast ARF2, was from S. Kornfeld (8). Proteins were expressed in
Escherichia coli and purified by standard procedures. For
depletion of dynamin, 1-ml aliquots of cytosol (10 mg/ml) were mixed
with 150 µg of either GST or GST-amphSH3 overnight at 4 °C.
GSH-Sepharose was then added to collect the protein complexes and then
removed by brief centrifugation. The supernatants were stored in small
aliquots at
For the separation of type I and type II PIP5Ks (25, 30, 31), 25 ml of
rat brain cytosol was dialyzed into 5 mM sodium phosphate,
pH 7.5, 0.25 M NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM 2-mercaptoethanol, 10% glycerol,
and 0.1 mM phenylmethylsulfonyl fluoride (buffer A) and
then loaded at 1.5 ml/min onto a phosphocellulose column (1.6 × 10 cm) pre-equilibrated in buffer A. After washing with 200 ml of
buffer A, bound protein was eluted with a 250-ml linear gradient of
0.25-1.25 M NaCl in buffer A, collecting 4-ml fractions. Fractions were analyzed on immunoblots after concentration by methanol/chloroform precipitation or assayed for PIP5K activity after
dialysis into buffer A lacking sodium chloride. For some experiments,
the PIP5KI and PIP5KII pools were concentrated with a Centricon 10. When assayed for PtdInsP production with lysosomes, the PIP5KI and
PIP5KII pools were exchanged into 25 mM Hepes-KOH, pH 7.2, 125 mM potassium acetate, 5 mM magnesium
acetate, 5 mM EGTA, 1 mM dithiothreitol over
NAP-5 columns.
Membrane Binding Assays--
Recruitment onto lysosomes was
performed as outlined previously (21). Briefly, reactions were
performed in 25 mM Hepes-KOH, pH 7.2, 125 mM
potassium acetate, 5 mM magnesium acetate, 5 mM EGTA, 1 mM dithiothreitol in a volume of 400 µl. All
assays contained 25 µg/ml of each of the protease inhibitors
indicated above. Lysosomes were added to final concentrations of 30-50
µg/ml and cytosol to 2-2.5 mg/ml as indicated in the legends. ATP,
an ATP regeneration system (1 mM ATP, 5 mM
creatine phosphate, 10 units/ml creatine kinase), apyrase, A-3,
neomycin, various alcohols, and PLD were added and mixed on ice. After
20 min at 37 °C, reactions were stopped by chilling in an ice-water
bath. Variations are noted in the figure legends. After centrifugation,
membrane-containing pellets and aliquots of the supernatants were
prepared for immunoblotting. Details of the conditions used for
SDS-PAGE and immunoblotting have been published elsewhere (5, 21). For
thin-section EM analysis, glutaraldehyde was added to the chilled
reactions to give a final concentration of 2% and, after 15 min on
ice, the membranes were collected by centrifugation and processed as
detailed elsewhere (21). The immunofluorescence-based morphological
binding assay, using digitonin-permeabilized NRK cells, was carried out on glass coverslips exactly as described (21). Cells were fixed with
3.7% formaldehyde in phosphate-buffered saline for 20 min and then
processed for indirect fluorescence microscopy.
Measurement of Phosphoinositide Kinase and PLD
Activity--
Synthesis of phosphoinositides on lysosomal membranes
was assayed in 25 mM Hepes-KOH, pH 7.2, 125 mM
potassium acetate, 5 mM magnesium acetate, 5 mM
EGTA, 1 mM dithiothreitol in a final volume of 50 µl.
Membranes were added to a final concentration of 0.5 mg/ml and cytosol,
when present, to 5 mg/ml. Incubations were initiated by the addition of
[
PLD activity was assayed in 50 mM Hepes-KOH, pH 7.5, 250 mM sucrose, 80 mM potassium chloride, 4.5 mM magnesium chloride, 3 mM calcium chloride, 3 mM EGTA, 1 mM dithiothreitol, protease inhibitors, and 0.5% 1-butanol in a final volume of 60 µl (32). Organelles were added to a final concentration of 160 µg/ml and activators were added as follows: a final concentration of 0.85 µM ARF1 together with 100 µM GTP Lipid Analysis--
Phosphoinositides were resolved on silica
gel HPTLC plates that had been previously immersed in 1% potassium
oxalate, 2 mM EDTA in 50% ethanol. Lipids were spotted
onto the plates after activation at 105 °C for several hours. The
developing solvent consisted of chloroform:acetone:methanol:acetic
acid:water (160:60:52:48:28). PtdOH and phosphatidylbutanol (PtdBut)
were resolved from PtdCho on heat-activated Silica Gel LK6 plates in a
solvent system of chloroform:methanol:acetic acid (13:5:1). After
chromatography, lipid standards were visualized with iodine vapor and
marked. Radiolabeled lipids were visualized by autoradiography. Plates containing 14C-labeled lipids were sprayed with Enhance
before exposure to film. Signals were quantitated by scintillation
counting after scraping the relevant portions of the plate into vials.
For the anion-exchange HPLC analysis, the relevant lipid spots were
scraped off the plates and deacylated directly on the silica with
methylamine reagent after addition of authentic
[3H]PtdIns(4)P or
[3H]PtdIns(4,5)P2 (NEN Life Science
Products). The water-soluble products were applied to a Partisil 10 SAX
column and eluted with a gradient of 0-1 M ammonium
phosphate, pH 3.8, as described elsewhere (27).
The Kinase Inhibitor A-3 Prevents AP-2 Recruitment onto
Lysosomes--
The recruitment of AP-2 onto purified lysosomes and
subsequent clathrin lattice assembly is absolutely dependent on
hydrolyzable ATP (21) (Fig. 1). This
makes it unlikely that coat assembly is initiated simply by the direct
association of AP-2 adaptors with sorting signals located on the
cytosolic domain of lysosomal glycoproteins or plasma-membrane proteins
which have made their way to the limiting membrane of the lysosome.
When added to gel-filtered cytosol, ATP supports coat assembly with an
EC50 of approximately 100 µM (Fig.
1A), suggesting that a phosphorylation event might be
involved rather than constant ATP hydrolysis to actively drive coat
assembly. Indeed, a broad specificity kinase inhibitor, the naphthalenesulfonamide A-3 (33), blocks clathrin assembly on lysosomes
(Fig. 1B). Compared with the recruitment of AP-2 and clathrin seen when purified lysosomes are incubated at 37 °C with brain cytosol and 500 µM ATP (lane e),
addition of equimolar (lane i) or higher (lane k)
concentrations of A-3 fully inhibits the translocation of the Correlation between PtdIns(4,5)P2 Formation and
Clathrin-coat Assembly--
Three lines of evidence suggest that
inositide phosphorylation might be important for the initiation of
clathrin-coat assembly on lysosomes. First, poorly hydrolyzable
analogues of ATP, AMP-PNF, and ATP Coat Assembly on Lysosomes Begins with AP-2 Adaptor
Recruitment--
The pleckstrin homology domain is a modular protein
fold that appears to regulate the translocation of several soluble
proteins onto membranes by binding to various phosphoinositides (39, 40). Because PtdIns(4,5)P2 is generated on the lysosome in
our assays, and because dynamin contains a pleckstrin homology
domain that specifically binds to PtdIns(4,5)P2 (41), it
was possible that we had reconstituted coat assembly in reverse.
Dynamin translocation onto PtdIns(4,5)P2-containing
lysosomes could trigger the recruitment of amphiphysin (29, 42) which,
in turn, could then recruit AP-2, synaptojanin and clathrin (43-45).
In fact, this reverse reaction is known to occur in vitro
(46). If dynamin recruitment does initiate clathrin-coat assembly on
lysosomes, then depleting this protein from brain cytosol should
abrogate coat formation in vitro. Dynamin was selectively
removed from rat brain cytosol using a GST-amphSH3 domain fusion
protein (29). For comparison, mock-depleted cytosol, treated with GST,
and AP-2-depleted cytosol (5), were also used. Examination of the
specificity (Fig. 2A) and
extent of depletion (Fig. 2B) confirms that dynamin and AP-2 removal is virtually complete but that the resulting depleted cytosols
still contain normal levels of several other major polypeptides known
to participate in clathrin coat formation.
Eliminating dynamin from cytosol does not alter clathrin-coat assembly
on lysosomes detectably. AP-2 recruitment is evident both from the loss
of the adaptor in the soluble fraction (Fig. 2B, lane e
compared with lane d) and from the simultaneous appearance of the adaptor complex in the lysosome pellet (Fig. 2C, lane
e compared with lane d). Clathrin behaves similarly.
This is identical to the results obtained with the mock-depleted
cytosol (lanes b and c). Removing AP-2 from the
donor cytosol, however, has a dramatic effect on coat formation. Very
little AP-2 and clathrin are found on the pelleted lysosomes (Fig.
2C, lane g compared with lanes c and
e) and no change in the amount of clathrin in the soluble
fraction is evident (Fig. 2B, lane g compared with lane f). This verifies that AP-2 binding is necessary for
subsequent clathrin recruitment. Although the pellets from incubations
with dynamin-containing cytosol do contain dynamin (Fig. 2C,
lanes c and g), equivalent amounts of dynamin are also
recovered in the pellets from incubations lacking lysosomes
(lanes b and f). This simply reflects the
propensity of dynamin to form extensive homo-oligomers, but makes it
difficult to discern whether dynamin is also being actively recruited
onto the clathrin-coated buds that form on the lysosomes.
These experiments establish that in our system clathrin-coat assembly
follows what is considered the physiological sequence, with adaptor
recruitment beginning the assembly process. The generation of
PtdIns(4,5)P2 may facilitate AP-2 binding and dynamin is
not required for the early stages of lattice assembly. This is in agreement with our earlier results showing that GTP Characterization of the Inositide Kinases Involved in
PtdIns(4,5)P2 Formation on Lysosomes--
PI4Ks are
divided into two distinct subfamilies, designated type II and type III
(23). Type II enzymes are predominantly membrane-associated, whereas
type III activity is mainly found in soluble extracts, although some
type III activity is also associated with the particulate fraction (38,
47). The type III enzymes are also completely inhibited by micromolar
concentrations of wortmannin (47-49) and, on this basis, can be
distinguished from the type II PI4K, which is wortmannin insensitive.
The generation of PtdIns(4)P on lysosomes is unaffected by up to 20 µM wortmannin (data not shown), suggesting that the
lysosomal PI4K is most likely a type II enzyme. This conclusion is
strengthened by the ability of a neutralizing anti-type II antibody,
mAb 4C5G (23), to inhibit PtdIns(4)P generation in a
dose-dependent fashion if the lysosomes are preincubated
with the antibody prior to the addition ATP (data not shown), and is in
accord with a study examining the subcellular distribution of PI4K
isoforms (38). It is also important to note that the insensitivity of
PtdIns phosphorylation to micromolar concentrations of wortmannin again
rules out that the labeled lipid generated on lysosomes is the product
of PI3K activity.
Two peaks of PIP5K activity are resolved after fractionating rat
brain cytosol on phosphocellulose (Fig.
3A). The first and major peak,
designated type I PIP5K (25, 30, 31), elutes with approximately 0.6 M NaCl and is composed of at least three distinct enzymes,
PIP5K types I
The minor peak of type II PIP5K activity elutes from phosphocellulose
with about 1.1 M NaCl (Fig. 3A). Again, there
are two main isoforms known, the ~53-kDa type II
Because type I PIP5Ks phosphorylate not only PtdIns(4)P but
3-phosphate-containing phosphatidylinositols as well (27, 52), we again
checked that the lipid produced by the type I pool is, in fact,
PtdIns(4,5)P2. HPLC analysis of the deacylated product also shows precise co-elution with the
[3H]GroPIns(4,5)P2 standard (data not shown).
PtdIns(4,5)P2 synthesis on lysosomes catalyzed by the
partially purified type I-PIP5K pool is dose dependent (Fig. 3E,
lanes c and e) and, importantly, is completely
abolished by including A-3 in the assay (lanes d and
f), just as is seen with whole cytosol (lanes a
and b). Taken together, our results show that a type II PI4K
located on the lysosome membrane phosphorylates PtdIns to produce
PtdIns(4)P, which, in turn, is phosphorylated by a soluble,
A-3-sensitive type I PIP5K to generate PtdIns(4,5)P2.
PtdOH Generation Precedes AP-2 Adaptor Recruitment--
The
in vitro activity of all known type I PIP5Ks is strongly
stimulated by PtdOH (25, 28). Two main pathways for the generation of
PtdOH are known. One occurs by the phosphorylation of diacylglycerol by
diacylglycerol kinase while a second is via the hydrolysis of the polar
head group of PtdCho catalyzed by PLD. Only trace amounts of PtdOH are
generated by phosphorylation in our assays (Fig. 1C) so to
investigate whether PLD activity is required to generate PtdOH
necessary to drive the synthesis of PtdIns(4,5)P2 by type I
PIP5K, we checked the effect of 1-butanol on the synthesis of this
lipid. PLD catalyzes a distinctive transphosphatidylation reaction
producing phosphatidylalcohols in the presence of primary alcohols.
Since phosphatidylalcohols are unable to substitute for the
biologically active PtdOH, primary alcohols inhibit
PLD-dependent reactions. When added at 1.5%, 1-butanol has
a dramatic effect on PtdIns(4,5)P2 formation (Fig.
4A, lane e compared with
lane d) while tertiary butanol is inert (lane f).
1-Butanol similarly prevents the formation of PtdIns(4,5)P2
catalyzed by the semipurified type I PIP5K
pool.2 Significantly, the
effect of 1-butanol on PtdIns(4)P synthesis is considerably weaker
(lanes a and d compared with lanes b
and e).
To determine whether PtdOH generated by PLD is important for
clathrin-coat assembly, we tested the effect of adding alcohols to our
recruitment assays. Adding 1-butanol to the standard assay inhibits the
recruitment of AP-2 onto lysosomes in a dose-dependent manner (Fig. 4B). At 1.5%, inhibition is near complete
while at the same concentration t-butyl alcohol has little
effect (Fig. 4B, lane n compared with lane d).
Analogous results are obtained with 1-propanol, which also abrogates
coat assembly, and 2-propanol, which has no effect.2 Taken
together, these experiments confirm the existence of a positive
feedback regulatory loop between PIP5KI and PLD (53) and indicate that,
in our system, PIP5KI activity is largely dependent on PtdOH. The
generation of PtdOH on the lysosome is a prerequisite for the
initiation of coat assembly, which, based on our results, is required
for the synthesis of PtdIns(4,5)P2.
PLD1-like Activity Is Associated with Purified Rat Liver
Lysosomes--
Two forms of PtdIns(4,5)P2-regulated
PtdCho-specific PLD, termed PLD1 and PLD2, have been identified in
mammals (54, 55). To verify that the PLD activity observed in our assay
is indeed lysosome-associated, we used an exogenous substrate assay
performed in the presence of 1-butanol. This enables us to follow the
stable product of transphosphatidylation, PtdBut. A low level of PtdCho cleavage occurs upon mixing purified lysosomes with the exogenous reporter micelles (Fig. 5A,
column b) indicating that PLD activity is present on lysosomes.
Activators of PLD1, ARF1, and PKC
For comparison then, we also analyzed purified Golgi membranes, which
are known to contain ARF-stimulated PLD1 activity (57). The catalytic
activity associated with Golgi responds to PLD1 activators very
similarly to that associated with lysosomes (Fig. 5A, columns
f-i). The relative purity of the two organelle preparations is
shown on immunoblots probed with antibodies against PtdOH Alone Drives Limited Clathrin Coat Formation--
Since
PtdOH is a potent biological mediator itself, the question arises
whether the critical agent generated by the positive feedback loop
between PLD and PIP5KI that is necessary to initiate clathrin-coat
assembly is PtdIns(4,5)P2, PtdOH, or both of these lipids.
If PtdIns(4,5)P2 is required only to stimulate
PLD1-dependent formation of PtdOH, then it should be
possible to promote nucleotide-independent coat assembly on lysosomes
in vitro using exogenous PLD. When 0.5 unit/ml S. chromofuscus PLD, which is not dependent on
PtdIns(4,5)P2 for
activity,3 is added to a
mixture of gel-filtered brain cytosol and lysosomes in the absence of
ATP, AP-2 does translocate onto the lysosome surface (Fig.
6, lane g). No recruitment is
seen in the absence of PLD (lane e). Adaptor binding is
about 3-fold less efficient with PLD than the recruitment seen in the
presence of ATP (lane g compared with lane c),
but is nevertheless accompanied by clathrin recruitment. Interestingly,
adding up to 8 units/ml of the bacterial PLD does not increase the
amount of coat components associated with the lysosomes in the absence
of ATP.3 Incubations containing PLD together with
apyrase or A-3 rule out the possibility that the bacterial PLD
facilitates coat recruitment by stimulating PIP5KI to generate
PtdIns(4,5)P2 using trace ATP remaining in the gel-filtered
cytosol. Neither AP-2 nor clathrin recruitment is markedly altered by
including either 10 units/ml apyrase or 100 µM A-3 (Fig.
6, lanes i and k compared with lane g).
Thin-section EM analysis verifies that the bacterial PLD indeed
promotes the assembly of identifiable clathrin coats (Fig. 7). Our purified lysosome preparations
consist predominantly of dense-core organelles and characteristic
bristle-like areas of assembled clathrin can occasionally be seen on
the lysosomes after incubation with gel-filtered cytosol and bacterial
PLD (panels a and b). While the coat appears
indistinguishable from the clathrin-coated buds that readily form on
lysosomes incubated with cytosol and ATP (panels c and
d), we do not find any evidence for coated bud formation in
the presence of PLD alone. The PLD treatment also results in
significant rupture of a proportion of the lysosomes, a phenomenon
rarely seen with ATP. Empty lysosomal membrane fragments and free
lumenal material are observed after the incubation with the bacterial
enzyme (panels a and b, arrows). We conclude that PtdOH plays an important role in initiating AP-2 and clathrin assembly
on lysosomes. Additional ATP- or
PtdIns(4,5)P2dependent steps are, however, required
for extensive lattice formation and invagination. This is in agreement
with our biochemical data showing only limited recruitment of
clathrin and AP-2.
Bacterial PLD Targets AP-2 to Lysosomes in Permeabilized NRK
Cells--
The rupture of lysosomes noted after adding PLD reiterates
that this enzyme hydrolyzes PtdCho rather indiscriminately. Because PtdOH has also been linked to the assembly of COPI-coated vesicles on
the Golgi (58), COPII coats at endoplasmic reticulum export sites (59,
60), protein export from the TGN (61-63) and to clathrin-coat assembly
(36), we examined whether the recruitment of AP-2 in the presence of
exogenous PLD shows compartmental specificity. Precise targeting of
AP-2 onto lysosomes is seen in permeabilized NRK cells incubated with
cytosol and ATP. An extremely high incidence of colocalization of AP-2
with the lysosomal glycoproteins lgp120 (Fig.
8, panels a and b)
and lgp852 is evident. In this system, AP-2 and clathrin
recruitment is also totally dependent on ATP. No AP-2 translocates onto
lgp120-positive structures in the absence of ATP (panels c
and d) unless PLD is added (panels e and
f). This again suggests that PLD appears to function
downstream of PIP5K. In addition, although the colocalization of AP-2
with lgp120 is less exact when PLD is used, the recruitment is still
compartmentally regulated. The staining pattern of the recruited
adaptor is clearly different from that of The results of this study provide a framework to begin to
understand the mechanisms that regulate the precise targeting of AP-2
onto membranes in greater molecular detail (Fig.
9). We find that both
PtdIns(4,5)P2 and PtdOH are important regulators of AP-2
recruitment and clathrin-lattice assembly on lysosomes. We show that
PLD1-like activity is associated with purified lysosomes, consistent
with the dispersed punctate distribution of transiently overexpressed
PLD1 observed by others (54, 64). Our results also demonstrate
directly, in the context of a biological membrane, that a positive
feedback loop exists between this enzyme and PIP5KI. PtdIns(4,5)P2 is obligatory for PLD activity and PtdOH, the
product of this activity, acts as an allosteric activator of PIP5KI
activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (4,500 Ci/mmol) was
from ICN and 1,2-dipalmitoyl-[1-14C]phosphatidylcholine
(114 mCi/mmol) was from Amersham. Apyrase, ATP, creatine kinase,
creatine phosphate, neomycin, phorbol 12-myristate 13-acetate,
dipalmitoyl phosphatidylcholine (PtdCho), phosphatidyethanolamine, PtdOH, phosphatidylinositol 4-phosphate (PtdIns(4)P),
PtdIns(4,5)P2, phosphoinositides, Streptomyces
chromofuscus PLD (type VII), protease inhibitors, and wortmannin
were purchased from Sigma. PtdIns(4)P, and PtdIns(4,5)P2
were also obtained from Roche Molecular Biochemicals. Synthetic
dipalmitoyl PtdIns(5)P was a gift from G. Prestwich. A-3 and
recombinant protein kinase C
(PKC
) were purchased from Calbiochem. Glutathione (GSH)-Sepharose 4B, NAP-5 columns, and Percoll
were from Pharmacia. Silica Gel 60 HPTLC plates were supplied by Merck
and P11 phosphocellulose and the silica LK6 TLC plates were from Whatman.
subunit of
AP-2, was provided by E. Ungewickell. Polyclonal serum from a rabbit
injected with a peptide corresponding to residues 11-29 of the mouse
µ2 sequence (22) was provided by J. Bonifacino. Cell lines
producing the clathrin heavy chain-specific mAb TD.1 and mAb AP.6, that recognizes the
subunit of AP-2, were provided by F. Brodsky. An
antibody specific for the clathrin light chain neuron-specific insert,
mAb Cl57.3, was obtained from R. Jahn. A dynamin-specific mAb (clone
41) was purchased from Transduction Laboratories and the neutralizing
antibody against type II PtdIns 4-kinase (PI4K), mAb 4C5G (23), was
obtained from UBI. Affinity purified rabbit anti-lgp120 antibodies and
mAb YA30, against lgp85, were provided by K. Akasaki.
Anti-
-mannosidase II antisera were from K. Moreman. Affinity-purified antibodies against the cation-independent mannose 6-phosphate receptor (MPR) have been described elsewhere (24). The
antibodies used to detect the PIP5Ks were affinity-purified anti-type I
PIP5K, prepared using purified erythroid PIP5KI (25), and polyclonal
anti-PIP5K type I
and anti-PIP5K type I
antibodies, each affinity
purified using the appropriate recombinant protein (26, 27). Serum from
rabbits injected with a peptide matching the carboxyl terminus of
PIP5KI
was provided by H. Ishihara and Y. Oka (28).
Affinity-purified anti-type II PIP5K antibodies were prepared using
recombinant PIP5KII
(27).
80 °C while the pellets were washed 4 times in
phosphate-buffered saline before solubilization in SDS sample buffer.
Immunodepletion of AP-2 from cytosol with AP.6-Sepharose was exactly as
described (5).
-32P]ATP (0.5-1 Ci/mmol) to a final concentration of
500 µM. After 10 min at 37 °C, the reactions were
terminated by addition of 3 ml of chloroform:methanol:concentrated HCl
(200:100:0.75), followed by vigorous mixing. Carrier phosphoinositides
(50 µg/tube) were added and then a biphasic mixture generated by
addition of 0.6 ml of 0.6 M HCl. After centrifugation at
200 × gave for 5 min, the lower organic
phase was removed, transferred to a new tube, washed twice with 1.5 ml
of chloroform, methanol, 0.6 M HCl (3:48:47) and then dried
under a stream of N2 gas at about 40 °C. Dried lipid
films were resuspended in chloroform:methanol:H2O (75:25:1) and spotted onto TLC plates. Formation of PtdIns(4,5)P2
from either commercial PtdIns(4)P or synthetic PtdIns(5)P was assayed
in a final volume of 50 µl in 50 mM Tris-HCl, pH 7.5, 10 mM magnesium acetate, 1 mM EDTA, 80 µM PtdInsP, 50 µM
[
-32P]ATP (5 Ci/mmol), and a source of enzyme. PtdInsP
micelles were prepared by resuspending the dried lipid at 5 mg/ml in 20 mM Tris-HCl, pH 8.5, 1 mM EDTA, and stored at
80 °C in small aliquots. Reaction mixtures were equilibrated to
~25 °C for 5 min before starting the assays with the addition of
ATP. After 6 min at ~25 °C, 3 ml of
chloroform:methanol:concentrated HCl (200:100:0.75) was added and the
lipids extracted as described above except that no carrier lipid was
added and the lower organic phase was only washed once.
S, and
0.1 µM PKC
with 1 µM phorbol
12-myristate 13-acetate. Exogenous substrate (mixed micelles of
phosphatidyethanolamine, [14C]PtdCho (52 mCi/mmol),
PtdIns(4,5)P2 at a molar ratio of 6.7:1.15:1) was added to
a final lipid concentration of 132 µM, prepared as described (32). Additions were made on ice followed by incubation at
37 °C for 1 h. Assays were stopped by addition of 1 ml of
chloroform:methanol:concentrated HCl (50:50:0.3) and 0.35 ml of 1 M HCl, 5 mM EDTA, followed by vigorous mixing.
After centrifugation at 370 × gmax for 5 min, 0.4 ml of the lower organic phase was removed and dried under a
stream of N2 gas. Dried lipid films were resuspended in
chloroform:methanol:concentrated HCl (50:50:0.3) and spotted onto TLC plates.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
µ2 subunits of the AP-2 complex and clathrin onto lysosomes. The
amount of these proteins recovered in the pellets (lanes i
and k) is equivalent to that found in the pellets from
incubations lacking lysosomes (lanes h and j). Coat recruitment is little changed by addition of 250 µM
A-3 (lane g), a competitive inhibitor with respect to
ATP.
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Fig. 1.
Clathrin-coat assembly and phosphoinositide
synthesis on lysosomes. A, reactions containing 30 µg/ml purified liver lysosomes, ~2.5 mg/ml gel-filtered rat brain
cytosol, and 0-1 mM ATP were prepared as indicated. After
20 min at 37 °C, the tubes were centrifuged and the pellets analyzed
by immunoblotting with anti-AP-2 -subunit mAb 100/2. AP-2 binding is
expressed as the percent of maximal obtained with an ATP-regeneration
system (1 mM ATP, 5 mM creatine phosphate, 10 units/ml creatine kinase) and the mean ± S.D. of four independent
determinations is shown. B, reactions containing 30 µg/ml
purified liver lysosomes, 2.5 mg/ml gel-filtered cytosol, an
ATP-regeneration system (ATPr), 500 µM
ATP, and 0-1 mM A-3 were prepared as indicated. After 20 min at 37 °C, the tubes were centrifuged
and the pellets analyzed by immunoblotting with anti-AP-2
-subunit
mAb 100/2, anti-AP-2 µ2-subunit serum, anti-clathrin heavy chain
(HC) mAb TD.1, anti-clathrin light chain (LC) mAb
Cl57.3 or anti-lgp85 mAb YA30. The mobility of
Mr standards is indicated on the left
and only the relevant portion of each blot is shown. Note that when ATP
is limiting, light chain-free clathrin appears to aggregate and
precipitate from the cytosol and is recovered in the pellet fractions
(lanes h-k). C, reactions containing 0.5 mg/ml
lysosomes, 5 mg/ml cytosol, 500 µM
[
-32P]ATP, and 1 mM A-3 were prepared as
indicated. After incubation at 37 °C for 10 min the lipids were
extracted and analyzed by TLC and autoradiography. The migration
positions of authentic phospholipid standards, visualized with iodine,
are indicated.
S, do not support coat assembly
(21) and neither derivative serves as a phosphate donor for
phosphoinositide synthesis (34). Second, neomycin, a polyamine
antibiotic that binds to PtdIns(4)P and PtdIns(4,5)P2 with
high affinity (35) and has been widely used to intercede in
phosphoinositide-regulated processes, inhibits clathrin-coat assembly
on purified lysosomes in a dose-dependent fashion.
Inhibition is apparent at 100 µM and, in the presence of
300 µM neomycin, the recruitment of both the AP-2 complex
and clathrin is completely blocked, indicating that the compound
inhibits an early stage of the assembly reaction (data not shown). A
similar observation has been made in a study of AP-2 recruitment onto endosomes (36). Third, A-3-mediated inhibition of AP-2 and clathrin recruitment (Fig. 1B) occurs together with a complete block
of PtdIns(4,5)P2 synthesis (Fig. 1C). Lysosomes
exhibit intrinsic PI4K activity (37, 38) and mixing purified lysosomes
with ATP allows this enzyme to phosphorylate endogenous PtdIns,
generating PtdIns(4)P in a temperature-sensitive reaction (Fig.
1C, lane b compared with lane a). Anion-exchange
HPLC analysis of the deacylated product of this lipid shows exact
co-elution with an internal [3H]glycerophosphorylinositol (GroPIns) 4-phosphate
standard (data not shown), verifying this lipid as PtdIns(4)P. Addition
of brain cytosol to an incubation containing lysosomes and ATP results in the synthesis of PtdIns(4)P and a second phospholipid, which comigrates with an authentic PtdIns(4,5)P2 standard (Fig.
1C, lane d). The deacylated product of this lipid elutes
coincidentally with a [3H]GroPIns(4,5)P2
standard (data not shown). Including 1 mM A-3, which
completely abrogates AP-2 and clathrin recruitment onto the lysosome
surface (Fig. 1B), totally inhibits the activity of the
soluble PIP5K (Fig. 1C, lane f) without affecting the PI4K activity. These experiments show that mixing lysosomes with cytosol and
ATP allows for a robust synthesis of PtdIns(4,5)P2 on the lysosome surface and suggest that this lipid might be important for the
initiation of clathrin coat formation on lysosomes.
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Fig. 2.
AP-2 binding initiates coat assembly.
A, Coomassie Blue-stained gels of the bound fraction
obtained after incubating cytosol with GST (lane a) or
GST-amphSH3 (lane b) and GSH-Sepharose, or with mAb
AP.6-Sepharose (lane d) or after incubating mAb
AP.6-Sepharose with buffer (lane c). Dynamin, synaptojanin
I, and the AP-2 subunits are indicated. B and C,
reactions containing 30 µg/ml lysosomes, 500 µM ATP,
and 2.5 mg/ml of the soluble depleted cytosol obtained after incubation
with GST, GST-amphSH3, or mAb AP.6 were prepared as indicated. After 20 min at 37 °C, membranes were recovered by centrifugation and
aliquots corresponding to 1/50 of each supernatant (B) or
1/3 of each pellet (C) were analyzed by immunoblotting with
anti- -subunit mAb 100/2, anti-µ2-subunit serum, anti-clathrin
heavy chain (HC) mAb TD.1, anti-clathrin light chain
(LC), mAb Cl57.3, or an anti-dynamin mAb.
S does not modulate adaptor recruitment in the lysosomal system (21).
, -I
, and -I
(26, 28, 50). The different type I
PIP5Ks are the products of separate genes (26, 28, 50) but, because the
central kinase domains of these enzymes are about 80% identical, all
these polypeptides are detected on immunoblots by affinity-purified
antibodies raised against type I PIP5K purified from erythrocytes (25)
(Fig. 3B). The identity of the ~90-kDa type I
, the
68-kDa type I
, and the 67-kDa type I
enzymes is confirmed on
duplicate blots probed with isoform-specific antibodies, however (Fig.
3B).
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Fig. 3.
PtdIns(4,5)P2 synthesis on
lysosomes catalyzed by type I PIP5K. A, rat brain
cytosol was loaded onto a phosphocellulose column and bound proteins
eluted with a linear gradient of 0.25-1.25 M NaCl. An
aliquot of every second fraction was assayed for PIP5K activity using
PtdIns(4)P micelles as a substrate. The two peaks of PIP5K activity
resolved, type I and type II, are indicated. B, aliquots of
every second fraction from a similar column run were analyzed on
immunoblots with affinity-purified anti-erythroid PIP5KI antibodies
(a), affinity-purified anti-PIP5KI antibodies
(b) affinity-purified anti-PIP5KI
antibodies
(c), anti-PIP5KI
serum (d), or
affinity-purified anti-PIP5KII antibodies (e). C,
reactions containing 0.5 mg/ml lysosomes, 5 mg/ml brain cytosol,
37.5-µl aliquots of either the PIP5KI or the PIP5KII pools and 500 µM [
-32P]ATP were prepared as indicated.
After 10 min at 37 °C, lipids were extracted and analyzed by TLC and
autoradiography. D, reactions containing 80 µM
PtdIns(4)P or synthetic PtdIns(5)P, 25-µl aliquots of either the
PIP5KI or the PIP5KII pool and 50 µM
[
-32P]ATP were prepared as indicated. After 6 min at
25 °C, lipids were extracted and analyzed by TLC and
autoradiography. Note that the mobility of the
PtdIns(4,5)P2 made from the synthetic PtdIns(5)P
differs from that of PtdIns(4,5)P2 made from PtdIns(4)P
because of differences in the acyl chain composition. E,
reactions containing 0.5 mg/ml lysosomes, 5 mg/ml brain cytosol,
35-µl aliquots of either unconcentrated (lanes c and
d) or 5-fold concentrated (lanes e and
f) PIP5KI pool, 500 µM
[
-32P]ATP, and 1 mM A-3 were prepared as
indicated. After 10 min at 37 °C, lipids were extracted and analyzed
by TLC and autoradiography. Note that the slightly aberrant migration
position of PtdIns(4,5)P2 in lane c of this
experiment was identical to the mobility of the carrier
PtdIns(4,5)P2 in that lane visualized with iodine
vapor.
and type II
,
both being detected with an affinity-purified anti-type II antibody
(Fig. 3B). To determine whether the type I, type II, or both
types of PIP5K can phosphorylate the PtdIns(4)P generated on lysosomes to form PtdIns(4,5)P2, pooled fractions enriched with each
activity from the phosphocellulose column were added to purified
lysosomes in the presence of [
-32P]ATP (Fig.
3C). Only the type I PIP5K pool produces
PtdIns(4,5)P2 (lane f). The type II pool is
inactive (lane h) although PtdIns(4)P is generated as a
potential substrate. This agrees with previous data showing that the
type II PIP5K does not phosphorylate intrinsic PtdIns(4)P in
phospholipid bilayers (30). In fact, unlike the type I PIP5Ks, the type
II pool shows a marked preference for PtdIns(5)P over PtdIns(4)P as a
substrate (Fig. 3D), confirming that these enzymes appear to
be predominantly PtdIns(5)P 4-kinases (51).
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Fig. 4.
Primary alcohols inhibit
PtdIns(4,5)P2 synthesis and coat assembly.
A, reactions containing 0.5 mg/ml lysosomes, 5 mg/ml
cytosol, 1.5% 1-butanol or t-butanol, and 500 µM [ -32P]ATP were prepared as indicated.
After 10 min at 37 °C, lipids were extracted and analyzed by TLC and
autoradiography. B, reactions containing 50 µg/ml
lysosomes, 2 mg/ml cytosol, 1 mM ATP, and 0-2% 1-butanol
or t-butanol were prepared as indicated. After 15 min at
37 °C, membranes were recovered by centrifugation and the pellets
analyzed by immunoblotting with anti-
-subunit mAb 100/2 or
anti-µ2-subunit serum. Note that the t-butanol experiment
comes from a separate blot on which the immunoreactivity in the
presence of 2% t-butanol did not differ significantly from
the signal obtained in the absence of the alcohol.
, augment the hydrolysis of the
exogenous PtdCho. Added alone, ARF1· GTP
S stimulates PLD
(column c) considerably better than PKC
and phorbol 12-myristate 13-acetate (column d) but, together, the two
proteins synergize to stimulate PLD activity maximally (column
e). Cleavage of PtdCho to produce PtdBut is totally dependent on
PtdIns(4,5)P2 and negligible PtdBut formation is seen when
liposomes lacking the phosphoinositide are used as a substrate (data
not shown). The production of PtdBut verifies the existence of PLD on
the lysosome surface and the specific activity of the activated
lysosomal enzyme (~2.5 nmol/mg/h) compares favorably with PLD1
activity measurements on other membrane preparations, although our data cannot be compared directly to assays quantitating choline head group
release because 1-butanol also inhibits the catalytic activity of PLD
(56).
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Fig. 5.
PLD1 activity associated with lysosomes and
Golgi membranes. A, reactions containing 0.16 µg/ml
lysosomes or Golgi membranes, 0.85 µM myristoylated ARF1
with 100 µM GTP S, 0.1 µM PKC
with 1 µM phorbol 12-myristate 13-acetate, 132 µM
phosphatidylethanolamine:[14C]PtdCho:PtdIns(4,5)P2-containing
substrate liposomes, and 0.5% 1-butanol were prepared as indicated.
After 60 min at 37 °C, lipids were extracted and analyzed by TLC and
fluorography. Quantitation of PtdCho hydrolysis from a representative
experiment of three (in which maximal stimulated PLD activity on
lysosomes (column e) and Golgi (column i) ranged
from 0.46 to 4.95 and 0.64 to 2.47 nmol/mg/h, respectively) is shown.
B, analysis of lysosome and Golgi membrane markers. Samples
of 25 µg each of a total rat liver homogenate or purified liver
lysosomes or liver Golgi membranes were fractionated by SDS-PAGE and
either stained with Coomassie Blue (a-c) or transferred to
nitrocellulose (d-f). Blots were probed with
anti-mannosidase II serum, affinity-purified anti-lgp120 antibodies or
anti-lgp85 mAb YA30.
-mannosidase II,
lgp120, and lgp85 (Fig. 5B). Lysosomes have no detectable mannosidase II but are heavily enriched with the lysosomal membrane proteins, as expected. The gross protein profiles of the two organelle preparations are also clearly different from each other (lanes b and c) and from the total liver homogenate
(lane a). These results show that like Golgi membranes,
purified rat liver lysosomes also contain membrane-associated PLD1-like
activity and the relative abundance of this enzyme on lysosomes makes
it unlikely that the activity comes from contaminating Golgi or
endoplasmic reticulum structures.
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Fig. 6.
Coat-protein recruitment evoked by bacterial
PLD. Reactions containing 30 µg/ml lysosomes, 2.5 mg/ml
gel-filtered cytosol, 1 mM ATP, 0.5 units/ml S. chromofuscus PLD, 10 units/ml apyrase, and 100 µM
A-3 were prepared as indicated. After 20 min at 37 °C, membranes
were recovered by centrifugation and the pellets analyzed by
immunoblotting with anti- -subunit mAb 100/2, anti-µ2-subunit
serum, anti-clathrin LC mAb Cl57.3, or anti-lgp85 mAb YA30. Note some
degradation of clathrin despite the protease inhibitors. Analysis of
the S. chromofuscus PLD by SDS-PAGE shows a near homogeneous
polypeptide of about 60 kDa and PLD assays confirm that the enzyme
hydrolyzes PtdCho to PtdOH and choline in a
PtdIns(4,5)P2-independent fashion (L. S. Arneson and
L. M. Traub, unpublished results).
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Fig. 7.
EM analysis of clathrin-coat assembly on
lysosomes. Reactions containing 120 µg/ml lysosomes, 3 mg/ml
gel-filtered cytosol, and either 0.5 unit/ml S. chromofuscus
PLD (a and b) or an ATP regeneration system
(c and d) were prepared on ice. After incubation
at 37 °C for 20 min the tubes were chilled, fixed, and then
processed for EM. Selected images that are typical of the coated
structures (arrowheads) formed under each condition are
shown. Note that no budding of the coated profiles
(arrowheads) is seen after PLD treatment and significant
rupture of the lysosomes, resulting in both free limiting membrane
fragments (fine arrows) and exposed lumenal material
(bold arrows) occurs. Bar, 100 nm.
-mannosidase II, a medial
Golgi marker (panels g and h). In most cells, the adaptor complex targets onto membranes that are spatially distinct from
the MPR-containing late-endosomal elements (panels i and j). While some overlap between the MPR-positive structures
and the recruited AP-2 is seen in a few cells (panels i and
j, arrowheads), careful examination does not
reveal convincing colocalization of the discrete labeled spots.
The similarity in staining may therefore reflect the close proximity of
the late endosome and lysosome compartments in some of these cells. We
do note that AP-2 recruitment onto remnants of the plasma membrane also
becomes evident in the presence of the bacterial PLD (panels f,
g, and i). Because PtdOH is likely to be generated on
all membranes in these experiments, the results establish that AP-2
does not simply associate with PtdOH-rich membranes; additional
determinants at the site of coat assembly appear necessary to initiate
adaptor recruitment.
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Fig. 8.
Bacterial PLD initiates adaptor recruitment
onto lysosomes. Digitonin-permeabilized NRK cells were incubated
with 2.5 mg/ml gel-filtered cytosol and 1 mM ATP
(a and b) or without ATP (c-j) and
with 0.5 unit/ml S. chromofuscus PLD (e-j). After
20 min at 37 °C the cells were washed, fixed, and then processed for
indirect immunofluorescence using a mixture of affinity-purified
anti-lgp120 antibodies (a, c, and e) and
anti- -subunit mAb AP.6 (b, d, and f) or mAb
AP.6 (g) and anti-mannosidase II serum (h) or mAb
AP.6 (i) and affinity-purified anti-CI-MPR antibodies
(j). The conditions for photography and printing were
identical for a-f. The images are typical of each treatment
and selected regions of colocalization of the lgp120 and AP-2 signals
are indicated (arrowheads) as are cells in which substantial
overlap between the AP-2 and MPR staining occurs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Schematic model illustrating the positive
feedback loop between PIP5KI and PLD1 that occurs at the lysosome
surface and how the products of this regulatory loop the appear to
effect clathrin-coat assembly on lysosomes.
PLD Activation and AP-2 Recruitment--
A clear role for
PtdOH in the construction of a clathrin-coated bud is evident from the
ability of bacterial PLD to induce AP-2 translocation and effect
limited coat assembly under conditions that avert
PtdIns(4,5)P2 synthesis. While we cannot rule out that residual PtdIns(4,5)P2 on the limiting membrane of the
lysosome contributes to AP-2 recruitment under these conditions, PtdOH must play an important role because, in the absence of ATP, no recruitment occurs without the exogenously added PLD. The data also
indicate that ongoing PtdIns(4,5)P2 formation is not
required to permit adaptor binding. Superficially, some of our results appear similar to those in a recent study examining the role of PLD
activation in the targeting of AP-2 onto endosomes (36). However,
GTPS is not at all required to induce coat formation in our system
(21). Because the hydrolysis of exogenous PtdCho by the lysosomal PLD
is potentiated by ARF·GTP
S, we assume the lysosome-associated
lipase to be PLD1-like. This corroborates the recent colocalization of
GFP-PLD1b, the major PLD1 isoform expressed in most rat tissues (65),
with LAMP-1-positive membranes (64). Nevertheless, the minimal effect
of GTP
S on clathrin-coat initiation suggests that activity of the
PLD1-like enzyme toward lysosomal membrane phospholipids does not
appear to be absolutely dependent on activated ARF. In fact, the
precise physiological roles of the in vitro activators of
PLD1, ARF, Rho, and PKCa, is still being debated (66).
PtdOH appears to play a general role in regulating the assembly of several types of coated membrane (36, 46, 58-63, 67), but it is unclear exactly how this is accomplished. One possibility is that direct interaction of certain phospholipids with coat proteins, in our case AP-2, initiates bud formation. It has recently been shown that clathrin-coated components can translocate onto synthetic liposomes, and commence the assembly of coated buds, in a nucleotide-independent fashion (46). The recruitment of AP-2 and clathrin from brain cytosol onto synthetic liposomes proceeds only poorly, however, and is considerably less efficient than the recruitment of dynamin, amphiphysin, and synaptojanin onto these same liposome preparations (46). Coat assembly equivalent to that seen when cytosol is mixed with synaptosome-derived membranes is only observed on the synthetic liposomes after fortifying the donor cytosol with ~0.5 mg/ml soluble AP-2 and clathrin extracted from purified clathrin-coated vesicles (46). This indicates that the interaction between AP-2 and lipids alone is a low-affinity phenomenon and, interestingly, no dramatic effect on AP-2 and clathrin binding or coat formation occurs on adding either phosphoinositides or PtdOH to the lipid vesicles (46). In permeabilized NRK cells treated with bacterial PLD under conditions that we expect PtdOH to be generated on all PtdCho-containing membranes, we show that AP-2 targets almost exclusively onto lysosomal glycoprotein-rich lysosomal membranes. This shows that lipids alone are not sufficient to nucleate coat assembly on any biological membrane. Nonetheless, we do find that the synthesis of relatively minor but potently active acidic phospholipids is pivotal in nucleating clathrin polymerization on lysosomes and the production of these lipids promotes exceptionally efficient coat formation in the presence of dilute cytosol. Our interpretation is that there are additional factors on the membrane that raise the affinity of AP-2 for the lysosomal membrane substantially. The growing consensus is that dedicated docking molecules, restricted to the bud site, probably provide the primary binding interface for adaptors on membranes (9). We favor the idea that PtdOH activates a putative AP-2-selective docking molecule (36, 68) in a manner analogous to the way ARF activates an AP-1-specific docking site at the TGN (5, 7, 8).
PtdIns(4,5)P2 and Clathrin-coat Assembly--
In the
absence of PtdIns(4,5)P2 production, we find that AP-2
binding to lysosomes is about 3-fold lower. At least two explanations for this result can be considered. First, as the bacterial PLD compromises the integrity of the lysosomal membrane, the reduced coat
assembly and the failure of the assembled lattice to invaginate might
simply reflect the damage inflicted on the membrane by the enzymatic
treatment. Alternatively, the data could suggests that, in addition to
serving as a co-factor for PLD1, PtdIns(4,5)P2 might also
contribute to the nucleation of coated structures directly. In fact,
evidence for multiple roles for PtdIns(4,5)P2 in
clathrin-mediated endocytosis has just been published (69). Our data
show that the lipids formed on lysosomes are PtdIns(4)P and
PtdIns(4,5)P2, neither of which affects the affinity of
AP-2 for tyrosine-based sorting signals (70). Therefore, direct
modulation of tyrosine-based sorting signals by polyphosphoinositides
is unlikely to account for increased binding of AP-2 to lysosomes (70).
PtdIns(4,5)P2 could anchor AP-2 to the lysosome surface
directly, since a high-affinity inositol polyphosphate-binding site is
located within the amino-terminal 50 residues of the subunit of the
AP-2 heterotetramer (71). Association of AP-2 with
PtdIns(4,5)P2 might orient the molecule for optimal
association with sorting signals and together, these two attachments
might transiently retain an adaptor on the membrane if release from the
putative docking site proceeds clathrin recruitment.
Retrograde Traffic from the Lysosome-- The physiological significance of clathrin-coat formation on lysosomes is somewhat controversial. While the evidence for an outward pathway of membrane flow is only indirect in mammalian cells, we reasoned that a retrograde route from the lysosome would be important to maintain overall membrane homeostasis and speculated that this would provide a suitable mechanism for recycling regulatory molecules, like v-SNARES (21). There is now very good evidence for retrograde movement from the Saccharomyces cerevisiae vacuole, the yeast equivalent of the lysosome. Strikingly, a phosphatidylinositol 3-phosphate 5-kinase, Fab1p, is intimately connected with the regulation of the size of the vacuole (72, 73). Within 30 min of shifting to the restrictive temperature, the vacuole of a fab1 temperature conditional mutant more than doubles in size, prompting the conclusion that phosphoinositide synthesis is a critical regulator of the size and integrity of the vacuole, probably by modulating transport into or out of this organelle (72). Direct evidence for protein flux through the vacuole has recently been obtained (74).
It is not known yet whether clathrin and adaptors are involved in the
return of proteins from the yeast vacuole and, certainly, the
AP-2-dependent clathrin-coat assembly we see on lysosomes in our in vitro assays is an exaggerated phenomenon.
Nevertheless, the yeast data, and the presence of both PI4K and PLD1
activity on the lysosome membrane suggest, but do not prove, that
clathrin-coat assembly on lysosomes occurs in a tightly regulated
manner (54). Some of the regulatory constraints appear to be lost in
the in vitro systems that we use. It is important to note,
however, that massive AP-2-containing clathrin-coat assembly does occur
on lysosomes under some conditions in vivo (21, 75). More
importantly, perhaps, our preliminary experiments analyzing
clathrin-coat assembly on preparations of highly purified synaptic
plasma membrane, and other independent studies (69), reveal that
PtdIns(4,5)P2 is also a critical regulator of AP-2
recruitment and clathrin polymerization at the cell surface. Our
current interpretation then is that the biochemistry that underlies
coat assembly on the lysosome is very likely to be similar to the
process that occurs at the cell surface.
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ACKNOWLEDGEMENTS |
---|
We are especially grateful to Mo Wilson and Phil Majerus for help with the HPLC analysis and to our many colleagues for readily providing essential reagents. We also thank Maureen Downs for technical support and Stuart Kornfeld, Ernst Ungewickell, and Matthew Drake for insightful comments and suggestions.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health post-doctoral training Grant T32 HL07088 (to L. S. A.) and Grant R01 DK53249 (to L. M. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to Monette Springer for continual encouragement, kindness, and generosity.
¶ To whom correspondence should be addressed: Div. of Hematology, Box 8125, Dept. of Internal Medicine, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-747-3711; Fax: 314-362-8813; E-mail: ltraub{at}im.wustl.edu.
2 L. M. Traub, unpublished results.
3 L. S. Arneson and L. M. Traub, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
TGN, trans-Golgi network;
AP, adaptor protein;
ARF, ADP-ribosylation factor;
GroPIns, glycerophosphorylinositol;
GST, glutathione S-transferase;
GTPS, guanosine
5'-O-(3-thio)triphosphate;
HC, heavy chain;
LC, light chain;
MPR, mannose 6-phosphate receptor;
PI4K, phosphatidylinositol 4-kinase;
PIP5K, phosphatidylinositol 4-phosphate 5-kinase;
PKC
, protein
kinase C
;
PLD, phospholipase D;
PtdBut, phosphatidylbutanol;
PtdCho, phosphatidylcholine;
PtdIns, phosphatidylinositol;
PtdInsP, phosphatidylinositol phosphate;
PtdIns(3)P, phosphatidylinositol
3-phosphate;
PtdIns(4)P, phosphatidylinositol 4-phosphate;
PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate;
PtdOH, phosphatidic acid;
SH3, Src homology domain 3;
ATP
S, adenosine 5'-(
-thio)triphosphate;
mAb, monoclonal antibody;
PAGE, polyacrylamide gel electrophoresis;
lgp, lysosomal glycoprotein;
HPLC, high performance liquid chromatography;
AMP-PNP, adenylyl
imidodiphosphate.
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