* Department of Biochemistry and Biophysics and the Hormone Research Institute, University of California, San Francisco,
California 94143-0534; and Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, Maryland 20892
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Abstract |
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In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric adaptor protein AP3 and a small molecular
weight GTPase of the ADP ribosylation factor (ARF)
family. We have now discovered a second pathway that
sorts the synaptic vesicle-associated membrane protein
(VAMP) into similarly sized vesicles. For this pathway
the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPS. The second pathway, however,
uses AP2 instead of AP3 and is brefeldin A insensitive.
The AP2-dependent pathway is inhibited by depletion
of clathrin or by inhibitors of clathrin binding, whereas
the AP3 pathway is not. The VAMP-containing, plasma membrane-derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled
plasma membrane preparations at 37°C. Dynamin-
interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from
endosomes. Thus, VAMP is sorted into small vesicles
by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.
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Introduction |
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NERVE terminals contain synaptic vesicles of uniform diameter that release their neurotransmitter
content by exocytosis. After exocytosis, the vesicle membrane is recycled from the plasma membrane to
form a vesicle, which quickly fills up with neurotransmitter. The recycling and refilling processes are very efficient,
allowing the nerve terminal to maintain remarkably high
rates of neurotransmitter release. Some vesicle recycling
might be by a neuron-specific form of exocytosis referred
to as "kiss-and-run" (Klingauf et al., 1998). Evidence that
a considerable fraction of the recycling process uses conventional, clathrin-mediated endocytotic machinery comes
from genetics, morphology, and subcellular fractionation. Mutations in the heterotetrameric adaptor complex, adaptor protein AP21 (Gonzalez-Gaitan and Jackle, 1997
), or
in the large GTPase, dynamin (van der Bliek et al., 1993
;
Herskovits et al., 1993
), block the reformation of vesicles.
Clathrin-coated vesicles are more abundant in rapidly
stimulated nerve terminals (Heuser and Reese, 1973
). Clathrin-coated vesicles isolated from nerve terminals are
enriched in synaptic vesicle membrane proteins (Maycox
et al., 1992
). Although there is some ambiguity about
whether synaptic vesicles are formed from the plasma
membrane, the endosome, or both (for review see De
Camilli and Takei, 1996
), there is little doubt that clathrin and dynamin are involved.
Synaptic vesicle formation involves both vesiculation
and sorting of synaptic vesicle membrane proteins. It is a
favorable experimental system for the study of vesiculation and sorting processes since the synaptic vesicle membrane is simple and almost completely characterized. This
laboratory has developed an in vitro system that reconstitutes the formation of neuroendocrine synaptic vesicle-like microvesicles, here referred to more simply as synaptic vesicles (SVs), by incubating labeled membranes from neuroendocrine PC12 cells in the presence of ATP and
brain cytosol (Desnos et al., 1995). The donor organelle
for this pathway is an endosome-like organelle (Lichtenstein et al., 1998
). To our surprise, depletion of clathrin,
AP2, and dynamin from brain cytosol had no detectable
effect on SV biogenesis (Faúndez et al., 1997; Horng, J.,
and R.B. Kelly, unpublished observations). Further analysis revealed that the coat that is recruited to the donor compartment in the presence of ATP and the small GTPase ADP ribosylation factor ARF1, was the heterotetrameric adaptor, AP3 (Faundez et al., 1998
). Mice that
lack one of the subunits of AP3 have neuronal abnormalities but are viable (Kantheti et al., 1998
). This suggests that there is a pathway of SV biogenesis in addition to the
one that involves endosomes and AP3.
In vivo evidence has been presented by Huttner and colleagues (Schmidt et al., 1997) that plasma membrane can
be a direct precursor of SVs in PC12 cells. Using a vesicle-associated membrane protein (VAMP) mutant that exhibited increased targeting to SVs, N49A/VAMP (Faúndez
et al., 1997), we set out to reconstitute vesicle budding
from plasma membranes in vitro. The results reported here show that vesicles with the sedimentation properties
of SVs, which contain multiple SV marker proteins such as
synaptophysin and SV2, and which exclude the Tf receptor
(TfR), can also be generated from the plasma membrane
in vitro. This alternative pathway of SV biogenesis has few
similarities with the endosome-derived pathway since it
uses AP2, clathrin, and dynamin-binding proteins. It thus
appears as if VAMP-containing SVs that exclude TfR can be formed in two different ways in PC12 cells. Comparing
two pathways in the same cell allows us to state with some
confidence that recruitment of clathrin or dynamin-related
proteins is not essential for the AP3-mediated pathway in
vitro, nor is ARF1 essential for the AP2-mediated pathway.
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Materials and Methods |
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125I and ECL reagents were obtained from Amersham Corp. (Arlington
Heights, IL). Iodogen came from Pierce Chemical Co. (Rockford, IL).
ATP, GTPS, creatine phosphate, creatine kinase, and Sephadex G25
spin columns were purchased from Boehringer Mannheim Corp. (Indianapolis, IN). Brefeldin A was purchased from Epicentre Technologies (Madison, WI). Percoll was obtained from Sigma Chemical Co. (St. Louis,
MO) and rat Tf was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Cell culture media and reagents were obtained from the University of California Cell Culture Facility (San Francisco, CA), with the exception of Geniticin (G418), which was obtained
from GIBCO BRL (Gaithersburg, MD). All the other reagent grade
chemicals were purchased either from Sigma, Fisher Scientific Co. (Fairlawn, NJ), or Calbiochem-Novabiochem Corp. (La Jolla, CA). Female
Sprague-Dawley rats were from Bantin and Kingman (Fremont, CA).
Cell Culture
PC12 cell lines were stably transfected to express rat VAMP, to which a T
antigen (TAg) epitope was attached at the COOH lumenal end (VAMP-TAg). Unless stated otherwise, the VAMP was the mutant N49A form
(Grote et al., 1995). Cells were grown in DME H-21 media supplemented
with 10% horse serum, 5% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, and with 250 µg/ml G418. The cells were treated for 24 h before
the experiments with 6 mM sodium butyrate to induce the expression of
the N49A/VAMP-TAg construct.
Recombinant Proteins
GST control fusion protein was generated by expression of the plasmid pGEX-3X (Pharmacia, Piscataway, NJ). GST-Dyn-PRD was constructed as described in Roos and Kelly (1998). The preparation of constructs encoding the COOH-terminal appendage domains of the
AP3,
GST-
3A (810-1094), GST-
3B (799-1081) and GST-
3Amt (
3A with
amino acids 820-822 mutated to alanine) has been described elsewhere
(Dell'Angelica et al., 1997
, 1998
). Polyhistidine-tagged clathrin heavy
chain fragment (hub) was a gift of F. Brodsky (University of California,
San Francisco, CA). Expression of GST fusion proteins followed the recommendations of the manufacturer. Fusion proteins were bound to glutathione-agarose (Sigma), eluted in glutathione elution buffer (20 mM
glutathione/50 mM Tris-HCl, pH 8.0/120 mM NaCl), concentrated in a
CentriPrep 10 concentrator (Amicon, Beverly, MA) and dialyzed against
intracellular buffer (38 mM potassium asparate, 20 mM potassium MOPS,
pH 7.2, 5 mM reduced glutathione, 5 mM sodium carbonate, 2.5 mM magnesium sulfate). Fusion protein concentration was determined using the
BCA reagent system (Pierce Chemical Co.) and stored at
20°C. Hub was
produced as described in Liu et al. (1995)
. In brief, hub-containing
BL21(DE3) bacteria (Novagen, Madison, WI) were induced with 0.8 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) for 3 h at 30°C. Protein was
purified from bacterial lysates in binding buffer (50 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole) by binding to a Ni2+ affinity resin, eluted
with binding buffer plus 245 mM imidazole, and then dialyzed against intracellular buffer.
Cell Labeling and Subcellular Fractionation
PC12 cells expressing the VAMP-TAg construct (N49A) were labeled
with 125I-KT3 mAb to the TAg epitope, following the methods of Desnos et al. (1995). In brief, confluent 15-cm dishes of cells were washed at 4°C
with labeling buffer (PBS supplemented with 3% BSA, 0.3 mM CaCl2,
0.3 mM MgCl2, and 1 mg/ml glucose), labeled with 125I-KT3 (5-10 µg/ml)
in the same buffer at 4°C for 1 h, and then transferred to 15°C or retained
at 4°C for an additional 1 h.
The same conditions were used to label cells with 125I-Tf, except that before the labeling procedure, cells were incubated for 1 h at 37°C in serum-free medium (DME-H21) to deplete their stores of Tf. The concentration of iron-loaded rat Tf in labeling buffer was 20 nM.
After labeling, the cells were extensively washed in the same buffer, scraped, and then sedimented at 800 g for 5 min. Cell pellets were gently resuspended in intracellular buffer containing protease inhibitors. Homogenizations were performed by eight passes through a ball bearing homogenizer (cell cracker; European Molecular Biology Laboratory, Heidelberg, Germany) with 12 µm clearance. A Percoll step gradient was used to obtain membranes free of cytosol. The step gradients were formed by 9 ml of 10% and 3 ml of 50% Percoll (100% Percoll is 1 vol of 10× intracellular buffer and 9 vol of Percoll with protease inhibitors). After collecting the membranes at the 10-50% Percoll interface, a protein assay of the membranes was performed using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories, Hercules, CA) using BSA as standard.
In Vitro Budding Assay
VAMP-TAg/N49A PC12 cells were labeled as described above. Aliquots
of 1 mg homogenate (or Percoll-washed membranes) were incubated for
30 min at 37°C or 4°C in the presence of an ATP-regenerating system
(1 mM ATP, 8 mM creatine phosphate, 5 mg/ml creatine kinase), and 1-4
mg/ml of rat brain cytosol prepared as described (Desnos et al., 1995). In
reactions containing peptides, mixtures were preincubated for 15 min at
4°C before warming up. The reactions were stopped by chilling to 4°C for
10 min before fractionation.
After the in vitro incubation a postnuclear supernatant was prepared by sedimenting first at 1,000 g for 5 min (S1) and then at 27,000 g for 35 min (S2). Vesicles in the S2 (150-250 µl, 2-5 mg/ml) were quantified by velocity sedimentation on 5-25% glycerol gradients prepared in intracellular buffer (218,000 g for 75 min at 4°C in a SW55 rotor; Beckman Instruments, Inc., Palo Alto, CA). Fractions (16 or 17) were collected from the bottom and counted on a gamma counter. About 1-2% of the plasma membrane label was recovered in SV-containing fractions of the glycerol gradient.
Immunoadsorption of SVs
SVs isolated by glycerol velocity gradient (fractions 8-10) were pooled
and aliquoted. Dynabeads M-450 (Dynal Inc., Great Neck, NY) preincubated with mAbs against syntaxin (Sigma); synaptophysin (Boehringer
Mannheim Corp.); SV2 (Buckley and Kelly, 1985); synaptobrevin (69.1);
synaptogyrin (80.1, provided by R. Jahn, Yale University, New Haven,
CT); or, as a control, mouse
-globulin (ICN Pharmaceuticals, Inc., Irvine,
CA) were added to each aliquot. After 16-h incubation at 4°C, the beads
were isolated, and then washed with PBS plus 1% BSA twice for 5 min.
Both isolated beads and the supernatant of each reaction were counted on
a gamma counter.
Immunodepletion of Clathrin and -Subunit of AP2
Clathrin heavy chains and AP2 complexes were quantitatively removed
from rat brain cytosol using the X22 mAb to clathrin heavy chain and AP6
mAb that binds -adaptin, respectively (Brodsky, 1985
). In brief, 60 µg of
antibody were bound overnight to 40 µl of packed protein G-Sepharose
in 0.5 ml of intracellular buffer. Unbound Ig was removed by extensive
washing in intracellular buffer, and then the antibody-bound affinity matrix was incubated with 0.8 mg of cytosol for 2 h at 4°C with gentle rocking.
The Sepharose beads were removed by centrifugation and the cytosol recovered for in vitro reactions. The beads were washed to remove cytosolic proteins. Bead-bound and free cytosolic clathrin or
-adaptin were determined by immunoblotting using an anti-clathrin heavy chain antibody or
an anti-
-adaptin antibody (Transduction Laboratories, Lexington, KY).
Blots were performed using an enhanced chemiluminescence (Amersham
Corp.) system. The cytosol protein concentration before and after the depletion remained constant.
Confocal Immunofluorescence Microscopy
Immunofluorescence procedures and confocal microscopy have been detailed elsewhere (Bonzelius et al., 1994). In brief, N49A/PC12 cells were
plated in poly-D-lysine-coated PermanoxTM slides (Nunc Inc., Naperville,
IL) 2 d before staining. Cells were loaded in vivo with KT3 antibodies (10 µg/ml) in labeling buffer and washed as with labeling buffer with or without 30 mM glycine, pH 2.4, as previously described (Grote and Kelly,
1996
). Then cells were fixed in 4% PFA in PBS, permeabilized in 0.02%
saponin in PBS, 2% BSA, 1% fish skin gelatin, and then incubated with
affinity-purified fluorescent-labeled goat anti-mouse IgG (Cappel Laboratories, Malvern, PA). Observation and image acquisition were performed in a Bio-Rad MRC600 confocal laser scanning microscope (Bio-Rad Laboratories).
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Results |
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Labeling the Plasma Membrane of N49A/PC12 Cells
To study vesicle formation from the plasma membrane, we
generated homogenates of PC12 cells in which only SV
proteins on the plasma membrane were labeled. The PC12
cell line used in the current study (N49A/PC12) was stably
transfected with a luminally tagged VAMP construct that
had a point mutation (N49A) in the cytoplasmic tail. This
VAMP derivative shows increased targeting to SVs compared with wild type (Grote et al., 1995), and even more
specific targeting to SVs than the del61-70 mutation used
in a previous study (Desnos et al., 1995
). To label the
plasma membrane, N49A/PC12 cells were incubated at
4°C with antibodies (KT3) against the lumenal epitope. Immunofluorescent staining was detected around the cell
periphery with no detectable staining of any intracellular
structures (Fig. 1 A). The peripheral labeling could be
stripped away with an acid wash (Fig. 1 B), indicating that,
at 4°C, the antibodies only labeled epitopes exposed on
the cell surface. In a control experiment, when N49A-transfected cells were labeled at 37°C for 40 min, the antibodies were internalized and an intense, acid strip-resistant intracellular staining could be observed (Fig. 1, C and
D). Restriction of label to the cell surface was confirmed
by biochemical subcellular fractionation experiments. Intact N49A cells were labeled at 4°C with iodinated KT3
followed by homogenization and fractionation on sucrose
density gradients. A peak of radioactivity was recovered at
38% sucrose, which represents labeled plasma membranes
(Clift-O'Grady et al., 1998
). Thus, incubating cells with antibody at 4°C only labels the cell surface.
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Vesicle Budding from the Plasma Membrane
To reconstitute in vitro vesicle budding from the plasma
membrane, we labeled intact N49A cells with iodinated KT3
at 4°C for 2 h. After washing away the unbound antibody,
the cells were homogenized and the homogenate incubated
at 37°C in the presence of rat brain cytosol and an ATP-regenerating system. After the incubation, the reaction
mix was centrifuged to remove large membranes and the
supernatant was analyzed by glycerol velocity sedimentation. As shown in Fig. 2 (circles), we observed radioactivity
in the SV-containing fractions (8-12) of the glycerol gradient, as well as in free antibody at the top of the gradient
(14 and above). SV generated by labeling N49A cells at
15°C followed by an in vitro reaction at 37°C (Desnos et
al., 1995) also migrate to the same glycerol fractions (Fig.
2, diamonds). The amount of radioactivity recovered in
the SV peak was always about five times less when labeled
plasma membrane was used, compared with labeled endosomes. The relatively weak signal was probably due to the
limited amount of VAMP on the cell surface compared
with the amount internalized at 15°C. The small size of the
signal, the shorter incubation time, and the use of a less
advantageous mutant VAMP (del61-70) may explain why
no budding from plasma membranes was observed in earlier experiments (Desnos et al., 1995
). Nonetheless, when expressed as recovery of membrane-bound radioactivity in
SVs, the plasma membrane and the endosomal assays
have comparable efficiency.
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Characterization of the Budding Reaction
In vitro vesicle budding is increased in efficiency by the
use of cytosol. As shown in Fig. 3 A, the amount of vesicles
generated in the budding reaction depended on the concentration of rat brain cytosol, suggesting that cytosolic
factors are required for vesicle formation from the plasma
membrane. A small vesicle peak was often observed in
control reactions with no added rat brain cytosol. Since
this background reaction is seen with washed membranes
it may be due to coating proteins that remain bound to the
plasma membrane after homogenization. As indicated by
Western blot, clathrin and -adaptin can be detected on
the washed membranes (not shown). Immunofluorescence
also supports the association of dynamin (Estes et al.,
1996
; Roos and Kelly, 1998
) and AP2 (Gonzales-Gaitan
and Jackle, 1997) with the plasma membrane at endocytotic "hot spots."
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A time course of vesicle budding from the in vitro reconstitution systems indicated that detectable vesicle production starts within 10 min of incubation and reaches a
plateau in ~40 min (Fig. 3 B), not significantly different
from kinetics of the in vitro SV budding from endosomes
(Desnos et al., 1995). Vesicle formation was reduced when
the ATP-regenerating system was omitted in the reaction
mix (Fig. 3 C). The presence of GTP
S in the reconstituted system greatly inhibited the generation of SVs, in
line with the possible involvement of dynamin or other
GTPases in endocytosis from the plasma membrane (Nuoffer and Balch, 1994
; Takei et al., 1995
, 1996
; Carter et al.,
1993
).
Newly Formed SVs Contain Multiple SV Proteins and Exclude Tf
Although neuroendocrine cells such as PC12 are not synapse-forming neurons, they have organelles that resemble
SVs. Three criteria that are conventionally used to describe the SVs in PC12 cells are their homogeneous size,
the presence of a full array of SV proteins, and the absence
of other recycling plasma membrane proteins such as the
TfR. To determine if SV markers other than VAMP are
present on the small vesicles, M-450 Dynabeads (sheep anti-mouse IgG) coated with mAbs against SV proteins
synaptophysin, synaptogyrin, or SV2 were used to immunoadsorb the vesicle fractions. As shown in Fig. 4 A, each
of these antibodies adsorbed most of the radioactivity associated with the vesicle fractions. As expected, an almost
complete adsorption was also achieved by an antibody against VAMP (synaptobrevin; Fig 4 A). However, antibodies against a t-SNARE protein, syntaxin, did not
bind vesicles above the level of controls containing mouse
-globulin confirming earlier observations (Salem et al.,
1998
). This result shows that the small vesicles formed
from the plasma membrane, as expected for SVs, contain a
spectrum of SV markers.
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To determine if the VAMP-containing vesicles that formed from the plasma membrane are devoid of TfR, PC12 cells were labeled at 4°C with 125I-Tf, homogenized, and then incubated in vitro with brain cytosol and ATP. No radioactivity was recovered in the SV peak region after glycerol velocity sedimentation of the reaction products (data not shown).
The absence of labeled Tf in SV fractions could be because the levels were too low to detect. To determine if
other Tf-containing vesicles were made, we analyzed the
reaction products differently using a sucrose velocity gradient protocol that resolves SVs from other membrane
populations (Lichtenstein et al., 1998). Intact N49A/PC12
cells were labeled with iodinated Tf at 4°C as before, washed, homogenized, and then the homogenate was incubated at 37°C under standard conditions. After the reaction, the plasma membrane was removed from the reaction mix by a lower speed, 5,000 g min centrifugation to
allow the analysis of membrane vesicles larger than SVs.
The supernatant of the reaction mixture was analyzed on a
10-45% sucrose density gradient. Using this new protocol, Tf-containing vesicles were recovered at 28% ± 0.5 sucrose (Fig. 4 B, closed circles), a density different from that
at which the KT3-containing vesicles were recovered
(23% ± 1.5 sucrose), as shown in Fig. 4 C (closed diamonds). Our results indicate that VAMP-containing SVs,
from which the TfR has been segregated, can bud from the
plasma membrane, suggesting that protein sorting may be
occurring directly at the plasma membrane.
Small amounts of label, especially in Tf, are recovered between 35% and 40% sucrose in the 5,000 g min supernatants from cells labeled at 4°C (Fig. 4 B). The label is presumably due to small fragments of plasma membrane that are not pelleted. They disappeared during the in vitro incubation, implying that they are potential precursors of the Tf-labeled vesicles at 28% sucrose. The nature of this putative precursor is not yet known.
SV Formation from the Plasma Membrane Requires AP2 and Clathrin But Not ARF1
Although the plasma membrane was the only labeled precursor added to the reaction mix it was possible that the plasma membrane was generating endosomes from which SVs were budding by an AP3 and ARF-dependent mechanism. To examine this possibility we took advantage of the sensitivity of the endosomal budding reaction to a fungal metabolite, brefeldin A (BFA).
ARF1, a small GTP-binding protein, is required for SV
biogenesis from endosomes (Faúndez et al., 1997). Its
function is to recruit the AP3 adaptor complex as a coat to
mediate vesicle budding (Faúndez et al., 1998). A hallmark of ARF1-mediated processes is their sensitivity to
BFA, which inhibits GDP-GTP exchange activity of ARF1
(Donaldson et al., 1992). As shown in Fig. 5 (closed circles), SV biogenesis from endosomes was inhibited by
BFA in a dose-dependent manner as described previously
(Faúndez et al., 1997). However, the vesicle production
from the plasma membranes was not inhibited in the presence of BFA
500 µg/ml. The difference in BFA sensitivity implies that coat recruitment to allow vesicle budding
from the plasma membrane does not require a cytoplasmic
ARF1. These observations are consistent with the previous reports showing that BFA inhibits vesicle formation
from the TGN, but not from the cell surface (Robinson
and Kreis, 1992
).
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We were unable to obtain any evidence that clathrin
(Faundez et al., 1997) or AP2 (Horng, J.-T., and R.B. Kelly,
unpublished) were required for SV formation from endosomes. In contrast, SV formation from the plasma membrane needs clathrin and AP2. When the cell-free reconstitution system was supplied with rat brain cytosol depleted
of clathrin heavy chain or AP2 (
-adaptin), the vesicle production was reduced (Fig. 6 A). The extent of depletion was confirmed by Western blotting of rat brain cytosol
with clathrin and
-adaptin-specific antibodies (Fig. 6 B).
The clathrin involvement was further tested using a bacterially expressed clathrin heavy chain fragment, often
called the hub domain. The clathrin hub fragment is the
minimal trimerization domain that can self-assemble into
clathrin triskelions in vitro (Liu et al., 1995
). It interferes with endogenous clathrin function by blocking clathrin
polymerization into polyhedral vesicle coats (Liu et al.,
1998
). An inhibitory effect on vesicle biogenesis was observed upon the addition of the hub fragment (Fig. 6 A).
These results suggest that the clathrin/AP2-coating complex is necessary to form KT3-containing vesicles from the plasma membrane.
|
In these depletion studies, inhibition of SV formation
was never complete. This was expected since SV formation was only stimulated about twofold by the addition of
cytosol (Fig. 2). As discussed above, membrane-associated
protein machinery may also contribute to the budding activity. To confirm that the inhibition was indeed due to
clathrin and AP2 depletion, coat proteins were isolated from brain-coated vesicles by conventional procedures
(Manfredi and Bazari, 1987) and added back to the reaction mix. When 80 µg/ml purified clathrin was added back
to clathrin-depleted cytosol, the budding activity recovered to 87% of that before clathrin depletion. Similarly,
100 µg/ml AP2 restored the activity to 94%.
Clathrin Is Needed for AP2, But Not AP3-mediated SV Formation In Vitro
Originally it was reported (Simpson et al., 1996) that clathrin did not codistribute with AP3 by immunofluorescence.
This was consistent with normal AP3-mediated sorting in
yeast cells lacking clathrin (Vowels and Payne, 1998
) and
our own data from in vitro reconstitution showing clathrin
independence (Faundez et al., 1998
). However, the COOH-
terminal appendage domain of the
3 subunit of AP3
binds clathrin, and AP3 and clathrin can be colocalized
on endosomes and TGN by immunoelectron microscopy (Dell'Angelica et al., 1998
).
Additional clathrin would not be needed in our in vitro assay if there were already sufficient clathrin on the donor membranes to generate budding. If that clathrin were recruited to the COOH-terminal appendage domains of AP3, then inhibition of budding might be seen in the presence of excess appendage domain. Incubating labeled endosomes with excess appendage domain, however, gave no inhibition of SV formation (Fig. 7 ES).
|
The absence of inhibition could be attributed to failure
to achieve inhibitory concentrations of appendage domain,
or the absence of clathrin recruitment from a membrane-bound pool. The first explanation is less likely because the
AP3 appendage domains at the same concentration (150 µM) inhibited SV production from labeled plasma membranes by ~70% (Fig. 7 PM). With 75 µM appendage domain, the inhibition was 40% and with 38 µM, the inhibition was not significant. A mutant, GST-3Amt, in which
residues DLD (820-822 of GST-
3A) are mutated to
three alanine residues, has no clathrin binding activity in
vitro (Dell'Angelica et al., 1998
), and does not show an inhibitory effect on SV formation. The appendage domain
of AP2 also inhibited (data not shown). These results are consistent with the evidence that the
3 appendage domain binds clathrin as efficiently as the
2 appendage domain of AP2 and so might inhibit AP2-mediated recruitment (Dell'Angelica et al., 1998
).
The data on appendage inhibition are consistent with the
other data on clathrin dependence of the AP2 pathway. A
concern, however, with the use of appendage domains as
inhibitors is the high concentration needed (150 µM) to
obtain significant inhibition. Earlier experiments using the
AP2 appendage domains reported a very low affinity between appendage and clathrin (Shih et al., 1995) consistent
with our findings. The low affinity might not be real but
might reflect inefficient folding of the recombinant proteins. It is encouraging that inhibition is not seen with mutant appendages that do not have binding activity (Fig. 7).
To resolve definitively whether clathrin is involved in
AP3-mediated budding, high affinity inhibitors of clathrin-mediated vesiculation are clearly needed.
The GTPase dynamin has been implicated in endocytosis from the plasma membrane (van der Bliek et al., 1993;
Vallee and Okamoto, 1995
; De Camilli and Takei, 1996
).
To explore how dynamin might function in SV formation,
a fusion of GST to the proline-rich domain (PRD) of dynamin (GST-Dyn-PRD) was made and used to screen for
brain-specific proteins that bound to it (Roos and Kelly, 1998
). Four major PRD-binding proteins were found in
brain extracts. To determine if dynamin or SH3-containing
proteins that bind to the Dyn-PRD were involved in SV
biogenesis from PC12 membranes, in vitro budding reactions were incubated with either GST (control) or GST-Dyn-PRD fusion proteins. The GST-Dyn-PRD protein
inhibited vesicle formation from plasma membranes by
>50%, but had no effect on budding from endosomes
(Fig. 8). We conclude that the proteins that bind dynamin
PRDs are likely to be involved in the AP2-mediated pathway of vesicle biogenesis, but not the endosomal, AP3-mediated pathway.
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Discussion |
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We have reconstituted the biogenesis of SVs from the plasma membrane of PC12 cells in vitro. Unlike the biogenesis of SVs from PC12 endosomes, the formation of plasma membrane-derived SVs requires AP2 and clathrin, but not GTP hydrolysis by ARF1. This mode of small vesicle biogenesis could be a model for the dominant pathway of SV biogenesis in neurons, which is likely to involve AP2 and clathrin on the basis of genetic, morphological, and cell fractionation evidence.
The presence of two distinct biosynthetic pathways in
the same cell type throws into sharper relief the differences between the AP2- and AP3-mediated pathways.
Most strikingly, AP2-mediated budding requires clathrin
recruitment under conditions that AP3 does not. The ability of clathrin triskelions to form pentagons and hexagons
has often led to the speculation, occasionally challenged (Kirchhausen et al., 1997), that the function of clathrin is
to impart curvature to vesicles budding from a donor
membrane. If the AP3 adaptor in the absence of clathrin
can form vesicle-sized structures in vitro, then the recruitment of clathrin is not essential for imparting curvature.
An alternative explanation is that clathrin recruitment is
not necessary because endosomes already have a large
pool of clathrin bound to the membranes. For this explanation to be plausible, clathrin recruitment from the membrane pool cannot be sensitive to the AP3 appendage domains. Efforts to make the endosomal budding system
dependent on clathrin by stripping the endosome precursors with high salt or Tris washes have not succeeded (Horng, J.-T., and R.B. Kelly, unpublished).
Conversely, the AP3-dependent endosomal pathway,
but not the AP2-dependent plasma membrane one, requires the addition of ARF1 in a GTP-bound form. Given
the involvement of small GTPases of the ARF family in
ER, Golgi, and endosomal budding events it is surprising
that recruitment of coat by the plasma membrane should be different. One possible explanation is that the AP2 recruitment mechanism is firmly bound to the plasma membrane. ARF6, for example, is firmly associated with the
plasma membrane and is insensitive to BFA (D'Souza-Schorey et al., 1995; Peters et al., 1995
).
The discovery of a second potential pathway of SV biogenesis helps us reconcile the findings of Schmidt et al.
(1997) with our own (Desnos et al., 1995
; Faundez et al.,
1997
, 1998
). By following the internalization of biotinylated synaptophysin, Schmidt et al. (1997)
could show that
SVs were generated from a plasma membrane precursor,
not an endosomal one as we had proposed. It is very likely
that the experimental conditions chosen by Schmidt et al.
(1997)
favored the discovery of the plasma membrane-
derived pathway, whereas ours favored the discovery of
the endosomal pathway. Using our techniques for monitoring vesicle formation conditions, almost all sSV biogenesis in vivo is sensitive to BFA and so occurs by the AP3
pathway.
Our earlier work had shown that PC12 endosomes containing TfRs were precursors of SVs (Lichtenstein et al.,
1998). The original goal of the present paper was to explore the formation of the endosomal precursor from
plasma membranes. To our surprise we found that vesicles
were formed from plasma membrane in vitro that were remarkably similar in size to SVs. To determine if these small vesicles were endosomal precursors we did the type
of double-labeling experiments shown in Fig. 4, B and C.
The result was clearcut: the small vesicles do not contain
Tf. If the small vesicles were endosomal precursors that
subsequently fuse with TfR-containing endosomes, sorting
at the plasma membrane would be a futile process. A more
likely possibility is that VAMP gets to endosomes by an alternative route. One possibility is the vesicles at 28% sucrose (Fig 4, B and C), which appear to contain both Tf
and VAMP.
The absence of detectable Tf in the plasma membrane-
derived microvesicles is also consistent with the observations of Schmidt et al. (1997). The simplest explanation of
our data and those of Schmidt et al. (1997)
is that there are
two separate pathways of internalization from the plasma
membrane, one that carries both SV proteins and carriers
such as the TfR and another that is exclusive for SV proteins. A specialized internalization pathway for SV proteins was first suggested by De Camilli and coworkers to
explain the differences between the endosomes in the axons and the cell bodies of neurons (Mundigl et al., 1993
),
an observation confirmed later in PC12 cells (Bonzelius et al.,
1994
). Recently evidence that more than one internalization pathway exists has also been obtained by comparing
the GLUT4 glucose transporter with the TfR, after internalization at 15°C (Wei et al., 1998
). A clathrin-independent pathway of internalization has been known for some
time (Sandvig and van Deurs, 1991
; Lamaze and Schmid, 1995
), but the pathway taken by VAMP-TAg described
here is both clathrin and dynamin dependent. If there are
two independent pathways of clathrin-mediated internalization from the plasma membrane, some mechanisms
must exist to segregate their cargo proteins selectively.
It is not clear why PC12 cells should have two pathways
for making SV-like organelles. One possibility is that the
two classes of vesicle have different protein compositions.
Recent evidence suggests that the AP3-mediated pathway
in brain is tightly linked to the formation of a subclass of
SVs that contain one of the zinc transporters, ZnT3 (Kantheti et al., 1998). Another possibility is that the two pathways are used at different levels of synaptic activity. A
third is that neurons of a certain class, for example with
more of a neuroendocrine function, or with large nerve
terminals, or with extensive endocytosis of neurotropin receptors, use the AP3-mediated pathway. Fortunately the
tools are now available to distinguish these possibilities.
![]() |
Footnotes |
---|
Address correspondence to R.B. Kelly, Hormone Research Institute, Box 0534, 513 Parnassus Ave., 1090 HSW, University of California, San Francisco, CA 94143-0534. Tel.: (415) 476-4095. Fax: (415) 731-3612. E-mail: rkelly{at}biochem.ucsf.edu
Received for publication 16 June 1998 and in revised form 9 October 1998.
We thank Dr. F. Brodsky (University of California, San Francisco, CA [UCSF]) for the generous gift of antibodies to clathrin (X22), AP2 (AP6), and of the polyhistidine-tagged hub fragment. We are also grateful to L. Spector for her help in the preparation of the manuscript.
This work was supported by grants to R.B. Kelly from Wheeler Center for the Neurobiology of Addiction, UCSF, and the National Institutes of Health (NIH; grant Nos. NS19878, NS15927, DA10154). J. Roos is supported by a postdoctoral fellowship from the American Cancer Society. V. Faundez was the recipient of an NIH Fogarty Fellowship.
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Abbreviations used in this paper |
---|
AP, adaptor protein; ARF, ADP ribosylation factor; BFA, brefeldin A; PRD, proline-rich domain; SV, synaptic vesicle; TAg, T antigen; Tf, transferrin; TfR, transferrin receptor; VAMP, vesicle-associated membrane protein.
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