1 Division of Gastroenterology, University of Tennessee Health Science Center,
Memphis, TN 38163, USA
2 Veterans Affairs Medical Center, Memphis, TN 38104, USA
3 VA Connecticut Healthcare, West Haven, CT 06516, USA
4 Department of Medicine, Yale University School of Medicine, New Haven, CT
06520, USA
* Author for correspondence (e-mail: cmansbach{at}utmem.edu)
Accepted 10 October 2002
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Summary |
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Key words: Pre-chylomicron transport vesicle, PCTV, COPII proteins, Vesicle budding, Lipid absorption, Sar1, Chylomicrons
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Introduction |
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In contrast to PCTV formation, the initial step in ER to Golgi transport of
nascent proteins is known and involves the ordered assembly of the COPII
proteins Sar1, Sec23/24 and Sec13/31 on the ER membrane
(Matsuoka et al., 1998).
Sec16, a membrane associated protein, may act as a scaffold for coat assembly.
The first step in vesicle formation is the attachment of Sar1, a GTPase, to
the ER membrane in its GTP-bound state. The binding of Sar1 to the membrane is
phosphorylation dependent (Aridor and
Balch, 2000
). Sec12, an integral membrane protein, acts as a
facilitator of GDP-GTP exchange on Sar1. In its active GTP bound form, Sar1
recruits the Sec23/Sec24 complex. It has been shown that all members of the
COPII coat are required for budding and that Sec23/24 mediates the selection
of VSV-G, a type 1 membrane protein, as cargo
(Aridor et al., 1998
). The
Sec13/31 protein complex is recruited last and is required for membrane
deformation and budding (Aridor and Balch,
2000
; Barlowe,
1998
; Salama et al.,
1997
).
Previous studies have demonstrated distinct features of protein and lipid
exiting from the ER. Protein transporting vesicles contain COPII proteins on
their surface and are 60-80 nm in diameter
(Matsuoka et al., 1998). PCTV
vesicles are larger (150-500 nm) than the protein vesicles to accommodate the
larger size of their cargo, chylomicrons (100-500 nm in diameter
(Zilversmit, 1967
). To
discriminate between vesicle types, they are identified here by their cargo:
PCTVs or protein vesicles containing lipid-rich chylomicrons or proteins,
respectively. Vesicles containing apoB-48 and nascent TAG generated in the
absence of COPII proteins are identified as lipid vesicles.
The present study demonstrates that PCTV generated in vitro from isolated enterocyte ER meets the criteria for a budded vesicle. The requirements for PCTV budding and the abilities of PCTV to fuse with isolated Golgi were also examined. PCTV generation from isolated ER was dependent on cytosol and ATP. These PCTV were competent to fuse with purified Golgi complexes. To examine the potential role of COPII proteins in PCTV budding, a double-labeling protocol was developed to directly compare budding of nascent protein and PCTV vesicles from enterocyte ER. Antibody depletion of the COPII protein Sar1 or antibody inhibition of Sar1 blocked budding of protein vesicles. By contrast, treatment with either Sar1 or Sec31 antibodies did not inhibit PCTV budding, but enhanced it. Notably, membrane-bound lipid vesicles generated in the absence of COPII proteins were not competent to fuse with isolated Golgi. These observations suggest that the presence COPII or associated proteins are required for PCTV fusion with the Golgi complex, and that membrane-bound lipid particles can bud from the ER in a COPII-independent manner.
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Materials and Methods |
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Antibodies and plasmid
Affinity-purified rabbit polyclonal anti-Sar1 and anti-Sec24 antibodies
were provided by J.-P. Paccaud (University of Geneva, Geneva, Switzerland).
Rabbit polyclonal antibodies against mammalian Sec31 (Sec31M) have been
characterized (Shugrue et al.,
1999). Polyclonal antibodies to Sec13 were a gift of Chris Kaiser
(MIT, Cambridge, MA). Rabbit polyclonal antibodies to syntaxin 5 were a kind
gift from W. E. Balch (Scripps Research Institute, La Jolla, CA). Goat
polyclonal anti-apolipoprotein B-48 (apoB-48), apolipoprotein AI (apoA-I), and
apolipoprotein A-IV (apoA-IV) antibodies were generously provided by P. Tso,
University of Cincinnati (Cincinnati, OH). Mouse monoclonal antibodies to
rBet1 and GOS28 were procured from StressGen Biotechnologies (Victoria,
Canada). Goat polyclonal anti-calreticulin and -calnexin antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit IgG
conjugated with agarose beads was purchased from Sigma. Goat anti-rabbit IgG,
goat anti-mouse IgG and goat anti-rabbit IgG conjugated with horseradish
peroxidase (HRP) were purchased from Santa Cruz Biotechnology.
His6-Sar1a in a PET 114 plasmid, originating in the laboratory of
W. E. Balch, was a generous gift of Brian Storrie (Virginia Polytech
Institute, Blacksburg, VA).
Isolation and metabolic labeling of enterocytes
Enterocytes were isolated as described
(Kumar and Mansbach, 1997)
from the small intestine of male Sprague Dawley rats (200-300
g, Harlan, Indianapolis, IN). 3H-TAG was
synthesized from 3H-oleate and sn-2-monooleoylglycerol by the cells
as described (Kumar and Mansbach,
1997
).
When protein/TAG labeling was required, cells were incubated in buffer A [137 mM NaCl, 1.5 mM EDTA, 11.5 mM KH2PO4, 8 mM Na2HPO4, 2.2 mM KCl, 0.5 mM dithiothreitol (DTT) and 10 mM glutamine] with 25 µCi of 14C-oleic acid plus 4 µmol of carrier oleate complexed to BSA, and 3H-leucine (50 µCi) at 37°C for 30 minutes and then placed on ice.
Preparation of ER and cytosol
ER was prepared from radiolabeled enterocytes as described
(Kumar and Mansbach, 1997).
Rat intestinal cytosol was prepared as described
(Kumar and Mansbach, 1997
).
The cytosol was dialyzed against Buffer B (25 mM Hepes, pH 7.2, 125 mM KCl,
2.5 mM MgCl2, 0.5 mM DTT, and protease inhibitors for 6 hours at
4°C, concentrated fivefold using a 50 ml Amicon Filter with a YM 10
membrane (Amicon, Beverly, MA). This cytosol was further concentrated on a
Centricon Filter (Amicon) with a 10 kDa cutoff to 20 mg protein/ml.
In vitro ER budding assay
ER fractions (500 µg protein) in Buffer C (0.25 M sucrose, 30 mM Hepes,
pH 7.2, 30 mM KCl, 5 mM MgCl2, 5 mM CaCl2, 2 mM DTT)
were incubated in a total volume of 500 µl containing 50 µl rat small
intestinal cytosol (0.8-1 mg protein), and Buffer C with an ATP regenerating
system (1 mM ATP, 5 mM phosphocreatine, and 5 U creatine phosphokinase) at
37°C for 30 minutes without Golgi acceptor. The reaction was terminated by
placing the reaction tubes on ice. The sucrose concentration of the reaction
mixture was adjusted to 0.1 M and the suspension was overlaid on a continuous
sucrose gradient and resolved as described
(Kumar and Mansbach,
1997).
Depletion of Sar1 from cytosol and ER
To remove Sar1 from cytosol, 20 µl of rabbit polyclonal anti-Sar1
antibody was incubated with 50 µl of intestinal cytosol (1 mg protein),
then anti-rabbit IgG conjugated to agarose beads were added. The
immunocomplexes were removed by centrifugation. Sar1-depletion was confirmed
by immunoblot. To remove Sar1 from the ER, ER was incubated with 2 M urea for
15 minutes at 4°C. The ER was isolated by centrifugation and washed twice
in PBS. Sar1 depletion was confirmed by immunoblot.
Removal of Rab proteins
To remove Rab proteins from the ER and cytosol, Rab-GDI was used
(Mukherjee et al., 2000). GDP,
1 mM, was pre-incubated with intestinal ER in Buffer C supplemented with the
ATP-regenerating system and protease inhibitors for 20 minutes at room
temperature followed by the addition of 12.5 µg of Rab GDI (Sigma) for 10
minutes at room temperature (Mukherjee et
al., 2000
). Removal of Rab proteins from cytosol was accomplished
by immunodepletion.
Transport of newly synthesized TAG and apoB-48 to the Golgi
PCTVs (150 µg protein) containing 14C-TAG and
3H-apoB-48 or only 3H-TAG were formed
(Kumar and Mansbach, 1997) and
incubated with Golgi membranes (300 µg protein), an ATP regenerating
system, 500 µg cytosolic protein and Buffer C for 30 minutes at 37°C.
The reaction was stopped by adding cold Buffer C and the Golgi fraction
obtained by separating the reaction on a sucrose step gradient
(Kumar and Mansbach, 1997
).
3H-TAG was isolated from the Golgi
(Coleman and Bell, 1976
).
To demonstrate delivery of PCTV cargo (3H-apoB-48 and
14C-TAG) to the Golgi lumen after fusion, a cis-Golgi enriched
fraction was incubated with or without carbonate buffer
(Kumar and Mansbach, 1997).
The released chylomicron fraction was isolated by centrifugation after adding
carrier rat chylomicrons (Mansbach and
Arnold, 1986a
). The chylomicron fraction was incubated with
anti-apoB antibody (Kumar and Mansbach,
1997
) and the apoB-48-antibody complex isolated, washed with PBS
and its radioactivity determined. 3H-TAG was extracted separately
from the chylomicron fractions (Coleman and
Bell, 1976
).
To investigate whether PCTV were attached but not fused with the Golgi,
3H-TAG-PCTV (150 µg protein) were incubated with Golgi (300
µg protein) in the fusion assay as above. The 3H-TAG-cis Golgi
were isolated using the discontinuous sucrose gradient
(Kumar and Mansbach, 1997).
The 3H-TAG-cis Golgi was incubated with or without trypsin (0.5
mg/ml) in Hepes pH 7.2 at 4°C for 1 hour (total vol. 1 ml). Soybean
trypsin inhibitor and protease inhibitor cocktail were added and the Golgi
isolated using the continuous sucrose gradient
(Kumar and Mansbach, 1999
).
The gradient was resolved, 0.5 ml fractions collected, and the total
3H-dpm determined. Fractions 18-21, containing the
3H-dpm, were concentrated using a Centricon filter, and 30 µg
protein loaded onto SDS-polyacrylamide gel electrophoresis (SDS-PAGE). GOS28
sensitivity to trypsin was tested by immunoblotting.
Sar1 rescue
Recombinant Sar1 was produced in E. Coli transfected with the
His6-Sar1a containing PET114 plasmid. The final product migrated as
a single band on SDS-PAGE (silver stain) with an apparent
Mr of 27-28 kDa. The band reacted with anti-Sar1 antibody
on immunoblot. To test Sar1 rescue of Sar1-depleted cytosol and ER, urea
washed ER (500 µg prot, containing 14C-TAG and
3H-protein) was incubated with cytosol (1 mg protein), recombinant
Sar1 (40 µg prot), an ATP regenerating system, and E600 (0.025%) in 500
µl total volume for 30 minutes at 37°C. The reaction was placed on a
sucrose density gradient as before and the gradient resolved.
14C-TAG-dpm and TCA precipitated 3H-protein-dpm were
determined in each tube.
Measurement of TAG and protein radioactivity
TAG radioactivity was quantitated as described
(Kumar and Mansbach, 1997).
Protein radioactivity was measured after precipitation with trichloroacetic
acid (TCA). In experiments in which both 3H- and
14C-radiolabels were used, the scintillation analyzer (Packard
TriCarb, Packard Instrument, Downer's Grove, IL) was set in the dual-isotope
mode.
Gel electrophoresis and immunoblots
Proteins were separated by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) and transblotted onto nitrocellulose membranes (Bio-Rad) as
described (Kumar and Mansbach,
1997). Proteins were detected by developing the blots using ECL
and exposing the developed blots to Biomax film (Eastman Kodak, Rochester,
NY).
Electron microscopy
To visualize budded vesicles by negative staining, glow-discharged Ni-grids
were coated with carbon and formvar
(Mukherjee et al., 2000). The
coated side of the grid was placed on top of a drop of PCTV,
1 mg protein,
and incubated for 2-3 minutes and rinsed with PBS and H2O. Samples
were stained with 0.5% aqueous uranyl acetate for 1 minute, blotted on filter
paper, air dried and examined under a JEOL 1200 EX electron microscope at
12,000x (JEOL, Peabody, MA).
To examine PCTVs at thin section electron microscopy (EM), PCTV were
collected from 3 to 4 experiments and concentrated using a Centricon filter
with a YM 10 membrane. The buds, 1 mg protein, were pelleted (2500
g for 10 minutes in an Eppendorf table top centrifuge at
4°C), fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide
buffered with 0.2 M imidazole, pH 7.2, and embedded in Spur medium. Sections
were cut using a glass knife, stained with uranyl acetate and lead citrate
(Tipton et al., 1989
) and
examined using a JEOL 1200 EX electron microscope at 5000x
magnification.
Immunolabeling of the PCTVs was performed as described
(Mukherjee et al., 2000).
Briefly, samples were incubated with 10% BSA containing mouse anti- (1:100)
and rabbit anti-syntaxin-5 (1:100) antibodies for 3-4 hours and subsequently
with goat anti-mouse IgG (1:50) conjugated with 10 nm colloidal gold and goat
anti-rabbit IgG (1:50) conjugated with 15 nm colloidal gold. The samples were
fixed in 1% glutaraldehyde in PBS for 10 minutes and stained with 0.5% aqueous
uranyl acetate for 1 minute, and examined under the JEOL 1200 EX electron
microscope (12,000x). Control studies were performed using anti-mouse
and rabbit pre-immune IgG and processed as described.
Measurement of lipid-P
Lipid phosphorus (lipid-P) was measured by extracting the phospholipids
from cellular fractions (Folch et al.,
1957), and measuring the lipid-P content by chemical means after
digestion of the phospholipids by H2SO4 as described
(Knox et al., 1991
).
Statistical analysis
Comparisons between means were carried out using a statistical package
supplied by GraphPad Software (Instat, GraphPad Software, San Diego, CA) using
a two-tailed t-test.
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Results |
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To observe vesicle formation, we examined concentrated putative PCTV fractions (fractions 1-7, Fig. 2A) by EM using negative staining. The results (Fig. 1A), show a field of pure vesicles ranging in size from 142 to 350 nm.
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Transmission electron microscopy of thin sections demonstrated that PCTVs
appear as 350-500 nm structures containing osmiophilic chylomicrons
(Fig. 1B). Some of these
structures appear elongated, which may be an artifact of the preparation
process, may be due to vesicle fusion or may indicate that these structures
are part of the vesicular tubular complex (VTC)
(Saraste and Svensson, 1991).
The latter postulate is supported by the presence of the VTC marker p58
(rodent homologue of ERGIC-53) (Saraste
and Svensson, 1991
) in this fraction (see below,
Fig. 5). Putative protein
vesicles (Fig. 1B, inset) were
found in the mid-portion of the gradient (collected and concentrated from
fractions 9-12, Fig. 2C). They
had an irregular coat, contained little electron dense material and were
smaller (70-117 nm diameter) than PCTVs.
|
Fig. 2A shows the distribution of 3H-dpm (85% of the dpm are TAG, the remainder are partial glycerides and FFA) across the sucrose gradient. 3H-dpm was maximal in the initial fractions, which suggests the presence of PCTVs. The generation of the light fraction required the addition of both ATP and cytosol to the ER (Fig. 2B). However, the generation of the low density fraction did not require added calcium (3H-lipid in PCTV fractions: 5 mM Ca=9259 3H-dpm; 1.8 mM Ca=11,714 3H-dpm; 0 mM Ca=13,603 3H-dpm; values are means of two experiments). The appearance of 3H-dpm was blocked by incubation at 4°C (not shown). To simultaneously examine protein and PCTV budding from intestinal ER preparations, we generated enterocyte ER with 3H-proteins and 14C-TAG. Fig. 2C shows newly synthesized proteins, as indicated by their 3H-dpm radioactivity, in the middle of the gradient (fractions 9-12). Little newly synthesized protein was found in fractions 1-3 occupied by PCTV. Most 3H-proteins were present in the densest portion of the gradient with the ER (the ER pellet data are not shown). By contrast, 14C-dpm associated with TAG was localized to the light fraction of the gradient as expected for PCTV with few 14C-dpm in the midportion of the gradient associated with the protein vesicles.
A characteristic of chylomicrons, which are contained in PCTVs, is the
presence of apoB-48 and apoA-IV (Kumar and
Mansbach, 1997). The immunoblot of PCTVs demonstrates the presence
of both apolipoproteins (Fig.
3A). ApoA-I is associated with mature chylomicrons but is not
present in PCTVs, which confirms previous studies
(Kumar and Mansbach, 1999
).
Calnexin and calreticulin, two ER-resident proteins
(Bergeron et al., 1994
;
Smith and Koch, 1989
), were
not detected in the PCTV fraction (Fig.
3B). Small amounts of calreticulin, but not calnexin, are also
present in the Golgi fraction. These data are consistent with the known
presence of calreticulin in the Golgi
(Zuber et al., 2000
) or could
be due to the crossreactivity of the carboxyl end of calreticulin (to which
the antibody was directed) with CALNUC, a Golgi-resident protein localized
primarily in the cis Golgi (Pin et al.,
1998
). The lack of calreticulin in the PCTVs suggests that ER
luminal resident proteins are not included as cargo in PCTVs.
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To examine the potential of PCTVs to fuse with the Golgi, PCTVs loaded with 14C-TAG and 3H-apoB-48 were incubated with unlabeled acceptor Golgi. In the absence of cytosol, there was little TAG or apoB-48 dpm associated with the Golgi (Fig. 4A). Similarly, when PCTVs were incubated with Golgi at 4°C, no increase in 3H-TAG dpm was found in the Golgi in excess of the 3H-TAG dpm present in the absence of cytosol (1618 vs. 1779 3H-dpm, mean of two experiments). When PCTV was incubated with Golgi in the presence of cytosol, 14C and 3H increased four- to fivefold in a Golgi fraction (Fig. 4A). Most of the 14C-TAG and 3H-apoB-48 were associated with the Golgi since the majority of the dpm remained with the Golgi membranes in the absence of carbonate (Fig. 4B). Carbonate treatment, which disrupts the Golgi membranes, released most of the 14C-TAG and 3H-apoB-48 to the top of the gradient where carrier chylomicrons were found (Fig. 4B). When the fusion reaction was performed at 4°C instead of 37°C, no shift of the TAG to the Golgi fraction was observed (with cytosol, 3936 3H-TAG-dpm; without cytosol, 1779 3H-TAG-dpm; and with cytosol incubated at 4°C, 1618 3H-TAG-dpm were transferred from PCTVs to the cis-Golgi during the 30 minute incubation; means of two experiments).
|
To release any PCTVs that might be attached by protein linkers but not
fused with the Golgi, PCTVs were incubated with Golgi, and then the Golgi was
re-isolated and treated with trypsin. After protease treatment, protease
inhibitors were added and the Golgi and other fractions were isolated on a
continuous sucrose gradient. The lack of protease-dependent PCTV release to
the light part of the gradient after protease treatment suggests that they are
not tethered to the Golgi complex (Fig.
4C). Further, the location of the 3H-dpm was consistent
with the distribution of the Golgi as shown by the specific Golgi SNARE, GOS28
(Subramanian et al., 1996), in
this portion of the gradient (Fig.
4C, inset). The reduced GOS28 signal on immunoblot confirmed the
efficacy of the trypsin treatment (Fig.
4C, inset). These data indicate that PCTV are fusion competent and
deliver prechylomicrons to the Golgi lumen.
To confirm that PCTVs were formed by budding from the ER and not its
fragmentation, several studies were performed. First, ER, PCTVs, protein
vesicles and Golgi proteins were immunoblotted with antibodies to syntaxin 5,
a membrane protein important in vesicle targeting to the Golgi
(Dascher et al., 1994), and
rBet1, a v-SNARE (Zhang et al.,
1997
). Signals were present in ER and Golgi fractions, but they
were much less prominent than those observed in PCTVs and protein vesicles
(Fig. 5A). The immunoblots
shown in Fig. 5A were loaded
with the same amount of protein in each lane. To assess the membrane content
of each fraction, lipid-P was assayed. The results were: PCTV=0.16; ER=9.5;
cis-Golgi=20.7; and trans-Golgi=53.3 µg lipid-P/mg protein (means of two
experiments). Thus the differences shown in the blots between ER, Golgi and
PCTV would be magnified if they were based on lipid-P rather than on protein.
Second, the distribution of key markers was examined in all sucrose density
gradient fractions collected after the addition of ER, cytosol and ATP and
compared with the originating ER and cytosol. As shown in
Fig. 6, Sar1, rBet1 and Sec31
all produced peaks that were coincident with both PCTVs and protein vesicles
(Fig. 2C). As expected, apoB-48
was found only in the light fraction of the gradient in the location of PCTVs.
Calnexin was not detected in either vesicle fraction but was present in the
original ER and cytosol (TM). These data strongly suggest that Sar1, Sec31 and
rBet1 were recruited to PCTVs as well as to protein vesicles and were not
products of ER fragmentation.
|
Next, we examined the protease sensitivity of the PCTV cargo protein, apoB-48. When PCTVs were treated with proteinase K there was no reduction in apoB-48 signal on immunoblot, compared with PCTVs not exposed to the proteinase (Fig. 5B). By contrast, when PCTVs were incubated with proteinase K and Triton X-100 to dissolve the vesicle membrane, the apoB-48 signal was eliminated. These studies provide biochemical evidence that PCTVs have an intact membrane that is acquired during budding.
To detect specific cargo proteins, fractions were probed for p58
immunoreactivity (Fig. 5A)
(Rowe et al., 1996). A
moderate signal was detected in the ER, Golgi and PCTV, and the most intense
reaction was in the area of the sucrose gradient expected for protein
vesicles. One interpretation of these findings is that both PCTVs and protein
vesicles contain p58.
Role of COPII proteins in PCTV-budding from ER
The budding of nascent protein vesicles from the ER requires only the COPII
proteins Sar1, Sec23/24 and Sec13/31
(Barlowe, 1998). As shown by
the immunoblots in Fig. 5C (see
Fig. 9 for Sec24), each COPII
protein was present in PCTVs. Sar1, Sec 13 and Sec31 appeared greater than
tenfold more concentrated in PCTVs compared with the ER. In the case of Sar1,
Sec13 and Sec31, the PCTVs were loaded at one-third the amount of protein as
the other fractions. The presence of the COPII proteins on the PCTV vesicles
suggested that COPII proteins might mediate their budding.
|
To test the functionality of COPII proteins in PCTV budding, cytosol (0.8 mg protein) was pre-incubated with anti-Sec31 antibodies (5 µl) at 4°C for 1 hour and the treated cytosol used in our budding assay. The results are shown in Fig. 7. Instead of the expected inhibition of budding, an increase in budding of approximately sixfold occurred. Addition of preimmune rabbit IgG had no effect (Fig. 7).
|
To remove any residual Sec31 from the ER membrane that might contribute to budding activity, the ER was washed with 2 M urea (data not shown). Although urea-treated ER was not as effective as native ER for budding, anti-Sec31-antibody-treated cytosol continued to induce greater amounts of vesicles than cytosol treated with pre-immune IgG (no cytosol, 5805±612 3H-dpm; with cytosol + pre-immune IgG, 15,110±1456 3H-dpm; with cytosol + anti-Sec31 antibody, 70,695±5927 3H-dpm; data from pooled fractions 1-7 of the sucrose gradient±s.e.m., n=4). These results show that lipid vesicle budding can occur in the absence of Sec31 activity, a protein that is required for COPII-dependent budding, but does not establish the functionality of these vesicles.
The effects of the COPII protein, Sar1
(Barlowe et al., 1993;
Matsuoka et al., 1998
), on
PCTV budding were next examined. Immunoblots of PCTVs, ER and intestinal
cytosol showed the presence of Sar1 in the cytosol, a smaller amount in PCTVs,
and very little in the ER (Fig.
5C). To examine Sar1 function, the protein was depleted from
cytosol (Fig. 9A) prior to the
budding assay. Similar to Sec31-antibody-treated cytosol, Sar1-depleted
cytosol (closed squares) markedly enhanced lipid vesicle budding
(Fig. 8A). Mock-depleted
cytosol was used as the source of native cytosolic protein (open squares,
Fig. 8A). When Sar1-depleted
cytosol was used, no Sar1 was detectable on lipid vesicles by immunoblot
(Fig. 9B), whereas it was
easily identified in native cytosol as well as native ER
(Fig. 9A). Because Sar1 was
present in native ER, the ER was washed with 2 M urea to remove the Sar1
(Fig. 9A), and tested for its
ability to generate lipid vesicles using cytosol treated with Sar1 antibodies
(Fig. 8B). An increase in
budding of approximately tenfold was observed over cytosol to which pre-immune
IgG had been added (Fig. 8B).
Notably, lipid vesicles formed in Sar1-depleted cytosol lacked not only Sar1,
but also Sec24, Sec31 and p58 (Fig.
9B). However, they were similar to PCTVs formed from non-depleted
cytosol in that they contained apoB-48 and apoA-IV
(Fig. 3A), they appear to
concentrate both apolipoproteins by comparison with the ER
(Fig. 3A), and lipid vesicle
apoB-48 was resistant to proteinase K treatment unless Triton X-100 was added
(Fig. 9C). That proteins were
removed from PCTVs by the antibody treatment was suggested by the increase of
lipid-P per milligram of protein in Sar1-depleted lipid vesicles (0.47 µg
lipid-P/mg protein) compared with PCTVs (0.16 µg lipid-P/mg protein, means
of two experiments). Importantly, syntaxin 5 and rBet1 were present in
lipid-rich vesicles formed in native or Sar1-depleted cytosol
(Fig. 9B). Finally, binding of
Sar1 to ER membranes was inhibited by H89 as described
(Aridor and Balch, 2000
)
(Fig. 9B). Proteins other than
Sar1 were not sought in PCTVs using H89-treated cytosol since we wished only
to be certain that H89 blocked Sar1 binding to PCTVs. Although H89 removed
Sar1 from PCTVs (Fig. 9B), the
treatment did not affect PCTV budding
(Table 1). Together, these
findings suggest that lipid vesicles that share characteristics with PCTVs can
form in the absence of COPII proteins. However, a major difference between
PCTVs formed in native cytosol and the lipid vesicles formed under
Sar1-depleted conditions is that the latter are no longer fusion competent
with the Golgi (Fig. 4D). One
possible explanation for this lack of fusion is the absence of p58 on lipid
vesicles generated in the absence of COPII proteins. The p58 protein is
required for recruitment of COPI proteins that mediate fusion with the Golgi
and, as shown here (Fig. 9B),
p58 is not present on lipid vesicles formed in Sar1-depleted cytosol
(Tisdale et al., 1997
).
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|
The lipid vesicles generated in the absence of Sar1 were examined at EM by negative staining. As shown in Fig. 10A, they were morphologically identical to the PCTVs seen in Fig. 1A. Thin section EM of the lipid vesicles (Fig. 10B) also demonstrated morphology similar to that shown in Fig. 1B in which budding occurred with native cytosol. Finally, to demonstrate that the lipid vesicles formed in the absence of Sar1 contained the v-SNAREs, rBet1 and syntaxin 5, on their surface, we used immunogold labeling as shown in Fig. 10C. The double-labeling technique demonstrated that both proteins were localized on these vesicles (Fig. 10Ci-iii). Control studies using pre-immune IgG showed no immuno-gold labeling (Fig. 10Civ).
|
To determine whether a Rab-GTPase was involved in PCTV budding, Rab-GTPase
was stripped from the ER membrane by pre-incubation with GDP followed by
Rab-GDI (Mukherjee et al.,
2000), as confirmed by immunoblot
(Fig. 5D), and stripped from
the cytosol by immunodepletion (Fig.
5D). Using this Rab-depleted system, PCTV budding continued at the
same rate as native ER and cytosol, but enhanced budding was not observed
(Table 1). These data are
consistent with our previous observations using anti-Rab1 and Rab2 antibodies
that were found not to inhibit the transport of TAG from ER to Golgi
(Kumar and Mansbach,
1997
).
Sar1 is required for protein vesicle budding but not for lipid
vesicle budding to occur
Since Sar1 depletion enhanced lipid vesicle formation, we determined
whether it affected protein vesicle formation. In experiments in which both
TAG and proteins were radiolabeled, when the ER was washed with urea to remove
Sar1 and the cytosol immunodepleted of Sar1, we confirmed
(Fig. 8C) the increase in
14C-TAG output seen in Fig.
8A,B. By contrast, using native cytosol, the 3H-protein
radioactivity increase over background seen in
Fig. 2C and confirmed in
Fig. 8D (closed squares) was
eliminated from the mid-portion of the gradient when Sar1 was depleted
(Fig. 8D, closed circles). The
findings indicate that no protein vesicle budding occurred in the absence of
Sar1. The Sar1 depletion did increase newly synthesized proteins in the
low-density part of the gradient where PCTVs are found
(Fig. 8D, closed circles). To
examine the specificity of the Sar1 antibody, exogenous recombinant Sar1 was
added to the reaction. To this end, Sar1 was immunodepleted from the cytosol;
this cytosol contained neither Sar1 (Fig.
9A) nor the anti-Sar1 antibodies (data not shown). Sar1 was
removed from the ER by urea washing. By using this Sar1-depleted cytosol and
ER, recombinant Sar1 reduced lipid vesicle output to the levels found using
native cytosol (Fig. 8E) and
restored protein vesicle output (Fig.
8F). These findings confirm the specificity of the Sar1 antibodies
and suggest that, similar to other systems, Sar1 is necessary for
COPII-dependent protein vesicle budding in the small intestine.
To further demonstrate that the intestine is similar to other tissues with
respect to the requirement of Sar1 for protein vesicle budding, we added the
kinase inhibitor H89, which prevents Sar1 recruitment to ER membranes
(Aridor and Balch, 2000). In
both the presence and the absence of H89, 14C-total dpm counts
above background levels, marking prechylomicron-TAG, were found in the light
portion of the gradient (Fig.
11A). The lack of effect of H89 on PCTV formation supports the
data shown in Table 1. As
expected (Table 1), GTP
S
had no effect on PCTV budding (Fig.
11A). By contrast, H89 completely blocked protein vesicle
formation (Fig. 11B). The
addition of GTP
S reduced protein vesicle formation by 25%
(Fig. 11B), consistent with
the findings of other workers (Barlowe et
al., 1994
; Rexach and
Schekman, 1991
).
|
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Discussion |
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Previous studies of COPII function have emphasized the general role of the
coatomer in mediating protein export from the ER. However, recent studies have
suggested that vesicles carrying specialized cargos may travel in vesicles
that are distinct from those which carry the bulk of nascent proteins. For
example, export of the yeast glycosylphosphatidylinositol (GPI) linked
protein, Gas1p, is inhibited by conditions that do not affect the export of
other nascent proteins. Thus, inhibition of ceramide synthesis, genetic
deletion of either Emp24 (a potential cargo receptor) or the -subunit
of COPI, all result in decreased ER export of Gas1p but not other proteins
(Schimmoler et al., 1995
;
Sütterlin et al., 1997
).
By using density centrifugation to separate vesicles budded from the ER, Gas1p
and Yps1 (another GPI-linked protein) were found in a population of ER-derived
vesicles that were distinct from those carrying the bulk of nascent secretory
proteins (Muñiz et al.,
2001
). Whether budding from the ER of vesicles containing
GPI-linked proteins requires COPI or COPII remains unclear. A second example
is the large elongated (>300 nm) vesicles that arise from the ER and
contain the rod-like structures of nascent procollagen. These are
morphologically distinct from the smaller (60-80 nm) COPII coated vesicles
that mediate the transport of most proteins from the ER. Further, the
procollagen transport vesicles exclude ts-045-G, a marker of bulk protein
movement from the ER (Stephens and
Pepperkok, 2002
). Another distinct cargo that moves from the ER to
the Golgi complex is lipid that is transported with surrounding lipoproteins.
In the intestinal mucosal cell these are known as PCTVs
(Kumar and Mansbach, 1999
). In
the liver, pre-very low-density lipoproteins (VLDL) transport vesicles are
likely to have a similar biogenesis and function as PCTVs. Little is known
about the mechanisms that regulate either the budding of these lipid-rich
particles or their fusion with the Golgi complex.
The present study describes an in vitro system for examining the generation
of lipid particles (PCTV) from isolated intestinal ER. The development of the
system was based on in vitro techniques that have been used to examine the
regulation of nascent proteins from the ER and studies of lipid trafficking
from our laboratory. One important conclusion of this study is that the PCTVs
we generated meet the criteria of a budded vesicle. The conclusion is
supported by the following: (1) EM studies that show intact vesicles of
appropriate size (350-500 nm) to contain chylomicrons (100-500 nm
(Zilversmit, 1967) enclosed in
a membrane bilayer; (2) inhibition by incubation at 4°C; (3) concentration
of the vesicle-membrane proteins syntaxin 5 and rBet1 in the PCTVs compared
with the ER; (4) protease-resistance of the chylomicron cargo protein apoB-48,
indicating a membrane-enclosed vesicle; (5) exclusion of ER resident proteins,
calnexin and calreticulin from PCTVs, similar to that observed by Rowe et al.
in protein vesicles (Rowe et al.,
1996
); and (6) fusion of isolated PCTVs with the Golgi. Thus,
PCTVs meet similar biochemical and functional criteria that have qualified the
nascent-protein-containing COPII vesicles as physiologic carriers. A second
conclusion is that PCTVs have distinct morphologic and biochemical features
when compared with vesicles that export the bulk of nascent proteins as their
major cargo. Thus, PCTVs are larger than protein vesicles (150-500 nm vs 60-80
nm), have an electron dense core, reflecting their lipid cargo, vs a lucent
core for protein vesicles, and contain apoB-48. However, the two classes of
vesicles do share some properties: both require ATP and cytosol for budding,
enrich selective ER membrane proteins, and have COPII proteins on their
surface.
The initial identification of Sec13, Sec24, Sec31 and Sar1 on PCTVs
suggested that COPII proteins might be involved in their budding. To test the
function of COPII proteins in chylomicron export from the ER we used
antibodies to two proteins required for protein vesicle budding, Sar1
(Barlowe et al., 1993) and
Sec31 (Tang et al., 2000
).
Treatment by Sar1 antibody completely blocked protein vesicle generation as
would be expected for this COPII-dependent process. However, treatment by
either Sar1 or Sec31 antibody increased 3H-TAG export from the ER
by approximately six- to tenfold. When all peripheral proteins, including Sar1
and Sec31, were removed from the ER by urea, Sar1- or Sec31-depleted cytosol
continued to support PCTV generation. These unexpected results suggested that
formation of PCTV might be COPII independent. To confirm this theory, we used
H89, which has been shown to block COPII assembly by preventing Sar1 from
attaching to the ER membrane (Aridor and
Balch, 2000
). H89 resulted in the same rate of lipid vesicle
budding as controls not treated with H89, but not at the increased rate seen
in our antibody inhibition studies. The explanation for the quantitative
differences between antibodies and H89 on PCTV budding is unclear. Further,
recombinant Sar1 protein completely reversed conditions in which Sar1 was
depleted from the cytosol and ER confirming the specificity of the depletion
experiments. Lipid vesicles generated in the absence of COPII proteins were
morphologically intact with cargo protein (apoB-48) that resisted protease
digestion, and continued to concentrate the membrane proteins rBet1 and
syntaxin 5. These findings provide compelling evidence that vesicles
containing nascent TAG can be generated by a COPII-independent mechanism. COPI
coat proteins are generally associated with retrograde movement from
intermediate compartments and the Golgi complex to the ER, and inter-Golgi
stack movement. There are reports of COPI coat proteins mediating budding from
ER (Bednarek et al., 1995
;
Sütterlin et al., 1997
).
However, the absence of COPI proteins from PCTV by immunoblot analysis makes
it less likely that they mediate PCTV generation. Preliminary studies from our
laboratory (S.A.S. and C.M.M., unpublished) suggest that distinct proteins are
concentrated on PCTVs compared with the ER. Although the identity of these
proteins is under study, it is possible that they have a role in the formation
of PCTVs.
COPII proteins are usually associated with small vesicles, making the
presence of COPII proteins on large PCTVs unexpected. COPII proteins have two
key functions, cargo selection and generation of a vesicle. Cargo selection is
mediated by the interaction of COPII proteins with the cytoplasmic domains of
ER transmembrane proteins. Interactions have been described between Sec23 and
di-acidic residues on these cytoplasmic extensions
(Nishimura et al., 1999). Such
interactions have been observed for proteins that cycle between the ER and
intermediate compartments (p58) and proteins destined for export to the plasma
membrane. More recently, valine at position -1 has been suggested as an export
signal for transmembrane proteins as vesicle cargo from the ER
(Nufer et al., 2002
), further
expanding the role of cytoplasmic extensions of transmembrane proteins as an
export signal.
The size of vesicle may be constrained by coat proteins. Thus, the diameter
of clathrin and COPII-coated vesicles are generated within a small range
(60-80nm). The selective interactions between Sec13/Sec31 heterodimers might
account for the uniform size of COPII vesicles that carry nascent proteins
(Lederkremer et al., 2001;
Matsuoka et al., 2001
).
However, COPII proteins have been found on large elongated (300 nm)
procollagen vesicles arising from the ER
(Stephens and Pepperkok,
2002
). This suggests that COPII proteins might be concentrated on
some populations of ER-derived vesicles without constraining their size. The
findings of the present study would be consistent with this conclusion.
Although the COPII proteins do not appear to be required for selection of a
lipid rich core, apoB-48 or budding of vesicles containing a lipid core, they
may participate in selecting a subset of PCTV cargo. This suggestion is
supported by two observations. First, when COPII proteins are excluded from
PCTV by inhibiting Sar1, p58 is no longer associated with PCTVs. However,
since rBet1, Syntaxin 5 and apoB-48 are concentrated in PCTVs formed in the
absence of COPII proteins, other mechanisms of cargo selection must remain.
Second, PCTVs generated in the absence of COPII proteins are no longer
competent to fuse with the Golgi complex. These findings suggest that COPII
proteins or COPII-recruited proteins are required for PCTV fusion with the
Golgi complex. A candidate for mediating fusion with the Golgi complex is p58,
a protein that is required for recruitment of proteins (likely to be COPI)
that mediate fusion of ER-derived vesicles with the Golgi complex
(Tisdale et al., 1997). Based
on in vitro studies, the COPII protein Sec23 mediates the enrichment of p58 in
vesicles (Kappeler et al.,
1997
). Under conditions that inhibit COPII assembly and attachment
of the Sec23/Sec24 complex, we find that p58 is no longer concentrated in
PCTVs. The speculation that p58 is required for fusion of PCTVs is supported
by our studies in which PCTVs formed in native cytosol are blocked from fusion
with Golgi membranes by the addition of anti-p58 antibodies (S.A.S. and
C.M.M., unpublished).
To explain the large increase seen in lipid vesicle budding when secretory
protein budding is inhibited, we postulate that the PCTV budding system
competes for available components of the COPII system that are required for
PCTV budding other than Sar1, Sec23/24 and Sec 13/31. These might include
Sec12 or Sec16. In this event, the availability of the COPII protein
components for PCTV budding could be rate limiting. Alternatively, COPII
proteins might exhibit an inhibitory effect on the PCTV budding mechanism.
Finally, the phospholipids required for protein vesicle budding to occur might
be diverted to supply the PCTV membrane. In this context, work from our
laboratory and others has shown that the amount of dietary phosphatidylcholine
available to enterocytes is proportional to the amount of lipid that can be
exported into the lymph as chylomicrons
(Kumar and Mansbach, 1997;
Mansbach and Arnold, 1986b
;
Tso et al., 1978
). Further, it
would appear that the enterocytes are not able to synthesize enough
phosphatidylcholine from choline to support normal chylomicron output into the
lymph but are able to if lyso-phosphatidylcholine is supplied
(Tso et al., 1978
).
Nevertheless, since lipid vesicles generated in the absence of COPII proteins
appear to be unable to fuse with the Golgi complex, their physiologic
relevance remains questionable.
Together, the data suggest that COPII proteins are found on PCTVs and demonstrate that COPII-interacting proteins might be needed to form a lipid vesicle that can fuse with the Golgi complex. Further, the data suggest that COPII proteins can assemble on vesicles with a lipid core that are larger than the traditional COPII vesicles of 60-80nm. However, the lipid vesicles formed in the absence of COPII proteins are unable to fuse with the Golgi complex. These observations suggest that COPII-independent mechanisms can support the budding of membrane-bound lipid vesicles from the ER, and that COPII or their associated proteins, which are required for PCTV fusion with the Golgi compartment, are not directed into such vesicles.
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Acknowledgments |
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