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INTRODUCTION |
In recent years, a large number of polypeptides that participate
in intracellular membrane trafficking have been characterized. These
proteins ensure temporal and spatial specificity of vesicular traffic
and include members of the Rab family (1, 2). The Rab proteins are
small monomeric GTPases that function in both the endocytic and
exocytic pathways. These essential proteins interconvert between a GDP-
and GTP-bound form that results in a cycle of membrane
association and release to the cytosol. When these proteins are
membrane-associated, they show a unique subcellular location, which
suggests that Rab proteins regulate compartment-specific transport
events. Most likely, this is due to their ability to recruit in a
step-specific manner soluble factors that facilitate protein-protein, membrane-membrane, or membrane-cytoskeletal
interactions (2).
Protein transport in the early secretory pathway requires Rab2 (3).
This protein immunolocalizes to pre-Golgi intermediates that contain
clusters of vesicles and tubules, termed vesicular tubular clusters
(VTCs) (4), and function as transport intermediates between the
endoplasmic reticulum (ER) and the Golgi complex. Moreover, VTCs are
the first site of segregation of the anterior and retrograde pathways
and thereby sort and recycle resident proteins from itinerant proteins
destined for secretion (5, 6). In our ongoing studies to define the
role of Rab2 within VTCs, we have found that a constitutively activated
form of Rab2 (Q65L) as well as Rab2 wild type promoted vesicle
formation from pre-Golgi intermediates (7). The released vesicles
contained
-COP and were enriched in a protein that recycles to the
ER, suggesting that Rab2 promoted formation of retrograde-directed carriers. Since protein transport between the ER and Golgi complex is
bidirectional, it is likely that vesicles containing Rab2 possess a
select set of molecules that labels the carrier as a
retrograde-directed shuttle. With this in mind, the polypeptide content
of the released vesicles was further analyzed with the goal of
identifying candidate Rab2 effectors. We found that the Rab2-generated
vesicles contained an ~38-kDa protein.
De Matteis et al. (8) reported that a 38-kDa protein
identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was ADP-ribosylated in a brefeldin A-dependent
manner. Brefeldin A blocks transport in both the exocytic and endocytic pathways and causes the Golgi complex to redistribute to the ER (9).
Chemical inhibitors of ADP-ribosylation antagonized brefeldin A-induced
effects, suggesting that mono-ADP-ribosylated proteins may play a role
in preserving Golgi structure and therefore a role in membrane
trafficking (10). In addition, Robbins et al. (11) reported
that a Chinese hamster ovary cell line harboring a mutant form of GAPDH
was defective in endocytosis. Based on these observations, we used
reagents specific to the enzyme and identified the 38-kDa
vesicle-associated protein as GAPDH.
GAPDH catalyzes the NAD-mediated oxidative phosphorylation of
glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate (12). The active
enzyme exists as a tetramer containing identical 37-kDa subunits.
Although this abundant protein (10-20% of total cellular protein) is
commonly known as a key enzyme in glycolysis, a number of intriguing
intracellular roles have been reported for GAPDH, including modulation
of the cytoskeleton, phosphotransferase/kinase activity, and the
promotion of vesicle fusion (13-16). Interestingly, all of these
activities are essential to the maintenance of normal secretory function.
In this study, we found that Rab2 stimulates GAPDH membrane binding. To
establish whether GAPDH is required for ER to Golgi transport, an
affinity-purified anti-GAPDH polyclonal was generated. This antibody
potently inhibits transport of VSV-G from the ER to the Golgi complex
and inhibits Rab2-stimulated recruitment of GAPDH to membrane. Although
vesicles released in response to Rab2 contain GAPDH, the antibody does
not block vesicle budding. These data are the first to show that GAPDH
is required for trafficking in the early secretory pathway. Our results
suggest that the GAPDH-negative vesicles formed in the presence of Rab2
and anti-GAPDH antibody are "dead end retrograde carriers" that are
unable to transport to and fuse with the target compartment, resulting
in the arrest of ER to Golgi transport.
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EXPERIMENTAL PROCEDURES |
Membrane-binding Reaction--
NRK cells were washed three times
with ice-cold phosphate-buffered saline (PBS). The cells were scraped
off the dish with a rubber policeman into 10 mM Hepes (pH
7.2) and 250 mM mannitol and then broken with 15 passes of
a 27-gauge syringe. The broken cells were pelleted at 500 × g for 10 min at 4 °C, and the supernatant was removed and
recentrifuged at 20,000 × g for 20 min at 4 °C. The
resulting pellet containing ER, pre-Golgi, and Golgi membranes was
resuspended in 10 mM Hepes (pH 7.2) and 250 mM
mannitol and employed in the binding reaction, as described previously
(5, 17). For some experiments, the membranes were washed with 1 M KCl in 10 mM Hepes, pH 7.2, for 15 min on ice
to remove peripherally associated proteins. Microsomes were recovered
by centrifugation at 20,000 × g for 20 min at 4 °C.
Membranes (30 µg of total protein) were added to a reaction mixture
that contained 27.5 mM Hepes (pH 7.2), 2.75 mM
MgOAc, 65 mM KOAc, 5 mM EGTA, 1.8 mM CaCl2, 1 mM ATP, 5 mM creatine phosphate, and 0.2 units of rabbit muscle creatine kinase. Recombinant Rab2, Rab2 13-mer, and affinity-purified anti-GAPDH were added at the concentrations indicated under
"Results," and the reaction mix was incubated on ice for 10-20
min. Rat liver cytosol (50 µg) and 2.0 µM GTP
S were
added, and the reactions were shifted to 37 °C and incubated for
5-10 min. The binding reaction was terminated by transferring the
samples to ice and then centrifuged at 20,000 × g for
10 min at 4 °C to obtain a pellet (P1). The supernatant (20,000 × g) was recentrifuged at 30 p.s.i. (~95,000 rpm)
for 30 min in an air centrifuge to recover released vesicles (P2). In
some cases, P2 was subjected to equilibrium density centrifugation, as
described previously (7, 18). P2 was mixed with 1 ml of 70% sucrose
and overlaid with 5 ml of a 30-50% sucrose. The gradient was
centrifuged at 50,000 rpm for 14 h at 4 °C, and 300-µl
fractions were collected from the bottom. The recovered fractions were
pelleted by ultracentrifugation (75,000 rpm for 30 min at 4 °C).
Both pellets from the binding assay (P1 and P2) were separated by
SDS-PAGE and transferred to nitrocellulose in 25 mM Tris
(pH 8.3), 192 mM glycine, and 20% methanol. The membrane
was blocked in Tris-buffered saline, which contained 5% nonfat dry
milk and 0.5% Tween 20, incubated with an affinity-purified polyclonal
antibody made to the EAGE peptide of
-COP (17) or a monoclonal
antibody to GAPDH (Biogenesis Inc., Sandown, NH) and then washed,
further incubated with a horseradish peroxidase-conjugated anti-rabbit
or anti-mouse antibody, developed with enhanced chemiluminescence (ECL)
(Amersham Pharmacia Biotech), and then quantitated by densitometry.
Purification of Recombinant Rab2 Protein and in Vitro
Prenylation--
Purification of recombinant Rab2 was performed as
described previously (7). Briefly, pET3A-Rab2 (Novagen, Madison, WI) was introduced into BL21 (DE3) pLysS (Novagen, Madison, WI). A 1-liter
culture was grown to an A600 of 0.4-0.5 and
induced with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h at
37 °C. The cells were centrifuged at 5000 × g for
10 min at 4 °C, and the cell pellet was resuspended in 50 mM Tris, pH 7.4, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM
benzamidine, 1 mM EDTA, and 1% Triton X-100 and then
homogenized by 20 passes with a Dounce tissue grinder. Lysozyme (400 µg/ml), DNase I (40 µg/ml), and 25 mM MgCl2
were added to the homogenate, allowed to digest for 30 min at 4 °C,
and then centrifuged at 22,000 × g for 30 min at
4 °C. The supernatant was applied to a 70-ml column containing Q
Sepharose Fast Flow (Amersham Pharmacia Biotech) equilibrated with
buffer A (50 mM Tris, pH 7.4, 10 mM
MgCl2, and 1.0 mM EDTA), washed with 2 bed
volumes of buffer A, and then eluted with a linear NaCl gradient
(0-400 mM) in buffer A. 3-ml fractions were collected, and
an aliquot of each fraction was separated by SDS-PAGE and immunoblotted
with a Rab2 polyclonal antibody. Rab2-enriched fractions were pooled,
concentrated, and applied to a 200-ml column containing Sephacryl S-100
(Amersham Pharmacia Biotech) and eluted with buffer A. Fractions
containing Rab2 were identified by SDS-PAGE and immunoblotting and then
pooled and concentrated. Typically, the protein prepared from this
procedure was ~90% pure. For use in the assays, the purified protein
was first prenylated in an in vitro reaction. The
isoprenylation reaction was performed in a total volume of 500 µl
that contained 5.0 µg of recombinant Rab2, 100 µg of geranylgeranyl
pyrophosphate (Sigma), 1 mM dithiothreitol, 250 µl of rat
liver cytosol, 10 mM MgCl2, 1 mM
ATP, 5 mM creatine phosphate, and 0.2 units of rabbit
muscle creatine kinase. The reaction was incubated for 1 h at
37 °C and then desalted through a 10-ml column of Sephadex G-25
(Amersham Pharmacia Biotech) to remove incompatible reagents that may
inhibit the in vitro assays. The fraction containing
prenylated Rab2 was collected and concentrated, and the protein
concentration was determined by Micro BCA Protein Assay Reagent
(Pierce). Routinely, 40-45% of the total Rab2 was prenylated as
determined by phase separation in Triton X-114 and 70-75%
active as determined by examining guanine nucleotide binding exchange properties (7).
Analysis of Transport in Vitro--
NRK cells were infected for
4 h with the temperature-sensitive VSV strain ts045 and then
biosynthetically radiolabeled with 100 µCi of Trans-35S
(specific activity of 1192 Ci/mmol, ICN Biomedicals, Irvine, CA) for 10 min at the restrictive temperature (39.5 °C) to accumulate the VSV-G
mutant protein in the ER. The cells were then perforated by swelling
and scraping and employed in the ER to cis/medial Golgi transport assay
as described (19). Briefly, transport reactions were performed in a
final volume of 40 µl in a buffer that contained 25 mM
Hepes-KOH (pH 7.2), 75 mM KOAc, 2.5 mM MgOAc, 5 mM EGTA, 1.8 mM CaCl2, 1 mM N-acetylglucosamine, an ATP-regeneration system (1 mM ATP, 5 mM creatine phosphate, and
0.2 IU of rabbit muscle creatine phosphokinase), 5 µl of rat liver
cytosol, and 5 µl of semi-intact cells (~5 × 107
cells/ml, ~25-30 µg of total protein) resuspended in 50 mM Hepes-KOH, 90 mM KOAc (pH 7.2). The
reactions were incubated at 32 °C and at the indicated time
(
t), supplemented with affinity-purified anti-GAPDH or
Rab2 (13-mer), incubated for a total of 60 min, and then transferred to
ice to terminate transport. Membranes were pelleted, solubilized in
buffer, and digested with endoglycosidase H (endo H). The samples were
analyzed by SDS-PAGE, and the fraction of ts045 VSV-G protein processed
to the endo H-resistant forms was quantitated by a Storm PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA).
Indirect Immunofluorescence--
NRK cells plated on coverslips
were permeabilized with digitonin (20 µg/ml), as outlined previously
(20). Coverslips with permeabilized cells were inverted and placed in
tissue culture wells that contained the transport mixture described
above, preincubated on ice for 20 min with or without 10 µg of
anti-GAPDH, and then incubated for 30 min at 32 °C. To terminate
transport, the cells were transferred to ice and fixed in 3%
formaldehyde/PBS for 10 min. Intracellular ts045 VSV-G was detected by
repermeabilization of the fixed cells with 0.05% saponin in PBS/normal
goat serum for 10 min, washed with PBS, and then incubated for 30 min
with a monoclonal antibody to VSV-G (P5D4) (Sigma). Cells were then washed with PBS, stained for 30 min with Texas Red anti-mouse antibody,
mounted, and viewed under a Zeiss Axiovert fluorescence microscope.
Generation of Anti-GAPDH Antibody--
Antibodies directed
against rabbit muscle GAPDH (Sigma) were produced in a New Zealand
White rabbit by subcutaneous injection with 1 mg of antigen emulsified
in complete Freund's adjuvant. Three biweekly immunizations of the
antigen were made in Freund's incomplete adjuvant. The serum was
applied to cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia
Biotech) to which rabbit muscle GAPDH was coupled for affinity
purification (21). The column was washed with five bed volumes of PBS,
eluted with 0.1 M glycine, pH 2.8, and then neutralized to
pH 7.2. The eluate was dialyzed against 25 mM Hepes, pH
7.2, 125 mM KOAc (25/125), and concentrated, and the
protein concentration was determined by the Micro BCA Protein Assay
Reagent (Pierce).
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RESULTS |
GAPDH Is Found on Vesicles Containing
-COP--
We
previously demonstrated that Rab2 promotes vesicle formation from
VTCs and that the released carriers contain Rab2,
-COP, p53/gp58,
and protein kinase C
/
(7, 22). To determine whether GAPDH was
also associated with these vesicles, we made use of a quantitative
binding assay. For this assay, microsomes were prepared from whole cell
homogenates and washed with 1 M KCl to remove peripherally
associated proteins. These membranes were preincubated in buffer for 20 min on ice in the presence of 300 ng of recombinant Rab2. This
concentration of Rab2 efficiently stimulates vesicle formation. The
reaction was then supplemented with rat liver cytosol and GTP
S and
incubated at 37 °C for 10 min to promote binding of soluble
components. To stop the reaction, membranes were pelleted by
centrifugation at 20,000 × g. The supernatant was
recovered from the reaction and then centrifuged at high speed (~95,000 rpm) to recover any vesicles released into the supernatant. This slowly sedimenting pellet was subjected to equilibrium density gradient centrifugation (7, 18). The gradient fractions were separated
by SDS-PAGE, and the presence of
-COP was determined by Western
blotting (Fig. 1). Consistent with our
previous results, membrane-associated
-COP peaked at ~42-43%
(w/w) sucrose, which is the expected density for coated vesicles. After
determining the distribution of
-COP, the blot was reprobed with a
monoclonal antibody to GAPDH (Fig. 1). This protein showed the same
gradient distribution as
-COP, suggesting that the enzyme is a
component of COPI-coated vesicles.

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Fig. 1.
GAPDH is associated with vesicles
containing -COP. Microsomes prepared from
NRK cell homogenates, as described under "Experimental Procedures,"
were preincubated with 300 ng of Rab2 for 10 min on ice. Cytosol and
GTP S were added, and the incubations were transferred to 37 °C
for 10 min to promote recruitment of soluble factors. Rapidly
sedimenting membranes were collected by centrifugation (20,000 × g for 10 min) to obtain a pellet (P1). The supernatant was
recentrifuged at 95,000 rpm for 30 min, and the resulting pellet (P2)
was subjected to equilibrium density centrifugation, as described under
"Experimental Procedures." The gradient was fractionated from the
bottom into 300-µl fractions, and the recovered fractions were
pelleted by ultracentrifugation (75,000 rpm for 30 min at 4 °C) and
then separated by SDS-PAGE and immunoblotted for -COP
(closed squares) and for GAPDH (open
circles). Vesicles released in response to incubation with
Rab2 contained both -COP and GAPDH. The results are representative
of three independent experiments.
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Rab2 Increases Membrane-associated GAPDH--
To learn whether
GAPDH was recruited from the cytosol to membrane in response to Rab2,
we measured membrane association of GAPDH in the microsomal binding
assay, described above. Salt-washed membranes were preincubated in
buffer for 20 min on ice in the presence or absence of increasing
concentrations of recombinant Rab2, which stimulate
-COP and protein
kinase C
/
membrane association (17, 22). The reaction was then
supplemented with rat liver cytosol and GTP
S and incubated at
37 °C for 10 min. Membranes were collected by centrifugation,
separated by SDS-PAGE, transferred to nitrocellulose, and then probed
with a monoclonal antibody to GAPDH. As shown in Fig.
2A, membranes incubated with
Rab2 showed a marked increase in GAPDH recruitment that occurred in a
dose-dependent manner and required GTP
S. The amount of
membrane-bound GAPDH increased ~10-fold when incubated with 50 ng of
Rab2. We then evaluated other Rab proteins in the microsomal binding
assay to determine whether recruitment of GAPDH was specific to Rab2
(Fig. 2C). Although Rab1 is required for ER to Golgi
transport, this protein failed to recruit GAPDH above the control
level. Likewise, the addition of 100 ng of Rab3 or 100 ng of Rab5 to
the binding assay had no effect on GAPDH binding.

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Fig. 2.
Rab2 stimulates recruitment of GAPDH to NRK
microsomes. Salt-washed NRK microsomes, prepared as described
under "Experimental Procedures," were preincubated with increasing
concentrations of recombinant Rab2 (A) or Rab2 13-mer
(B), 100 ng of the indicated recombinant Rab protein
(C), or a 50 µM concentration of the indicated
peptide (see Table I), for 20 min on ice. Cytosol and GTP S were then
added, and the membranes were incubated for 10 min at 37 °C. After
separation by SDS-PAGE and Western blotting, the transfer was probed
with a monoclonal antibody to GAPDH, washed, further incubated with
horseradish peroxidase-conjugated secondary antibody, and developed
with ECL. The amount of recruited GAPDH was quantitated by
densitometry. The results are the mean ± S.D. of four independent
experiments performed in duplicate.
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To determine whether GAPDH is directly recruited to membrane by Rab2 or
requires an additional cytosolic(s) component, the binding assay was
supplemented with a peptide made to the amino terminus of Rab2
(residues 2-14) (23). Like Rab2, this peptide has previously been
shown to require GTP
S, ADP-ribosylation factor, and protein kinase C
to stimulate
-COP recruitment to membrane (17). By using the peptide
(Rab2 13-mer), which binds directly to microsomes, it is not necessary
to generate purified Rab2-GDP dissociation inhibitor complexes to
deliver Rab2 to the membrane. Microsomes were preincubated with or
without increasing concentrations of Rab2 (13-mer) for 15 min to allow
binding of the peptide to membranes, in the absence of cytosol.
Purified GAPDH and GTP
S were then added and incubated for 10 min at
37 °C, after which the membranes were analyzed by SDS-PAGE and
immunoblotted to assess the level of membrane-bound GAPDH. As we
observed with Rab2 protein, the Rab2 peptide efficiently recruited
GAPDH to membrane in a dose-dependent manner (Fig.
2B). We made use of a battery of peptides employed in our
previous studies to learn whether GAPDH recruitment was specific to the
Rab2 (13-mer) (Table I). In contrast to
Rab2 (13-mer), the control peptidomimetics failed to stimulate GAPDH membrane binding at a comparable concentration (Fig. 2C).
These combined results suggest that the enhanced recruitment of GAPDH to membrane was specific to the Rab2 amino terminus.
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Table I
Peptides tested in microsomal binding assay
The membrane binding assay was performed as described under
"Experimental Procedures."
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Anti-GAPDH Arrests ER to Golgi Transport--
To assess the
biochemical role of GAPDH in transport of cargo between the ER and the
Golgi complex, we generated a polyclonal antibody to the enzyme. The
affinity-purified antibody detected purified GAPDH (Fig.
3A, lane
a) and an ~38-kDa protein that comigrated with GAPDH from
NRK cell lysates (Fig. 3A, lane b) and
from rat liver cytosol (Fig. 3A, lane
c). Since the antibody appeared to be specific to GAPDH, we
tested the reagent in an in vitro transport assay to
determine whether the antibody had an effect on the early secretory
pathway. For this assay, tissue culture cells are first infected with
ts045 VSV-G (VSV-G), a virus that synthesizes a protein with a
thermoreversible defect resulting in ER retention at 39.5 °C (24).
The plasma membrane of these cells is perforated to release soluble
content but retain functional ER and Golgi stacks (19). Incubation of
the perforated cells at the permissive temperature of 32 °C
initiates export of VSV-G from the ER in the presence of cytosol and
ATP. This semi-intact cell assay measures transport of VSV-G protein
from the ER to the cis/medial Golgi compartment by following the
processing of the two N-linked oligosaccharides to endo
H-resistant forms.

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Fig. 3.
Affinity-purified anti-GAPDH blocks ER to
Golgi transport, in vitro. A, rabbit
muscle GAPDH (2 µg) (lane a), NRK cell lysate (25 µg)
(lane b), and rat liver cytosol (25 µg) (lane
c) were separated by SDS-PAGE and then transferred to
nitrocellulose for Western blot analysis. The blot was probed with
affinity-purified anti-GAPDH prepared as described under
"Experimental Procedures." B, anti-GAPDH inhibits
transport of ts045 VSV-G from the ER to the Golgi complex. Semi-intact
NRK cells were incubated with the indicated concentration of
affinity-purified anti-GAPDH for 20 min on ice in a transport mixture
as described under "Experimental Procedures." The cells were then
transferred to 32 °C for a total of 60 min. The fraction of VSV-G
processed to the endo H-resistant forms (% of Total) was
determined after analysis by SDS-PAGE and fluorography. C,
anti-GAPDH inhibits transport downstream of Rab2 function but prior to
delivery of cargo to the cis Golgi complex. Semi-intact cells were
incubated at 32 °C in a transport mixture described under
"Experimental Procedures." At the indicated time ( t),
the control (open circle) was transferred to ice,
or 10 µg of anti-GAPDH (closed square) or 75 µM Rab2 13-mer (closed triangle)
was added and incubated with the cells for a total of 60 min. The
fraction of VSV-G processed to endo H-resistant forms was determined as
described above. Results shown are representative of three independent
experiments.
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Preincubation of cytosol with affinity-purified antibody to GAPDH led
to a dose-dependent inhibition of ER to Golgi transport (Fig. 3B). The processing of VSV-G to endo H-resistant forms
was reduced by 50% when the assay was supplemented with 2 µg of
antibody. Transport of VSV-G was almost completely arrested in the
presence of 10-15 µg of antibody. We then determined the kinetics of
inhibition of anti-GAPDH relative to the block imposed by the addition
of Rab2 (13-mer). Semi-intact ts045-infected NRK cells were incubated at 32 °C and at the indicated time (
t), transferred to
ice (control), or supplemented with 10 µg of anti-GAPDH or with 75 µM Rab2 (13-mer) and then incubated for a total of 60 min. This protocol allows any VSV-G that has migrated past the GAPDH-
or Rab2-requiring step to continue migration to the cis Golgi
compartment. Therefore, the fraction of VSV-G processed to endo H
resistance will depend on the amount of the reporter molecule that has
transported beyond the GAPDH- and the Rab2-sensitive steps at the time
of their addition. In control cells, VSV-G endo H-resistant forms were
detected after a 15-min lag (Fig. 3C). Within ~20 min of incubation,
50% of the VSV-G protein was processed, indicating migration to the
Golgi complex (Fig. 3C). In contrast, cells treated with
anti-GAPDH were blocked in transport ~5-7 min before the processing
of VSV-G to endo H-resistant forms, and by 20 min of incubation >60%
of the VSV-G had transported through the anti-GAPDH-sensitive step. This temporal site of inhibition by anti-GAPDH occurs immediately following the block that occurs when cells are incubated with Rab2
(13-mer). In both cases, the block in transport by anti-GAPDH and Rab2
(13-mer) preceded the processing of VSV-G to the endo H-sensitive form
by ~5-10 min. It appears that GAPDH acts downstream of Rab2 but is
required prior to delivery of cargo to the cis-Golgi.
To define the morphological site of VSV-G accumulation in response to
anti-GAPDH, NRK cells were infected with ts045 VSV-G for 2 h at
the nonpermissive temperature to restrict VSV-G to the ER. The cells
were then permeabilized and incubated in the presence or absence of
anti-GAPDH for 40 min at 32 °C, and the distribution of VSV-G was
determined by indirect immunofluorescence. Control cells efficiently
transported the reporter molecule to the Golgi complex (Fig.
4B). Consistent with the
biochemical data, cells incubated with anti-GAPDH failed to transport
VSV-G to the Golgi complex. VSV-G accumulated in a collar-like
structure composed of small vesicular elements that ringed the nucleus.
This staining pattern is similar to that observed when cells are
incubated with a constitutively activated form of Rab2 (Rab2 Q65L),
which causes vesiculation of VTCs (7). In the absence of VTCs, there is no target compartment for the newly budded ER vesicles to transport to
and fuse, resulting in the accumulation of the reporter molecule in the
ER and ER-derived carriers. These morphological results suggest that,
like Rab2 Q65L, GAPDH plays a role in protein recycling from
pre-Golgi intermediates.

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Fig. 4.
NRK cells incubated with anti-GAPDH fail to
transport ts045 VSV-G to the Golgi complex. NRK cells grown
on coverslips were infected with ts045 VSV-G for 3 h at 39.5 °C
to retain ts045 VSV-G in the ER (A). The cells were rapidly
shifted to ice, permeabilized with digitonin, and then incubated in a
complete transport mixture in the absence (B) or presence of
10 µg of anti-GAPDH (C) for 40 min at 32 °C. The
distribution of ts045 VSV-G was determined as described under
"Experimental Procedures." The arrows indicate reporter
protein transported to the Golgi complex.
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Anti-GAPDH Blocks Recruitment of GAPDH to Membrane--
The
affinity-purified anti-GAPDH antibody was further characterized in the
microsomal binding assay to determine the mechanism by which the
reagent blocks ER to Golgi traffic. We next examined whether the
antibody arrested transport by interfering with Rab2-mediated recruitment of GAPDH to membrane. Alternatively, the antibody may
interfere with a GAPDH-mediated membrane-associated event that is
essential for transport. To address the first possibility, cytosol was
preincubated with anti-GAPDH on ice for 20 min, and then GTP
S and
salt washed microsomes were added in the presence or absence of
recombinant Rab2 and incubated for 10 min at 37 °C. As observed
previously, Rab2-treated membranes showed enhanced recruitment of GAPDH
to membrane (Fig. 5A).
However, the ability of Rab2 to stimulate membrane association of GAPDH
was blocked when cytosol was pretreated with anti-GAPDH. Membrane-bound
GAPDH was reduced to nearly the control level in the presence of 10 µg of anti-GAPDH (Fig. 5A). The effect of the antibody
could be neutralized. We performed the binding assay as above, in the
presence or absence of pure GAPDH. Fig. 5B shows that 5 and
10 µg of GAPDH acted as competitive inhibitors of the antibody and
restored the ability of Rab2 to stimulate GAPDH recruitment. These
results indicate that anti-GAPDH blocks GAPDH membrane binding and
provide further evidence that the affinity-purified antibody
specifically recognizes the enzyme.

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Fig. 5.
Anti-GAPDH blocks Rab2 GAPDH recruitment to
membrane. A, salt-washed NRK cell microsomes prepared
as described under "Experimental Procedures" were preincubated with
or without 100 ng of recombinant Rab2 for 10 min on ice. Cytosol or
cytosol pretreated with 4 or 8 µg of anti-GAPDH and GTP S was then
added, and the reactions were incubated for 10 min at 32 °C. The
membranes were pelleted, separated by SDS-PAGE, and transferred to
nitrocellulose, and the blot was probed with a monoclonal antibody to
GAPDH. After incubation with horseradish peroxidase-conjugated
secondary antibody, the blot was developed with ECL, and the amount of
membrane-associated GAPDH was quantitated by densitometry.
B, salt-washed NRK microsomes that were preincubated in the
presence or absence of 100 ng of Rab2 for 20 min on ice received either
untreated cytosol or cytosol supplemented with 5 or 10 µg of rabbit
muscle GAPDH and 8 µg of anti-GAPDH and then shifted to 32 °C and
incubated for 10 min. The level of membrane-associated GAPDH was
quantitated, as described above.
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GAPDH Is Not Required for Vesicle Formation--
After finding
that anti-GAPDH prohibited Rab2-stimulated GAPDH recruitment to
membrane, we wanted to determine whether the enzyme was required for
vesicle formation. To address this question, we performed the binding
assay as above supplemented with an inhibitory concentration of
anti-GAPDH (8 µg). The low speed supernatant was recovered and then
recentrifuged at high speed to recover released vesicles, and the
resulting pellet (P2) was subjected to SDS-PAGE and Western blotting.
As we previously observed, Rab2 stimulated formation of vesicles
containing
-COP (Fig. 6A)
and GAPDH (Fig. 6B). We found that microsomes treated with
anti-GAPDH released vesicles containing
-COP into the supernatant
(Fig. 6A). However, these vesicles did not contain GAPDH
(Fig. 6B). Although GAPDH is recruited to VTCs by Rab2 and
is found on COPI carriers, the enzyme is not required for vesicle
budding.

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Fig. 6.
Anti-GAPDH does not interfere with
Rab2-stimulated vesicle budding. Microsomes prepared from NRK cell
homogenates, as described under "Experimental Procedures," were
preincubated with 300 ng of Rab2 for 10 min on ice. Cytosol and GTP S
were added, and the incubations were transferred to 37 °C for 10 min
to promote vesicle budding. Rapidly sedimenting membranes were
collected by centrifugation (20,000 × g for 10 min) to
obtain a pellet. The supernatant was recentrifuged at 95,000 rpm for 30 min, and the resulting pellet (P2) was then separated by SDS-PAGE and
immunoblotted for -COP (A) and for GAPDH (B).
Vesicles released in response to incubation with Rab2 and anti-GAPDH
contained -COP and a trivial amount of GAPDH.
|
|
 |
DISCUSSION |
The ability to sort anterograde from retrograde transported
proteins and retrieve escaped ER-resident proteins exists within pre-Golgi intermediates and throughout the Golgi stack (25, 26).
However, the mechanism(s) for this segregation event(s) is unknown.
Because protein traffic is bidirectional, it is likely that vesicles
possess a distinct molecule or set of molecules that labels the vesicle
as either a forward shuttle or a recycling intermediate. Membrane
proteins that possess the dilysine motif (KKXX) at their
carboxyl terminus are examples of targeting/sorting molecules that play
a role in vesicle retrieval (27-29).
Our previous studies on Rab2 demonstrated a role for this protein in
driving the formation of COPI vesicles enriched in the recycling
protein p53/gp58 (KKXX motif) but lacking
anterograde-directed cargo. In this study, the polypeptide composition
of the vesicles was further analyzed with the thought that proteins
associated with the carriers might be part of the retrieval machinery
that functions in targeting/docking/fusion of recycling vesicles to the
ER. GAPDH was detected on COPI vesicles and found to be actively recruited from the cytosol to membrane by Rab2.
GAPDH is a tetrameric enzyme consisting of four chemically identical
subunits. The structure and enzymatic activity of this protein have
been thoroughly investigated (12). In addition to its catalytic
function, GAPDH is involved in diverse biological activities. One of
these activities includes a role in endocytosis. Robbins et
al. (11) reported that a single amino acid change in GAPDH
(Pro234) rendered cells defective in endocytosis. Chinese
hamster ovary cells expressing the mutant GAPDH showed decreased
accumulation and altered distribution of an endocytic tracer. To
determine whether GAPDH activity was also required in the secretory
pathway, an affinity-purified polyclonal antibody to GAPDH was
introduced into an assay that reconstitutes ER to Golgi transport. The
reagent efficiently inhibited transport, indicating that GAPDH was an essential component of the trafficking machinery. GAPDH activity is
required after Rab2 recruitment to VTCs but prior to cargo delivery to
the cis Golgi compartment. Because anterograde and retrograde transport
are coupled, the block in ER to Golgi transport is most likely due to
the formation of "dead end" retrograde carriers (GAPDH negative)
that cannot transport and fuse to the ER. In this situation, components
are not retrieved from VTCs that are necessary for cargo transport from
the ER, resulting in the arrest of VSV-G transport.
Interestingly, the GAPDH from the Chinese hamster ovary mutant cells
described above, also showed altered microtubule (MT) binding
properties (11). GAPDH is known to modulate the cytoskeleton by
promoting actin polymerization and MT bundling (13, 30). This change in
the cell architecture could have profound consequences, since MTs
control both organelle positioning and transport of vesicular and
tubular elements, an activity that requires the motor proteins kinesin
and dynein. Kinesin has been found on pre-Golgi intermediates and
constitutively cycles between the ER and Golgi complex, implicating
cytoskeletal involvement in retrograde trafficking (31). In the
secretory pathway, GAPDH may function to promote the interaction of
Rab2-generated retrograde vesicles with MTs. This would allow the
vesicle to be sorted from anterograde traffic and redirected to the ER.
GAPDH might work in unison with MT by providing energy and coordinating
motile processes for vesicle movement. Although MT and actin assembly
do not appear to be directly regulated by small GTPases, Rab proteins
may provide a molecular link for vesicle movement to the appropriate
target. In that regard, Peranen et al. (32) showed a
striking change in actin and microtubule organization when cells
overexpressed a constitutively activated form of Rab8.
We found that membrane-associated GAPDH was not essential to vesicle
budding, yet protein trafficking was arrested in the presence of
anti-GAPDH. This results suggests that the GAPDH associated with
Rab2-generated vesicles may be required for fusion of the retrograde
carrier to the target compartment. In support of this interpretation,
Lopez Vinals et al. reported that rabbit muscle GAPDH was a
potent fusogen of negatively charged liposomes (33). Later studies by
Glaser and Gross (34) found that an isoform of GAPDH isolated from
rabbit brain cytosol promoted fusion of vesicles containing
plasmenylethanolamine, cholesterol, and phosphatidylserine. The
absolute requirement for plasmenylethanolamine in the liposome was
thought to be due to the ability of this lipid to adopt an inverted
hexagonal phase, which promotes membrane fusion. Although fusion
activity in this system was calcium-independent, the investigators pointed out that calcium might serve to modulate GAPDH activity. Subsequent studies from this laboratory have recently shown that GAPDH
catalyzes membrane fusion between isolated pancreatic secretory granules and fractionated plasma membrane in vitro (35). In contrast to the above studies, Hessler et al. using human
neutrophil cytosol have found a calcium-dependent fusogenic
activity toward vesicles containing
phosphatidylethanolamine/phosphatidic acid (15). The fusogen in this
system was also identified as GAPDH. It may be that the ability of
GAPDH to promote fusion in a calcium-dependent versus calcium-independent manner simply reflects the
participation of a unique GAPDH isoform. The different isoforms might
function within intracellular compartments that provide the appropriate lipid content. This argument is consistent with the finding that multiple GAPDH isoforms exist within a cell/tissue (11, 34).
We propose that Rab2 functions in the early secretory pathway to
regulate vesicular traffic through a subcompartment within pre-Golgi
intermediates where recycling proteins have been sorted and
concentrated away from anterograde-directed cargo. In this model, Rab2
initiates a cascade of events that results in recruitment of soluble
factors including GAPDH to ultimately release vesicles enriched in
recycling components. The GAPDH associated with the retrograde vesicle
may interact with microtubules to direct movement or, alternatively,
promote fusion of the vesicle to the ER. Experiments are in progress to
distinguish between these possibilities.