From the Department of Biochemistry, University of
Kentucky College of Medicine, Lexington, Kentucky 40536 and the
¶ Roy M. and Phyllis Gough Huffington Center on Aging, Departments
of Molecular and Cellular Biology and Dermatology, Baylor College of
Medicine, Veterans Affairs Medical Center, Houston, Texas 77030
Received for publication, October 16, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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The role of The soluble
N-ethylmaleimide-sensitive factor (NSF)
1 attachment
protein family ( Biochemical assays indicate that Materials--
Antibodies to the following proteins were
obtained commercially: anti-
The cDNA encoding the full-length Gaf-1 (KIAA0857) was obtained
from the Kazusa DNA Research Institute, Japan (27). Recombinant proteins were produced by inserting coding regions for Construction of the Yeast Two-hybrid cDNA
Library--
Poly(A)+ mRNA (5 µg) from IIB-Mel-J
cells (28, 29) was used to prepare cDNAs fragments with an
EcoRI site at their 5' ends and a XhoI site at
their 3' ends by standard procedures. The cDNA fragments of
0.5-3.0 kilobase pairs were cloned into the HybriZAP yielding 3 × 106 independent plaques, more than 90% of which contain
a cDNA insert. A phagemid cDNA library with the cDNAs fused
to the Gal4 activation domain was isolated from the Yeast Two-hybrid System--
The yeast strain PJ-X, which
carries HIS-3, ADE-2, and LacZ
reporter genes, was used for library screening. The cDNA encoding Expression of GFP Fusion Proteins in HEK-293 Cells--
All
cells were grown in DMEM with 10% fetal bovine serum (FBS),
penicillin, and streptomycin at 37 °C in 10% CO2. To
produce stable transfectants, HEK-293 cells were plated at 2 × 106 cells/10-cm plate 1 day prior to transfection, which
was done using the calcium phosphate precipitate method. Two days after transfection, the cells were replated under selective conditions (100 µg/ml Geneticin, Life Technologies, Inc.) and stable, drug-resistant cells were recovered after a month of continuous selection. The subsequent mixture of stable transfectants was used for experiments without further cloning. For transient transfection experiments, HEK-293 or HepG-2 cells were first plated onto laminin-coated coverslips, transfected as above, and then examined by epifluorescence microscopy 2 days after transfection.
Immunofluorescence Analysis--
Cells were plated onto
laminin-coated coverslips (5 µg/ml) at least 1 day prior to analysis.
Coating of the coverslips was essential for the reproducible appearance
of the reticular phenotype seen in the GFP- Miscellaneous Methods--
Metabolic labeling of HepG-2 cells
was done in methionine-free DMEM with 10% FBS supplemented with 500 µCi/ml [35S]methionine (400 Ci/mmol, ICN, Costa Mesa,
CA) for 8 h. Metabolic labeling with
[32P]PO4 (carrier-free, ICN, Costa Mesa, CA)
was done in phosphate-free DMEM, 10% FBS, supplemented with 200 µCi/ml for 6 h. In both cases the labeled cells were harvested
on ice and then solubilized with 0.5% Triton X-100 in 25 mM Hepes/KOH, pH 7.0, 38 mM KCl, 108 mM NaCl, 1 mM dithiothreitol, 10 mM
EGTA, 1 mM o-phenanthroline, 1 mM
leupeptin, 1 mM benzamidine, 40 µg/ml chymostatin, 40 µg/ml antipain, 0.12 unit/ml aprotinin, 1 µM pepstatin,
and 1 mM sodium o-vanadate. The radiolabeled
proteins were detected by either autoradiography (for
32PO4 label) or autofluorography (for
35S label) using Amplify (Amersham Pharmacia Biotech) as
fluor. Subcellular fractionation of HepG-2 cells was performed
according to the methods of Lutsenko and Cooper (32). Cells were
harvested on ice and disrupted by 10 passes through a Balch-style ball
homogenizer (33). The fractions were then recovered by differential
centrifugation and analyzed by Western blot. The mitochondrial
fraction, P2, was further analyzed by protease digestion using 0.1 µg/ml proteinase K for 20 min on ice. After addition of phenylmethane
sulfonyl fluoride, to a final concentration of 1 mM, to
stop the reaction, the digested mitochondria were subjected to analysis
by Western blot (34).
Identification of
Initial binding studies with recombinant proteins suggested that the
interaction between
The clones for Gaf-1 were incomplete and contained only the C terminus
of a larger protein. A search of the data base identified three
homologous mouse expressed sequence tags, all of which lacked an N
terminus. One human clone, KIAA0857 (accession no. AB020664) identified
by Nagase et al. (27) contained an open reading frame that
encoded a 733-amino acid protein; however, when the first methionine
(with appropriate Kozak sequence (Ref. 37)) was used as the start site,
the open-reading frame encoded a protein of 653 amino acids (38). This
protein's C terminus (amino acids 490-653) was identical to and in
frame with the Gaf-1 clone recovered in our screen. Subsequent Western
blotting studies with anti-C-terminal Gaf-1 antibodies detected a
single 75-kDa protein (Figs. 2 and 3). From this we concluded that the
KIAA0857 clone encoded the full-length Gaf-1 protein. Sequence analysis
(using Prosite) of Gaf-1 showed it to be a hydrophilic protein with
numerous potential recognition sites for cAMP-dependent
protein kinase, protein kinase C, and casein kinase II. There is a
P-loop motif commonly found in nucleotide-binding proteins and a
calcium-binding, C2, motif similar to that found in protein kinase C
and synaptotagmins. Although the N-terminal domain may be significant
for Gaf-1 binding to membranes (see below), no ATP binding has been
detected (data not shown). Gaf-1 appears to be identical to a protein
called pp75, which was identified in a two-hybrid screen using the
SS-A/Ro autoantigen as bait (38).
Characterization of the Gaf-1- Gaf-1 Distribution and Localization--
Northern blot analysis by
Nagase et al. (27) and Wang et al. (38) suggested
that Gaf-1 was ubiquitously expressed in mammalian tissues as is
The subcellular distribution of Gaf-1 was first addressed by indirect
immunofluorescence. Initial experiments showed that Gaf-1 staining
resulted in a punctate and tubular pattern that resembled mitochondria
but not endosomes or lysosomes (Fig. 4). This localization was confirmed by costaining with the mitochondrial specific vital stain, Mitotracker. In HEK-293 (Fig. 4,
A-C), Chinese hamster ovary (Fig. 4, D-F),
HepG2 (Fig. 4, G-I), HeLa, and Vero cells (data not shown),
Gaf-1 immunostaining colocalized with Mitotracker staining (Fig. 4,
A, D, and G). No immunostaining was
seen when the antibody was first incubated with recombinant Gaf-1 or
when primary antibody was omitted (data not shown). Interestingly, anti-
To confirm the subcellular distribution of Gaf-1, HepG2 cells were
subjected to subcellular fractionation by differential sedimentation
(32). The five membrane fractions and one supernatant fraction were
probed with various antibodies to evaluate their purity and the
distribution of Gaf-1 and
The Gaf-1 sequence indicates that it is a hydrophilic protein; however,
the fractionation experiments above suggest that it is
membrane-associated. To further characterize Gaf-1, total cellular membranes, from HepG2 cells, were extracted with 1 M KCl,
100 mM NaCO3, or 1% Triton X-100, fractionated
by sedimentation and then supernatant and pellet fractions were probed
for Gaf-1. Gaf-1 is partially released from membranes by high salt and
completely released by Na2CO3 and Triton X-100
(Fig. 5B). These data demonstrate that Gaf-1 is peripherally
associated with membranes but is not an integral membrane protein.
Since Gaf-1 was associated with mitochondria, we next asked whether it
was on the outer mitochondrial membrane. For this experiment, freshly
prepared P2 fraction (Fig. 5A) was incubated with proteinase
K and then probed for Gaf-1 and cytochrome c oxidase by
Western blotting. Although cytochrome c oxidase, a protein
exposed to the inter mitochondrial membrane space, is protected from
proteinase K, Gaf-1 is almost completely sensitive to the protease
(Fig. 5C). In summary, these data show that Gaf-1 is
peripherally associated with the cytosolic face of the outer
mitochondrial membrane.
To further analyze this phenotype, stable transformants were generated
using selection with G418. These transformants have retained the
reticular phenotype for more than 10 months in culture. Mitochondria
appear to colocalize with the reticular distribution of GFP-
Given the appearance of the GFP fusion protein distribution, it seemed
likely that it might colocalize with one of the cytoskeletal elements
in HEK-293 cells. Stable transformants expressing GFP-
The colocalization of In this report, we have attempted to identify novel cellular roles
for The subcellular distribution of Gaf-1 was surprising based on the
proposed roles for Ectopic protein expression studies were used to determine the cellular
role of The reticular distribution of In summary, our data clearly demonstrate that /
-SNAP (Soluble NSF Attachment
Protein) in vesicular trafficking is well established; however, the
function of the ubiquitously expressed
-SNAP remains unclear. To
further characterize the cellular role of this enigmatic protein, a
two-hybrid screen was used to identify new,
-SNAP-binding proteins
and to uncover potentially novel functions for
-SNAP. One such
SNAP-binding protein, termed Gaf-1 (
-SNAP associate factor-1)
specifically binds
- but not
-SNAP. The full-length Gaf-1 (75 kDa) is ubiquitously expressed and is found stoichiometrically
associated with
-SNAP in cellular extracts. This binding is distinct
from other SNAP interactions since no
-SNAP or NSF coprecipitated
with Gaf-1. Subcellular fractionation and immunofluorescence analysis
show that Gaf-1 is peripherally associated with the outer mitochondrial membrane. Only a fraction of
-SNAP was mitochondrial with the balance being either cytosolic or associated with other membrane fractions. GFP-
-SNAP and the C-terminal domain of Gaf-1 both show a
reticular distribution in HEK-293 cells. This reticular structure
colocalizes with Gaf-1 and mitochondria as well as with microtubules
but not with other cytoskeletal elements. These data identify a class
of
-SNAP interactions that is distinct from other members of the
SNAP family and point to a potential role for
-SNAP in mitochondrial dynamics.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-,
-, and
-SNAP) is thought to function in vesicular transport by mediating the binding of the NSF to
the SNAP receptors (SNAREs) (reviewed in Refs. 1-4). In this sequence,
SNAPs are important adaptors, which recruit NSF to the SNARE
heterotrimer, thereby forming the so-called 20 S complex (5-7). SNAPs
stimulate NSF's ATPase (8, 9), which is required to disassemble
cis-SNARE heterotrimers, thereby activating the individual
SNAREs for trans complex formation and subsequent membrane
fusion (10-12). Based on sequence analysis,
-SNAP is the least
homologous member of the SNAP family (25% identical to
-SNAP; Refs.
13 and 14).
-SNAP, due to its high sequence identity (83% identical
to
-SNAP) and its similar activity, is thought to function as a
brain-specific isoform of the more ubiquitous
-SNAP (13, 15).
-SNAP, like
- and
-SNAP, can partially restore intercisternal
transport activity to salt-extracted Golgi membranes (16, 17), although
in this assay,
-SNAP has a much lower specific activity. All three
SNAPs facilitate NSF binding to membranes and to hydrophobic surfaces
(16) and, once bound, can stimulate NSF's ATPase activity (8).
-SNAP had the greatest effect in this assay, perhaps because it
binds better to the plastic surface used. In other systems,
-SNAP
augments neurotransmission from squid giant axons (18) and
catecholamine release from chromaffin cells (19).
-SNAP is also
required for Golgi fragment reassembly following illiquinone treatment
(20). However, no role for
-SNAP is detected in endoplasmic
reticulum to Golgi transport (21). These data suggest a role for
-SNAP in membrane trafficking processes, but, unlike
-SNAP, its
requirement in these processes is less well documented.
-SNAP may not serve the same
function as does
- or
-SNAP.
-SNAP can activate the ATPase activity of NSF when it is initially bound to a hydrophobic surface (8), and can stabilize the 20 S complex formation (5). However,
-SNAP cannot replace
-SNAP in 20 S complex formation assays nor
does it compete with
-SNAP for membrane binding
(22)2 Unlike
-SNAP,
-SNAP is not released from endosomes in an
NSF/ATPase-dependent manner (23) consistent with an
alternative type of binding interaction between
-SNAP and membranes.
This is further supported by data showing that
-SNAP binding to
membranes has a different binding kinetic and temperature profile than
did
-SNAP binding (22). In the present study, we have attempted to
further characterize
-SNAP by identifying
-SNAP-binding proteins.
Using a yeast two-hybrid screen, we have detected two proteins that
specifically interact with
-SNAP: cytosolic thiolase (24) and a
protein denoted
-SNAP associated factor-1 (Gaf-1). Only Gaf-1
significantly colocalized and coimmunoprecipitated with
-SNAP.
Additional studies using over-expression of GFP-
-SNAP resulted in
the appearance of a reticular network of
-SNAP that partially
colocalized with Gaf-1 and mitochondria. This network also colocalized
with
-tubulin in these cells and partially with
-tubulin, but not
with vimentin or cytokeratins. These findings describe a new
interaction for the ubiquitous
-SNAP, which is unlike other SNAP
family members, and suggest a potentially novel function for
-SNAP
in connecting mitochondria with cytoskeletal elements.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin (clone B-5-1-2),
-tubulin
(clone TUB 2.1), and
-tubulin (clone GTU-88), anti-actin (clone
AC-40), anti-vimentin (clone V9), anti-cytokeratin (mixture of clones
C-11, PCK-26, CY-90, KS-1A3, M20, andA53-B/A2), and
anti-Golgi 58K (25) (Sigma), anti-bovine ubiquinol-cytochrome
c oxidoreductase 1 subunit (cytochrome c oxidase,
clone 13G12-AF12 BB11; Molecular Probes, Eugene, OR), anti-cathepsin D
(Biomeda, Foster City, CA), and anti-EEA1 (Transduction Laboratories,
Lexington, KY). Dr. T. Vanaman (University of Kentucky, Lexington, KY)
generously provided anti-plasma membrane Ca2+ ATPase
(PMCA). Dr. Anthony Sinai (University of Kentucky) generously provided
the anti-calnexin antibody (26). The anti-
-SNAP antibody has been
described previously (22). Polyclonal antibodies recognizing Gaf-1
(amino acids 490-653) and cytosolic thiolase were generated under the
supervision of the Department of Laboratory Animal Resources (University of Kentucky). Secondary fluorescent antibodies to mouse and
rabbit IgG were from Vector Laboratories (Burlingame, CA). The
biotin/avidin kit for immunofluorescence amplification was from Vector
Laboratories. Laminin and collagen for coating coverslips were from Sigma.
-SNAP,
-SNAP, Gaf-1, C-terminal fragment of Gaf-1 (C-Gaf-1), or thiolase into pProEX-HTa (Life Technologies, Inc.) for His6-tagged
protein expression or pGEX-KG (Amersham Pharmacia Biotech) for
glutathione S-transferase fusion proteins. GFP fusion
protein expression constructs were produced using the pEGFP-C1 vector
from CLONTECH (Palo Alto, CA). Mouse tissues were
homogenized and solubilized in SDS-PAGE sample buffer.
Glutathione-agarose beads were from Sigma. All other materials were of
reagent grade.
HybriZAP
library by in vivo excision with Exassist helper phage
according to the manufacturer's recommendation (Stratagene, La Jolla, CA).
-SNAP was cloned into the pGBT9 "bait" vector and confirmed by dideoxy nucleotide sequencing. Expression of the bait fusion protein (Gal4 BD-
-SNAP) was confirmed by Western blotting using anti Gal4-DBD monoclonal antibody (CLONTECH). PJ-X
transformants containing the "bait" plasmid pGBT9-
-SNAP were
transformed with a two-hybrid cDNA library from IIB-Mel-J melanoma
cells using the lithium acetate method (30). Transformants were plated
on a selective synthetic dextrose medium lacking adenine, tryptophan,
and leucine. Colonies that grew in the presence of 3-aminotriazole (5 mM) and in the absence of tryptophan, leucine, adenine, and
histidine were further analyzed for
-galactosidase activity by a
liquid assay (31). cDNA inserts derived from triple positive
(Ade+, His+, LacZ+) yeast colonies
were tested for bait specificity by retransformation with different
Gal4-DBD fusion proteins and pAS2-1 expressing the Gal4-DBD only.
-SNAP-expressing cells
(see Figs. 6 and 7). Similar results were also seen when collagen (5 µg/ml) was used. For Mitotracker Red (CM-H2-Xros,
Molecule Probes, Eugene, OR) staining, cells were incubated in 0.5 µM dye for 1 h prior to fixation in cold (
80 °C) methanol. Mitotracker can stain endoplasmic reticulum when
used at higher concentrations, but such spurious staining has not been
seen when 0.5 µM Mitotracker is used. For immunostaining, the cells, plated on coverslips, were fixed with cold methanol and then
blocked with PBS containing 5% goat, 5% horse serum for 20 min.
Incubations with primary and fluorescent secondary antibody dilutions
(in blocking solution) were done for 20 min with four intervening
washes. For immunofluorescence analysis using the anti-
-SNAP
antibody, a biotinylated secondary antibody and fluorescent avidin kit
(Vector Laboratories) was used to amplify the signal. The coverslips
were mounted with Vectashield (Vector Laboratories) and examined with
an Eclipse E600 epifluorescence microscope (Nikon, Melville, NY). The
images were digitized with a Spot CE camera (Diagnostic Instruments,
Sterling Heights, MI) using the Metaview software package (Universal
Imaging Corporation, West Chester, PA). Figs. were created using
Photoshop 5.5 (Adobe, San Jose, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-SNAP-binding Proteins--
To uncover
potential roles for
-SNAP, we sought to first identify novel
-SNAP-binding proteins using a yeast two-hybrid screening approach.
The cDNA encoding full-length, human
-SNAP (35) was cloned into
the pGBT9 "bait" plasmid. This construct had no internal
transcription activity when cotransfected with the pGAD424 vector. A
screen of 1 × 107 Trp+Leu+
transformants yielded 61 colonies that grew in selective medium and
expressed
-galactosidase. A search of GenBankTM revealed that 22 of
the insert sequences were cytosolic thiolase (EC 2.3.1.9), and 32 were
the C-terminal domain of a protein we have called
-SNAP-associated
factor-1 (Gaf-1). The clones recovered for cytosolic thiolase were
full-length and identical to the clone previously reported by Song
et al. (24). Re-transformation assays showed that thiolase
and Gaf-1 fusion proteins interact specifically with the
-SNAP bait
because no interaction was detected with either Gal4-DBD alone or
Gal4-DBD-p53 (data not shown).
-SNAP and thiolase was not stable (lane 7, Fig.
1). Binding was not augmented by thiolase
substrates such as coenzyme A (50 µM) and acetoacetyl-CoA
(15 µM) or by a known thiolase activator, potassium ions
(up to 200 mM) (data not shown). Since
-SNAP had no
effect on recombinant thiolase activity as measured using the methods
of Fukao et al. (36) (data not shown), further
characterization of thiolase was not continued. C-Gaf-1 (amino acids
490-653) stably bound to GST-
-SNAP (Fig. 1, lane
10). This binding was specific; C-Gaf-1 did not bind to GST
or to GST-
-SNAP (Fig. 1, lanes 8 and
9). Since the Gaf-1/
-SNAP interaction was appreciably
more stable, we continued the characterization of Gaf-1 protein.
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Fig. 1.
Analysis of
-SNAP-interacting proteins by in vitro
binding. In vitro binding experiments done with
recombinant proteins generated from clones recovered in the two-hybrid
screen. Lanes 1-3 represent the total amounts of
protein added to the binding reactions. 20 µg of
His6-thiolase or His6-C-Gaf-1 were immobilized
on Ni2+-nitrilotriacetic acid-agarose beads and then
incubated, as indicated, with 10 µg of GST, GST-
-SNAP, or
GST-
-SNAP. The binding reactions were then washed five times in
binding buffer. The bound proteins were eluted with SDS-PAGE sample
buffer and analyzed by Western blotting.
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Fig. 2.
Gaf-1 binding to
-SNAP in vitro and in
vivo. Panel A, GSH-agarose beads alone or
bound to GST-
-SNAP (5 µg) or GST-
-SNAP (5 µg) were incubated
with His6-Gaf-1 (5 µg) at room temperature for 1 h.
The beads were washed with PBS, 0.5% Triton X-100 and the bound
material was eluted with SDS-loading buffer and subjected to Western
blotting by using antibody against C terminus of Gaf-1.
Panel B, GST-
-SNAP precipitates endogenous
Gaf-1 from liver cell extract. GSH-agarose beads alone and GST-
-SNAP
(5 µg) and GST-
-SNAP (5 µg) bound beads were incubated in the
presence of 100 µg of liver extract at room temperature for 1 h.
After the beads were washed, the bound material was eluted with
SDS-loading buffer and subjected to Western blotting by using antibody
against Gaf-1. Panel C, coimmunoprecipitation of
Gaf-1 and
-SNAP. 6 µg of pre-immune IgG or anti-C-terminal Gaf-1
antibody were incubated with 100 µg of liver extract and the
immunocomplexes were recovered on protein G-agarose beads. The beads
were washed, and the bound material was eluted with SDS-loading buffer
and subjected to Western blotting using antibodies against Gaf-1 and
-SNAP. Panel D, metabolic labeling experiment.
HepG2 cells were labeled with [35S]methionine and the
cell extract (200 µg, 1,000,000 cpm) was subjected to
immunoprecipitation using antibodies against Gaf-1 or pre-immune
antibody. The bound material was eluted with SDS-loading buffer, loaded
on SDS-PAGE gel, and detected by autofluorography.
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Fig. 3.
Gaf-1 is ubiquitously expressed.
Panel A, tissue distribution of Gaf-1. 100 µg
of extracts from different mouse tissues were loaded on SDS-PAGE gel
and subjected to Western blotting using antibody against the C terminus
of Gaf-1. Panel B, 100 µg of extracts from the
indicated cell lines were subjected to Western blotting using the same
antibody. Panel C, antibody against C-Gaf-1 is
specific. 100 µg of liver extract was subjected to Western blotting
using anti-C terminus of Gaf-1 antibody (control) and 200 µg of
His6-Gaf-1 pre-absorbed anti-C-Gaf-1 antibody.
-SNAP
Interaction--
Full-length recombinant Gaf-1 was produced in
Escherichia coli and tested for binding to
-SNAP. In Fig.
2A full-length Gaf-1, like the C-terminal fragment,
specifically bound to GST-
-SNAP but not to GST or to GST-
-SNAP.
Endogenous Gaf-1 also specifically bound
-SNAP. Pull-down
experiments, using detergent-solubilized extract from mouse liver
incubated with recombinant GST, GST-
-SNAP, or GST-
-SNAP, show
that Gaf-1 bound only to GST-
-SNAP (Fig. 2B). As a
control, the precipitated complexes were probed for NSF, which was
found associated with GST-
-SNAP but not with GST-
-SNAP (data not
shown). In Fig. 2C,
-SNAP was coimmunoprecipitated from
liver extracts with an anti-Gaf-1 antibody. Neither
-SNAP nor NSF
was present in these immunoprecipitates (data not shown). Finally, when
Gaf-1 was immunoprecipitated from [35S]methionine-labeled
HepG2 cells, only two major bands coprecipitated with the 75-kDa Gaf-1
protein (Fig. 2D). The lower band below 45 kDa is consistent
with the size of
-SNAP. The band at 66 kDa and a minor band at 110 kDa are yet to be identified. When Gaf-1 was immunoprecipitated from
HepG2 cells metabolically labeled with
[32P]PO4, no phosphorylated Gaf-1 or
-SNAP
was recovered (data not shown). From these experiments it is clear that
Gaf-1 specifically and stoichiometrically interacts with
-SNAP
in vitro as well as in vivo; however, neither
protein appears to be phosphorylated in HepG2 cells.
-SNAP (13). To confirm this we used an antibody against the
C-terminal domain of Gaf-1 to probe extracts from various mouse tissues
and cell lines. As shown in Fig. 3 (A and B),
Gaf-1 is detected as a single 75-kDa band in all tissues and cell lines
that were tested. Interestingly, unlike
-SNAP (13), there are some
differences in the level of Gaf-1 expression in the various tissue
tested, with adipose, heart, liver, and kidney showing the highest
expression. This immunodetection was specific since pre-incubation of
the antibody with recombinant Gaf-1 eliminated the signal (Fig.
3C).
-SNAP antibodies also faintly stained the mitochondria in HepG2
cells, although, because of the poor quality of the antibody, avidin-biotin amplification was needed to visualize the reticular pattern (Fig. 4, K-M). These immunofluorescence data
indicate the Gaf-1 and at least a portion of
-SNAP are localized to
the mitochondria in a number of cell-types.
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Fig. 4.
Gaf-1 is present on mitochondria.
HEK-293 (panels A-C), Chinese hamster
ovary (panels D-F), and HepG2
(panels G-L) cells were plated onto
laminin-coated coverslips 24 h prior to analysis by
immunofluorescence. One hour prior to fixation with cold ( 80 °C)
methanol, the cells were incubated with 0.5 µM
Mitotracker (panels A, D,
G, and J) for 1 h at 37 °C. After
fixation the cells were immunostained with anti-Gaf-1
(panels B, E, and H) or
anti-
-SNAP (panel K) antibody and then probed
with fluorescent secondary reagents, fluorescein isothiocyanate-labeled
anti-rabbit IgG (panels B, E, and
H) and biotinylated anti-rabbit IgG followed by fluorescein
isothiocyanate-labeled avidin (panel K). The images were
collected digitally and the overlapping images (panels
C, F, I, and L) were
generated using Adobe Photoshop 5.5. Initial images were recorded at
150-fold magnification in panels A-I and
250-fold magnification in panels J-M.
-SNAP (Fig.
5A). P1 contains nuclei and
unbroken cells. P2 contains mitochondria, and consistently cytochrome
c oxidase was found in that fraction. Cathepsin D, a marker
for lysosomes, was found in P3, and calnexin, a marker for endoplasmic
reticulum, was found in P3 and P4. Golgi (Golgi 58K; Ref. 25) and
endosomal (EEA1; Ref. 39) markers were found in P4, and PMCA, a marker
for plasma membrane, was found in P5. Cytosolic enzymes such as
cytosolic thiolase were found in the 100,00 × g
supernatant (S). Although the majority of Gaf-1 was present in the
mitochondrial P2, only a fraction of
-SNAP (~20%) was present in
this fraction. These data confirm the mitochondrial localization of
Gaf-1 determined by immunofluorescence. They additionally show that
-SNAP is present in multiple fractions, and not just the
mitochondria.
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Fig. 5.
Gaf-1 is peripherally associated with the
outer mitochondrial membrane. Panel A, HepG2
cells were fractionated by sequential, differential centrifugation.
Fifty micrograms of protein from each fraction were subjected to
Western blotting using antibodies against Gaf-1, cathepsin D, EEA1,
calnexin, Golgi 58K, cytosolic thiolase, -SNAP, PMCA, and
cytochrome c oxidase. Panel B, 50 µg
of the P2 fraction were washed separately with 1 M KCl, 100 mM Na2CO3 (pH 10.0), PBS, 0.5%
Triton and the membranes were sedimented at 100,000 × g for 30 min. The supernatants and the pellets were
subjected to Western blotting using antibody against Gaf-1.
Panel C, 50 µg of P2 fraction were treated with
0.1 µg/ml proteinase K in the absence or presence of 0.5% Triton
X-100 on ice for 20 min. The reaction was stopped by addition of
phenylmethylsulfonyl fluoride (1 mM) and analyzed by
Western blotting using antibodies to Gaf-1 and cytochrome c
oxidase.
-SNAP and
-Tubulin Coexist in a Complex--
To determine
the possible roles of the Gaf-1/
-SNAP interaction, we undertook a
series of transfection experiments. Initially HEK-293 cells were plated
onto laminin-coated coverslips and transiently transfected with
constructs that would express
-SNAP, C-Gaf-1 (amino acids 490-653),
N-Gaf-1 (amino acids 1-218), and full-length Gaf-1, all fused to GFP.
Healthy transformants were detected for both the
-SNAP and C-Gaf-1
constructs, but cells positive for N-Gaf-1 or full-length Gaf-1
expression were rounded and did not survive past 12 h after
transfection. Consistently, after multiple attempts we have not been
able to make stable transformants using either construct (GFP-Gaf-1 or
GFP-N-Gaf-1). Further analysis will be required to determine the exact
effect of Gaf-1 and N-Gaf-1 expression. Overexpression of
-SNAP
(Fig. 6, A, D, and
G) and C-Gaf-1 (Fig. 6M) gave a more interesting
phenotype that was quite unexpected. In both cases, many of the
positive cells (~30%) showed a reticular distribution of the GFP
fusion protein that resembled cytoskeletal elements or endoplasmic
reticulum (however, there was no colocalization with calnexin; data not
shown and see Fig. 5A). This type of reticular distribution was not
seen for GFP alone, for GFP-
-SNAP or for any other GFP fusion
proteins that we have expressed in HEK-293 cells (i.e.
-SNAP, SNAP-23, VAMP-3, VAMP-8; Ref. 40 and data not shown). Western
blotting analysis showed that GFP-
-SNAP expression in the
transformants was at least 10-fold higher than endogenous levels (data
not shown). One feature of this distribution is that it is dependent on
the matrix used to coat the coverslips. The reticular phenotype was seen in cells plated onto laminin- and collagen-coated coverslips but
not when cells were plated directly onto acid-washed glass coverslips.
This significance of the effect of the plating matrix is still under
investigation.
View larger version (52K):
[in a new window]
Fig. 6.
Overexpression of
-SNAP forms a reticular structure that colocalizes
with
-tubulin. Stable HEK-293
transformants expressing GFP-
-SNAP were analyzed by
immunofluorescence and vital staining. Cells were initially plated on
laminin-coated coverslips, fixed in cold methanol, and immunostained
with the indicated antibodies and relevant Texas Red-labeled secondary
reagents. In panels A-C, cells were
immunostained with anti-
-tubulin monoclonal antibodies. In
panels G and H, cells were
immunostained with anti-Gaf-1 polyclonal antibodies and in
panels J-L, cells were stained with
anti-
-tubulin monoclonal antibodies. In panels
D-F, the cells were pre-incubated with 0.5 µM
Mitotracker; and in panels N and O,
the cells were pre-incubated with 16 and 33 µM nocodazole
for 9 h, respectively. Panel M represents
the distribution of the GFP-C-Gaf-1 in HEK-293 cells. The images were
collected digitally and the overlapping images (panels
C, F, I, and L) were
generated using Adobe Photoshop 5.5. Initial images were recorded at
150-fold magnification.
-SNAP.
In Fig. 6 (D-F), the Mitotracker staining pattern in the
stable transformants was coincident with the GFP-
-SNAP distribution
and appeared to overlay the meshwork; however, the distribution of
mitochondria was not as extensive as that for GFP-
-SNAP. The
distribution of Gaf-1 in these cells was also not as extensive as that
of GFP-
-SNAP. Gaf-1 distribution was punctate and appeared to align
with selected regions of the GFP-
-SNAP network, as did the
Mitotracker staining. These data suggest that GFP-
-SNAP is binding
to (or causing the formation of) a cellular meshwork to which
mitochondria can associate. It should be noted that in no case was
Mitotracker and Gaf-1 staining seen in regions that did not also
contain GFP-
-SNAP.
-SNAP were
initially treated with either nocadazole (16 and 33 µM
for 9 h; Fig. 6, N and O) or acrylamide (10 mM for 9 h; data not shown) under conditions that
would disrupt microtubules or intermediate filaments, respectively
(41). Although the cells appeared rounded, there was no significant
degradation of the reticular distribution of the GFP fusion protein. To
extend this analysis, stable transformants were immunostained with
antibodies to various cytoskeletal proteins. GFP-
-SNAP did not
overlap with anti-actin, anti-cytokeratin, or anti-vimentin
immunostaining patterns (data not shown). There was partial overlap
between GFP-
-SNAP and the staining pattern for
-tubulin (Fig. 6,
J-L), but this overlap was not complete. Although
-tubulin is generally centrosomal, there have been reports that
-tubulin can form tubules, which are resistant to nocadazole and to
temperature (42). Since the distribution of GFP-
-SNAP did not
exactly colocalize with any of the other proteins and was resistant to
nocodazole, we immunostained stable transformants with anti-
-tubulin
antibodies. In Figs. 6 (A-C) and 7 (A-F), the
reticular distribution of GFP-
-SNAP colocalized almost completely with the immunostaining for
-tubulin. Although the most dominant distribution of
-tubulin in untransfected HEK-239 cells is
centrosomal (43), a faint reticular pattern of immunostaining (as seen
in Fig. 6B and 7 (B and E)) is clearly
detectable in a significant population of the cells. By Western
blotting, there was no cross-reactivity between the anti-
-tubulin
antibody and
- or
-tubulin.
-tubulin and GFP-
-SNAP suggests that the
two proteins could exist in the same complex; however, data presented
earlier (Fig. 2) suggest that only
-SNAP and Gaf-1 were stably
associated. To gain further insight, the pull-down and
coimmunoprecipitation experiments of Fig. 2 were again used but under
conditions that would stabilize microtubules (80 mM Pipes, pH 6.8, 1 mM MgCl2, 1 mM
EGTA, 1 mM GTP, 1 mM dithiothreitol, 5%
glycerol, 1% Triton X-100 (Ref. 44)). In Fig.
7G (left
panels), when GST-
-SNAP is incubated with cell extracts,
both Gaf-1 and
-tubulin associate with the recombinant protein. The
specificity of this interaction is demonstrated by the fact that
-tubulin but not
-tubulin binds to GST-
-SNAP and neither Gaf-1
nor
-tubulin significantly bind to GST-
-SNAP. In a further
experiment (Fig. 7G, right panel),
-tubulin, but not
-tubulin, coimmunoprecipitates with Gaf-1 when
the anti-Gaf-1 antibody is used. Neither protein is found associated
with pre-immune immunoglobulin. Since it was previously shown that
-SNAP coimmunoprecipitates with Gaf-1 (Fig. 2C), these
data imply that Gaf-1,
-SNAP, and
-tubulin can be part of the
same complex.
View larger version (53K):
[in a new window]
Fig. 7.
-Tubulin and
Gaf-1/
-SNAP form a complex. Stable
HEK-293 transformants expressing GFP-
-SNAP were analyzed by
immunofluorescence. In panels A-F, cells were
initially plated on laminin-coated coverslips, fixed in cold methanol,
and immunostained with anti-
-tubulin antibodies and relevant Texas
Red-labeled secondary reagents. Samples were examined under appropriate
illumination for GFP (A and D) and Texas Red
(B and E), and the images were recorded as in
Fig. 6. Panels C and F represent the merge of the
two images. Panel G, extracts from HEK-293 cells
were prepared under conditions that would maintain microtubule
integrity (44). 500 µg of the extracts were incubated with either GST
fusion proteins (GST-
-SNAP or GST-
-SNAP) or antibodies
(anti-Gaf-1 or pre-immune Ig). The complexes were then recovered by
precipitation with either GSH-agarose or protein G beads and analyzed
by SDS-PAGE and Western blotting. The lane at the
left of each set represents 100 µg of the starting
extract.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-SNAP. Initially,
-SNAP was shown to play a role in vesicular
transport (16); however, in the present study, we demonstrate a
potential role for
-SNAP in control of mitochondrial organization.
Using yeast two-hybrid screening, we have identified Gaf-1 as a
specific
-SNAP-binding protein (Figs. 1 and 2). Consistently, immunoprecipitation experiments show that
-SNAP is the major Gaf-1-associated protein in HepG2 cells. Gaf-1 is ubiquitously expressed in mammalian tissues based on Northern (27, 38) and Western
blotting analysis (Fig. 3). Potential orthologues can be identified in
Drosophila, zebrafish, and Dictyostelium based on
partial sequences in the data base and Western blotting. There are no
apparent orthologues in the yeast genome; this is also true for
-SNAP (14). Gaf-1 (also called pp75) was previously identified via
two-hybrid screening using the SS-A/Ro auto-antigen as bait (38). In
those studies, the Gaf-1/pp75 interaction with SS-A/Ro, in
vivo, was very weak (perhaps analogous to the thiolase interaction
reported here) and required cross-linking to demonstrate (38). From the
data presented in this report, Gaf-1/pp75 appears to be predominantly a
-SNAP-binding protein.
-SNAP in membrane trafficking (16). The majority
of Gaf-1 is present on the mitochondria, as demonstrated by
immunofluorescence colocalization in five different cells (Fig. 4) and
also confirmed by subcellular fractionation of HepG2 cells (Fig. 5).
Based on protease sensitivity (compared with protease resistance of
cytochrome c oxidase), Gaf-1 is peripherally associated with
the outer mitochondrial membrane where it apparently faces the cytosol
(Fig. 5). At present it is difficult to determine the exact
stoichiometry of the Gaf-1/
-SNAP complex. Based on the relative
sizes of the two proteins and the intensities of the methionine-labeled
bands coprecipitated in Fig. 2D, the ratio could be ~1 to
1. With this in mind, most of the mitochondrial Gaf-1 might be bound to
-SNAP. Most of the
-SNAP, however, is not associated with Gaf-1
on the mitochondria (see Fig. 5). This other pool of
-SNAP may be
used to facilitate membrane trafficking events (16, 18-20) or other
cellular functions.
-SNAP. Due to the poor quality of the anti-
-SNAP antibodies (see Fig. 4K), GFP fusion constructs were used to
more effectively analyze the distribution of cellular
-SNAP. In
HEK-293 cells, overexpression of GFP-
-SNAP or GFP-C-Gaf-1 (490-653,
which is apparently the
-SNAP binding region) either transiently or stably leads to the formation of a reticular network of fluorescent protein in about 30% of the transfected cells (Figs. 6 and 7). This
reticular network resembles the staining patterns for cytoskeletal elements such as microtubules or intermediate filaments. To address this, GFP-
-SNAP-expressing cells were analyzed by immunofluorescence analysis using a wide range of antibodies directed to cytoskeletal components. The reticular pattern of GFP-
-SNAP distribution
colocalized partially with
-tubulin. Surprisingly, GFP-
-SNAP
distribution completely colocalized with
-tubulin (Figs. 6 and 7).
The reticular distribution, however, did not colocalize with actin,
vimentin, or cytokeratins. This apparent interaction with
-tubulin
was confirmed using pull-down and coimmunoprecipitation studies under conditions that stabilize microtubules (Ref. 44 and Fig.
7G). It is clear from the data in Fig. 7 that the
interaction between
-tubulin and Gaf-1/
-SNAP is not as robust as
the interaction between Gaf-1 and
-SNAP; however, it appears to be
specific since
-tubulin does not associate with the complex.
-tubulin is surprising since
-tubulin is generally found associated with the microtubule
organizing center (45-47). However, it should be noted that the
majority of
-tubulin (80%) is not centrosomal but appears to be in
cytosolic complexes (48, 49).
-Tubulin has been seen associated
along the walls of microtubules during mitosis (50, 51). Consistently, the GFP-
-SNAP reticular distribution is not seen in serum-starved cells (presumably arrested in G0), but reappears 4-6 h
after the addition of serum-containing
media.3 Tubules of
-tubulin have also been reported in cells overexpressing
-tubulin
(42). These "
-tubules" were present in about 30% of the
transfected cells and were resistant to cold and to nocodazole just as
the GFP-
-SNAP structures shown here (Fig. 6, N and
O). Untransfected HEK-293 cells show the centrosomal
immunostaining pattern that is standard for
-tubulin (Ref. 43 and
data not shown); however, a faint reticular distribution of
-tubulin
is detectable by immunofluorescence in a significant number of
untransfected cells. It is clear that the binding of GFP-
-SNAP to
this reticular,
-tubulin-containing structure enhances its
visibility and thus allows its detection. However, at this stage it is
not clear whether GFP-
-SNAP expression causes the formation of the
structure or is localizing to an extant structure. One feature of the
reticular phenotype is its apparent dependence on cellular attachment
to laminin or collagen. Although this could just be based on better cell spreading on the coated coverslips, attachment to laminin can
induce structural changes in the distribution of microtubules (52-54).
-SNAP/Gaf-1 complexes
can transiently interact with a
-tubulin-containing, reticular
network. Based on the Gaf-1 localization, at least part of this complex
is associated with the mitochondria. This suggests a potential role for
this complex in the attachment of mitochondria to the cytoskeleton. In
such a model, Gaf-1 would serve as the mitochondrial attachment site
for the complex and
-SNAP might serve as an adaptor connecting Gaf-1
(on mitochondria) to the
-tubulin-containing microtubule scaffold.
Such speculation implies novel functions for two proteins,
-SNAP and
-tubulin, which have previously been shown to be important for other
cellular processes including vesicular trafficking (14, 16, 18-20) and microtubule nucleation (reviewed in Refs. 55 and 56) respectively. Future studies are aimed at testing this hypothesis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Katsuko Tani, Professor Mitsuo Tagaya, and Dr. Franz Bruckert for sharing unpublished results. We acknowledge the help of the Kazusa DNA Research Institute and Dr. Takashiro Nagase. We also thank Dr. Janet Shaw and Dr. Anthony Sinai for their helpful suggestions. As always, we are indebted to the Whiteheart laboratory: Elena Matveeva, Tara Rutledge, and Todd Schraw, for their contributions to this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant HL56652 from the NHLBI, National Institutes of Health (to S. W. W.), Grant 0020471B from the American Heart Association, Ohio Valley Affiliate (to D. C.), and Grant AG-3663 from the NIA, National Institutes of Health (to E. E. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ The first two authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of
Biochemistry, University of Kentucky College of Medicine, 800 Rose St., Lexington, KY 40536. Tel.: 859-257-4882; Fax: 859-323-1037; E-mail: whitehe@pop.uky.edu.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M009424200
2 S. W. Whiteheart and J. E. Rothman, unpublished results.
3 E.-J. Lim and S. W. Whiteheart, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NSF, N-ethylmaleimide-sensitive factor;
SNAP, soluble
N-ethylmaleimide-sensitive factor attachment protein;
SNARE, SNAP receptor;
GFP, green fluorescent protein;
GST, glutathione
S-transferase;
Gaf-1, -SNAP-associated factor;
PMCA, plasma membrane Ca2+ ATPase;
PBS, phosphate-buffered
saline;
EEA1, early endosome antigen 1;
DMEM, Dulbecco's modified
Eagle's medium;
DBD, DNA-binding domain;
PAGE, polyacrylamide gel
electrophoresis;
FBS, fetal bovine serum;
Pipes, 1,4-piperazinediethanesulfonic acid;
C-Gaf-1, C-terminal fragment of
Gaf-1;
N-Gaf-1, N-terminal fragment of Gaf-1.
![]() |
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