Gaf-1, a gamma -SNAP-binding Protein Associated with the Mitochondria*

Dong ChenDagger §, Weidong Xu§, Ping HeDagger , Estela E. Medrano, and Sidney W. WhiteheartDagger ||

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The role of alpha /beta -SNAP (Soluble NSF Attachment Protein) in vesicular trafficking is well established; however, the function of the ubiquitously expressed gamma -SNAP remains unclear. To further characterize the cellular role of this enigmatic protein, a two-hybrid screen was used to identify new, gamma -SNAP-binding proteins and to uncover potentially novel functions for gamma -SNAP. One such SNAP-binding protein, termed Gaf-1 (gamma -SNAP associate factor-1) specifically binds gamma - but not alpha -SNAP. The full-length Gaf-1 (75 kDa) is ubiquitously expressed and is found stoichiometrically associated with gamma -SNAP in cellular extracts. This binding is distinct from other SNAP interactions since no alpha -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 gamma -SNAP was mitochondrial with the balance being either cytosolic or associated with other membrane fractions. GFP-gamma -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 gamma -SNAP interactions that is distinct from other members of the SNAP family and point to a potential role for gamma -SNAP in mitochondrial dynamics.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The soluble N-ethylmaleimide-sensitive factor (NSF) 1 attachment protein family (alpha -, beta -, and gamma -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, gamma -SNAP is the least homologous member of the SNAP family (25% identical to alpha -SNAP; Refs. 13 and 14). beta -SNAP, due to its high sequence identity (83% identical to alpha -SNAP) and its similar activity, is thought to function as a brain-specific isoform of the more ubiquitous alpha -SNAP (13, 15). gamma -SNAP, like alpha - and beta -SNAP, can partially restore intercisternal transport activity to salt-extracted Golgi membranes (16, 17), although in this assay, gamma -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). gamma -SNAP had the greatest effect in this assay, perhaps because it binds better to the plastic surface used. In other systems, gamma -SNAP augments neurotransmission from squid giant axons (18) and catecholamine release from chromaffin cells (19). gamma -SNAP is also required for Golgi fragment reassembly following illiquinone treatment (20). However, no role for gamma -SNAP is detected in endoplasmic reticulum to Golgi transport (21). These data suggest a role for gamma -SNAP in membrane trafficking processes, but, unlike alpha -SNAP, its requirement in these processes is less well documented.

Biochemical assays indicate that gamma -SNAP may not serve the same function as does alpha - or beta -SNAP. gamma -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, gamma -SNAP cannot replace alpha -SNAP in 20 S complex formation assays nor does it compete with alpha -SNAP for membrane binding (22)2 Unlike alpha -SNAP, gamma -SNAP is not released from endosomes in an NSF/ATPase-dependent manner (23) consistent with an alternative type of binding interaction between gamma -SNAP and membranes. This is further supported by data showing that gamma -SNAP binding to membranes has a different binding kinetic and temperature profile than did alpha -SNAP binding (22). In the present study, we have attempted to further characterize gamma -SNAP by identifying gamma -SNAP-binding proteins. Using a yeast two-hybrid screen, we have detected two proteins that specifically interact with gamma -SNAP: cytosolic thiolase (24) and a protein denoted gamma -SNAP associated factor-1 (Gaf-1). Only Gaf-1 significantly colocalized and coimmunoprecipitated with gamma -SNAP. Additional studies using over-expression of GFP-gamma -SNAP resulted in the appearance of a reticular network of gamma -SNAP that partially colocalized with Gaf-1 and mitochondria. This network also colocalized with gamma -tubulin in these cells and partially with alpha -tubulin, but not with vimentin or cytokeratins. These findings describe a new interaction for the ubiquitous gamma -SNAP, which is unlike other SNAP family members, and suggest a potentially novel function for gamma -SNAP in connecting mitochondria with cytoskeletal elements.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Antibodies to the following proteins were obtained commercially: anti-alpha -tubulin (clone B-5-1-2), beta -tubulin (clone TUB 2.1), and gamma -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-gamma -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.

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 gamma -SNAP, alpha -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.

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 lambda  HybriZAP library by in vivo excision with Exassist helper phage according to the manufacturer's recommendation (Stratagene, La Jolla, CA).

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 gamma -SNAP was cloned into the pGBT9 "bait" vector and confirmed by dideoxy nucleotide sequencing. Expression of the bait fusion protein (Gal4 BD-gamma -SNAP) was confirmed by Western blotting using anti Gal4-DBD monoclonal antibody (CLONTECH). PJ-X transformants containing the "bait" plasmid pGBT9-gamma -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 beta -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.

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-gamma -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-gamma -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).

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of gamma -SNAP-binding Proteins-- To uncover potential roles for gamma -SNAP, we sought to first identify novel gamma -SNAP-binding proteins using a yeast two-hybrid screening approach. The cDNA encoding full-length, human gamma -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 beta -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 gamma -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 gamma -SNAP bait because no interaction was detected with either Gal4-DBD alone or Gal4-DBD-p53 (data not shown).

Initial binding studies with recombinant proteins suggested that the interaction between gamma -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 gamma -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-gamma -SNAP (Fig. 1, lane 10). This binding was specific; C-Gaf-1 did not bind to GST or to GST-alpha -SNAP (Fig. 1, lanes 8 and 9). Since the Gaf-1/gamma -SNAP interaction was appreciably more stable, we continued the characterization of Gaf-1 protein.


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Fig. 1.   Analysis of gamma -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-alpha -SNAP, or GST-gamma -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.

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).


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Fig. 2.   Gaf-1 binding to gamma -SNAP in vitro and in vivo. Panel A, GSH-agarose beads alone or bound to GST-alpha -SNAP (5 µg) or GST-gamma -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-gamma -SNAP precipitates endogenous Gaf-1 from liver cell extract. GSH-agarose beads alone and GST-alpha -SNAP (5 µg) and GST-gamma -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 gamma -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 gamma -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.

Characterization of the Gaf-1-gamma -SNAP Interaction-- Full-length recombinant Gaf-1 was produced in Escherichia coli and tested for binding to gamma -SNAP. In Fig. 2A full-length Gaf-1, like the C-terminal fragment, specifically bound to GST-gamma -SNAP but not to GST or to GST-alpha -SNAP. Endogenous Gaf-1 also specifically bound gamma -SNAP. Pull-down experiments, using detergent-solubilized extract from mouse liver incubated with recombinant GST, GST-gamma -SNAP, or GST-alpha -SNAP, show that Gaf-1 bound only to GST-gamma -SNAP (Fig. 2B). As a control, the precipitated complexes were probed for NSF, which was found associated with GST-alpha -SNAP but not with GST-gamma -SNAP (data not shown). In Fig. 2C, gamma -SNAP was coimmunoprecipitated from liver extracts with an anti-Gaf-1 antibody. Neither alpha -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 gamma -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 gamma -SNAP was recovered (data not shown). From these experiments it is clear that Gaf-1 specifically and stoichiometrically interacts with gamma -SNAP in vitro as well as in vivo; however, neither protein appears to be phosphorylated in HepG2 cells.

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 gamma -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 gamma -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).

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-gamma -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 gamma -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-gamma -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.

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 gamma -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 gamma -SNAP (~20%) was present in this fraction. These data confirm the mitochondrial localization of Gaf-1 determined by immunofluorescence. They additionally show that gamma -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, gamma  -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.

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.

gamma -SNAP and gamma -Tubulin Coexist in a Complex-- To determine the possible roles of the Gaf-1/gamma -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 gamma -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 gamma -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 gamma -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-alpha -SNAP or for any other GFP fusion proteins that we have expressed in HEK-293 cells (i.e. alpha -SNAP, SNAP-23, VAMP-3, VAMP-8; Ref. 40 and data not shown). Western blotting analysis showed that GFP-gamma -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.


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Fig. 6.   Overexpression of gamma -SNAP forms a reticular structure that colocalizes with gamma -tubulin. Stable HEK-293 transformants expressing GFP-gamma -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-gamma -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-alpha -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.

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-gamma -SNAP. In Fig. 6 (D-F), the Mitotracker staining pattern in the stable transformants was coincident with the GFP-gamma -SNAP distribution and appeared to overlay the meshwork; however, the distribution of mitochondria was not as extensive as that for GFP-gamma -SNAP. The distribution of Gaf-1 in these cells was also not as extensive as that of GFP-gamma -SNAP. Gaf-1 distribution was punctate and appeared to align with selected regions of the GFP-gamma -SNAP network, as did the Mitotracker staining. These data suggest that GFP-gamma -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-gamma -SNAP.

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-gamma -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-gamma -SNAP did not overlap with anti-actin, anti-cytokeratin, or anti-vimentin immunostaining patterns (data not shown). There was partial overlap between GFP-gamma -SNAP and the staining pattern for alpha -tubulin (Fig. 6, J-L), but this overlap was not complete. Although gamma -tubulin is generally centrosomal, there have been reports that gamma -tubulin can form tubules, which are resistant to nocadazole and to temperature (42). Since the distribution of GFP-gamma -SNAP did not exactly colocalize with any of the other proteins and was resistant to nocodazole, we immunostained stable transformants with anti-gamma -tubulin antibodies. In Figs. 6 (A-C) and 7 (A-F), the reticular distribution of GFP-gamma -SNAP colocalized almost completely with the immunostaining for gamma -tubulin. Although the most dominant distribution of gamma -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-gamma -tubulin antibody and alpha - or beta -tubulin.

The colocalization of gamma -tubulin and GFP-gamma -SNAP suggests that the two proteins could exist in the same complex; however, data presented earlier (Fig. 2) suggest that only gamma -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-gamma -SNAP is incubated with cell extracts, both Gaf-1 and gamma -tubulin associate with the recombinant protein. The specificity of this interaction is demonstrated by the fact that gamma -tubulin but not alpha -tubulin binds to GST-gamma -SNAP and neither Gaf-1 nor gamma -tubulin significantly bind to GST-alpha -SNAP. In a further experiment (Fig. 7G, right panel), gamma -tubulin, but not alpha -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 gamma -SNAP coimmunoprecipitates with Gaf-1 (Fig. 2C), these data imply that Gaf-1, gamma -SNAP, and gamma -tubulin can be part of the same complex.


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Fig. 7.   gamma -Tubulin and Gaf-1/gamma -SNAP form a complex. Stable HEK-293 transformants expressing GFP-gamma -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-gamma -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-gamma -SNAP or GST-alpha -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

In this report, we have attempted to identify novel cellular roles for gamma -SNAP. Initially, gamma -SNAP was shown to play a role in vesicular transport (16); however, in the present study, we demonstrate a potential role for gamma -SNAP in control of mitochondrial organization. Using yeast two-hybrid screening, we have identified Gaf-1 as a specific gamma -SNAP-binding protein (Figs. 1 and 2). Consistently, immunoprecipitation experiments show that gamma -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 gamma -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 gamma -SNAP-binding protein.

The subcellular distribution of Gaf-1 was surprising based on the proposed roles for gamma -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/gamma -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 gamma -SNAP. Most of the gamma -SNAP, however, is not associated with Gaf-1 on the mitochondria (see Fig. 5). This other pool of gamma -SNAP may be used to facilitate membrane trafficking events (16, 18-20) or other cellular functions.

Ectopic protein expression studies were used to determine the cellular role of gamma -SNAP. Due to the poor quality of the anti-gamma -SNAP antibodies (see Fig. 4K), GFP fusion constructs were used to more effectively analyze the distribution of cellular gamma -SNAP. In HEK-293 cells, overexpression of GFP-gamma -SNAP or GFP-C-Gaf-1 (490-653, which is apparently the gamma -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-gamma -SNAP-expressing cells were analyzed by immunofluorescence analysis using a wide range of antibodies directed to cytoskeletal components. The reticular pattern of GFP-gamma -SNAP distribution colocalized partially with alpha -tubulin. Surprisingly, GFP-gamma -SNAP distribution completely colocalized with gamma -tubulin (Figs. 6 and 7). The reticular distribution, however, did not colocalize with actin, vimentin, or cytokeratins. This apparent interaction with gamma -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 gamma -tubulin and Gaf-1/gamma -SNAP is not as robust as the interaction between Gaf-1 and gamma -SNAP; however, it appears to be specific since alpha -tubulin does not associate with the complex.

The reticular distribution of gamma -tubulin is surprising since gamma -tubulin is generally found associated with the microtubule organizing center (45-47). However, it should be noted that the majority of gamma -tubulin (80%) is not centrosomal but appears to be in cytosolic complexes (48, 49). gamma -Tubulin has been seen associated along the walls of microtubules during mitosis (50, 51). Consistently, the GFP-gamma -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 gamma -tubulin have also been reported in cells overexpressing gamma -tubulin (42). These "gamma -tubules" were present in about 30% of the transfected cells and were resistant to cold and to nocodazole just as the GFP-gamma -SNAP structures shown here (Fig. 6, N and O). Untransfected HEK-293 cells show the centrosomal immunostaining pattern that is standard for gamma -tubulin (Ref. 43 and data not shown); however, a faint reticular distribution of gamma -tubulin is detectable by immunofluorescence in a significant number of untransfected cells. It is clear that the binding of GFP-gamma -SNAP to this reticular, gamma -tubulin-containing structure enhances its visibility and thus allows its detection. However, at this stage it is not clear whether GFP-gamma -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).

In summary, our data clearly demonstrate that gamma -SNAP/Gaf-1 complexes can transiently interact with a gamma -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 gamma -SNAP might serve as an adaptor connecting Gaf-1 (on mitochondria) to the gamma -tubulin-containing microtubule scaffold. Such speculation implies novel functions for two proteins, gamma -SNAP and gamma -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, gamma -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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