ARTICLE |
Correspondence to: Juan S. Bonifacino, Cell Biology and Metabolism Branch, NICCHD, Bldg. 18T, Rm. 101, NIH, Bethesda, MD 20892.
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Summary |
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Furin is a dibasic endopeptidase responsible for the proteolytic maturation of many precursor proteins in the secretory and endocytic pathways of mammalian cells. The levels of furin expression in most cells are very low, and this has hampered attempts to identify the intracellular compartments in which endogenous furin is localized. We have used a specific antibody reagent to a sequence in the carboxy terminus of furin to perform immunofluorescent staining of mammalian cell lines. This antibody was sensitive enough to detect staining for furin in various cell lines. For the most part, furin staining was confined to a juxtanuclear structure characteristic of the Golgi complex. Analyses by video microscopy and confocal microscopy showed that the distribution of furin was distinct from that of mannosidase II, a marker of the Golgi stack, and most closely resembled that of TGN38, a marker of the trans-Golgi network. Therefore, our results suggest that endogenous furin is predominantly localized to the area of the Golgi complex, most likely within the trans-Golgi network. (J Histochem Cytochem 45:3-12, 1997)
Key Words: Furin, Golgi, TGN, Immunolocalization, Video microscopy
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Introduction |
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Furin is a transmembrane, dibasic endoprotease responsible for the proteolytic processing of a wide variety of precursor proteins in the secretory pathway (reviewed by
Since the initial discovery of the existence of furin in mammalian cells, there has been considerable interest in determining its subcellular localization. However, attempts to immunolocalize endogenous furin have been hampered by the low abundance of the protein in most cells (
The Golgi complex of mammalian cells is organized into three morphologically and functionally distinct regions: the cis-Golgi network (CGN), the Golgi stack, and the trans-Golgi network (TGN) (reviewed by
It remains to be established the extent to which the localization of furin constructs that are overexpressed, modified with epitope tags, or both reflects the actual distribution of endogenous furin within cells. The only published report on the localization of endogenous furin has produced results that differ from those obtained using recombinant furin constructs (
To address this apparent discrepancy, we decided to reevaluate the localization of endogenous furin using several antibody reagents that bind to different furin epitopes (
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Material and Methods |
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Cells
The cell lines used in this study are listed in Table 1. B6 and RD4 cells were the kind gift of Dr. Joe Harford (NIH). AtT20 cells were kindly provided by Dr. Yoke Peng Loh (NIH). All of the other cells lines were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Biofluids; Rockville, MD) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin, except for CHO-K1 cells, which were grown in RPMI 1640 medium with the same supplements. RBL cells stably transfected with a furin-HA construct were described previously (
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Antibodies
The antibody to furin, Cfur, was obtained by immunizing a rabbit with a peptide corresponding to the carboxy terminus of furin (SDSEEDEGRGERTAFIKDQSAL, residues 772-793). Other antibodies used were as follows: the rabbit antiserum JH4 to rat TGN38 (
Metabolic Labeling
Untransfected RBL cells or RBL cells stably transfected with a furin-HA construct (
Immunoprecipitation
The labeled cells were thawed at 4C and lysed by incubation for 15 min at 4C in 1% (w/v) Triton X-100 0.5% (w/v) sodium deoxycholate, 0.3 M NaCl, and 50 mM Tris-HCl, pH 7.4 (lysis buffer). The lysates were cleared by centrifugation at 15,000 x g for 15 min at 4C, filtered through 0.45-µm Millex-HA filters (Millipore; Bedford, MA), and incubated for 2 hr at 4C with the Cfur antibody (5 µl per sample) bound to protein A-Sepharose beads. The beads were washed three times with 0.1% (w/v) Triton X-100, 0.3 M NaCl, and 50 mM Tris-HCl, pH 7.4, and then three times with PBS. The washed beads were boiled in sample buffer as described by
Glutathione-S-transferase (GST) Fusion Proteins
The sequence encoding the cytoplasmic tail of mouse furin (
Immunofluorescent Staining and Conventional Microscopy
Cells were grown on glass coverslips to 30-70% confluency and fixed for 15 min at room temperature (RT) in 2% (w/v) formaldehyde in PBS. After washing in PBS, the cells were incubated for 45 min at RT with primary antibodies diluted in PBS containing 0.1% saponin, washed in PBS to remove excess antibodies, and then incubated for 30 min at RT with fluorescently labeled secondary antibodies. After a final wash with PBS, the coverslips were mounted on microscope slides with Fluoromount G (Southern Biotechnology Associates; Birmingham, AL). Samples were examined with a Zeiss Photomicroscope equipped with a x63 Planapo lens (Carl Zeiss; Oberkochen, Germany) and photographed with Kodak Tri-X-pan ASA 400 film.
Video Microscopy/Image Analysis
Cells stained with two different fluorochromes were examined with a modular light microscope (custom made by Yona Microscopes; Columbia, MD) fitted with an infinity-corrected lens (x100, 1.3 numerical aperture; Zeiss). A 100-W mercury lamp connected to fiberoptic cables was used as the light source. Neutral density, excitation, emission, and di-chroic filters (Omega Optical; Brattleboro, VT) selected the appropriate spectra for fluorescein, rhodamine, and indocarbocyanine optics.
Biological Detection Systems imaging software (v 1.6, now Oncor Imaging; Oncor Instruments, San Diego, CA) was used to acquire images via an interface with an electronic control box (MAC 2000; Ludl Electronic Products; Hawthorne, NY) which regulated a filter wheel containing the optical filters and the shutter. The microscope has two image detection systems. The first was used to locate and focus on the cells of choice. It included a CCD (charge-coupled device)-containing video camera (CCD-72; Dage-MTL, Michigan City, IN) and a quick capture card (Data Translation; Marlboro, MA). The second system was used to shunt images to the computer. A temperature-regulated cooled-CCD (model NU-200; Photometrics, Tucson, AZ) was used to capture digital images with a KAF-1400 chip (Eastman-Kodak; Rochester, NY).
All digital image processing was accomplished by use of IPLab Spectrum software (Signal Analytics; Vienna, VA). An appropriate background image (illuminated with fluorescein, rhodamine, or indocarbocyanine) was first subtracted from the images, which were then converted into 8 bits (256 gray levels) and combined into 24-bit color images. Image enhancement approaches for data presentation included contrast stretching, smoothing, and sharpening filtering.
Confocal Microscopy
In the experiment shown in Figure 5D-F, double-stained specimens were examined with a Zeiss Laser Scan Microscope LSM 410 fitted with a x63 Plan-Apochromat lens.
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Results |
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The Cfur rabbit polyclonal antibody was raised to a carboxy-terminal sequence of furin which is highly conserved among mammals (
Having established that the Cfur antibody was capable of recognizing furin specifically, we screened a total of 18 cultured cell lines for expression of endogenous furin by immunofluorescence microscopy utilizing the Cfur antibody. In agreement with the metabolic labeling-immunoprecipitation experiment described above, no staining for furin could be detected in untransfected RBL cells (Figure 2A). In contrast, staining for endogenous furin could easily be detected in 12 other cell lines derived from different animal species and tissues (Figure 2B-H; Table 1). In all of these cells, furin staining was mainly localized to a juxtanuclear structure characteristic of the Golgi complex, although a few stained vesicular structures were also seen in some cells. In addition to these structures, we also observed faint punctate staining throughout the cell. Staining of the Golgi-like structure was particularly bright NRK cells. Therefore, all of the subsequent experiments were performed using this cell line.
To establish whether the pattern of staining with the Cfur antibody was specific, glutathione-S-transferase (GST) or a GST-furin tail fusion protein were used as competitors in the immunofluorescence microscopy experiments (Figure 3). Binding of Cfur to the GST-furin tail fusion protein was expected to prevent specific staining for endogenous furin. Addition of normal GST produced a staining pattern indistinguishable from that of cells incubated with Cfur alone (Figure 3; compare 3A and 3B). In contrast, incubation in the presence of the GST-furin tail construct completely abrogated staining of the Golgi-like structure without affecting the background punctate staining (Figure 3C). This observation demonstrated that staining of the Golgi-like structure was specific, whereas the punctate staining was not. Therefore, the results of these immunofluorescence microscopy experiments were consistent with the localization of endogenous furin to a region of the Golgi complex, as reported earlier for recombinant furin constructs (
We next attempted to further define the localization of furin by immunoelectron microscopy. Unfortunately, we failed to observe specific staining using Cfur, probably due to the low abundance of furin and to the low sensitivity of the immunoelectron microscopy techniques we used. It was therefore not possible to establish whether endogenous furin, like the transfected furin constructs, was localized to the TGN. However, we thought that it would be possible to obtain more information from immunofluorescent staining experiments by comparing the localization of furin with other markers of the Golgi complex, as previously done for other Golgi proteins (
Several controls were performed to verify the validity of this approach. First, it was important to determine that there was no register shifting in the images obtained using the two different fluorochromes. This was controlled by coating Staphylococcus aureus particles with rabbit IgG and then with a mixture of fluorescein- and rhodamine-conjugated anti-rabbit antibodies. The double-stained particles were added to each coverslip before mounting them on slides. In most cases, no shifts were observed for the double-stained particles. In the few instances when a shift did occur (by two or three pixels), the images were aligned using image analysis software.
The second control consisted of staining NRK cells with 53FC3, a mouse monoclonal antibody to mannosidase II, a marker of cis-medial cisternae of the Golgi stack (
To test whether this image analysis technique was capable of distinguishing between different regions of the Golgi complex, cells were double stained for mannosidase II and TGN38, a TGN marker (
Next, we compared the localization of mannosidase II and furin (Figure 5). We observed that although the compartments containing mannosidase II (green) and furin (red) were in the same area of the cell, their structures were clearly distinguishable, even when cells were examined in a single channel (Figure 5A and Figure 5B). Superimposition of the two images revealed some regions of overlap (yellow), although for the most part the proteins were separated (green or red) (Figure 5C). The fact that some structures were either bright red or bright green suggests that the separation is not due to different intensities of the two fluorescent signals but reflects a spatial segregation of the two proteins.
Finally, we compared the localization of furin with the localization of TGN38, a TGN marker (
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Discussion |
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The results of our study demonstrate that endogenous furin is predominantly localized to a juxtanuclear structure characteristic of the Golgi complex. This conclusion is consistent with previous morphological studies on the localization of recombinant furin (
The results of immunofluorescence double-staining experiments show that the distribution of furin overlaps to a large extent with that of TGN38 and less with that of mannosidase II. These observations suggest that endogenous furin is predominantly localized to the TGN or to a compartment that is closely apposed to it. The potential localization of endogenous furin to the TGN has important implications for the physiology of precursor protein cleavage. The observations presented here suggest that most secreted and cellular proteins undergo cleavage at the TGN while in transit to the plasma membrane. Therefore, proteolytic processing by endogenous furin does not require diversion of the precursor proteins away from the secretory pathway, as would be the case if furin were exclusively localized to endosomal-lysosomal vesicles. The detection of endogenous furin protein in cells derived from different animal species and tissues agrees with previous studies of furin mRNA expression (
The double-staining method used in our study is similar to others that have been used before to examine the localization of various Golgi proteins (
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Acknowledgments |
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We thank Carolyn Smith, John Presley, and Carl Baker for their help in printing Figure 4 and Figure 5. We are also grateful to Jennifer Lippincott-Schwartz and Michael Marks for critical review of the manuscript.
Received for publication May 9, 1996; accepted September 3, 1996.
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