Journal of Histochemistry and Cytochemistry, Vol. 45, 965-974, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Immunolocalization of Rap1 in the Rat Parotid Gland: Detection on Secretory Granule Membranes

Nisha J. D'Silvaa, Dennis H. DiJulioa, Carol M. Beltona, Kerry L. Jacobsona, and E. L. Watsona,b
a Department of Oral Biology, University of Washington, Seattle, Washington
b Department of Pharmacology, University of Washington, Seattle, Washington

Correspondence to: Nisha J. D'Silva, Dept. of Oral Biology, Box 357132, U. of Washington, Seattle, WA 98195.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The objective of this study was to localize rap1 in the rat parotid gland. Rap1 is a small GTP-binding protein that has been linked to phagocytosis in neutrophils and various functions in platelets. In this study, we used [{alpha}-32P]-GTP-blot overlay analysis, immunoblot analysis, and immunohistochemistry to identify rap1 in rat parotid gland. The immunohistochemical techniques included immunoperoxidase and widefield microscopy with image deconvolution. Rap1 was identified in the secretory granule membrane (SGM), plasma membrane (PM), and cytosolic (CY) fractions, with the largest signal being in the SGM fraction. The tightly bound vs loosely adherent nature of SGM-associated rap1 was determined using sodium carbonate, and its orientation on whole granules was assessed by trypsin digestion. Rap1 was found to be a tightly bound protein rather than a loosely adherent contaminant protein of the SGM. Its orientation on the cytosolic face of the secretory granule (SG) is of significance in postulating a function for rap1 because exocytosis involves the fusion of the cytoplasmic face of the SG with the cytoplasmic face of the PM, with subsequent release of granule contents (CO). Therefore, the localization and high concentration of rap1 on the SGM and its cytosolic orientation suggest that it may play a role in the regulation of secretion. (J Histochem Cytochem 45:965-973, 1997)

Key Words: rap1, small GTP-binding protein, rat parotid gland, widefield microscopy with image deconvolution


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

There are two groups of signal-transducing GTP-binding proteins, heterotrimers and monomers. The heterotrimeric GTP-binding or G-proteins transduce signals between cell-surface receptors and intracellular effectors (Hepler and Gilman 1992 ) and, therefore, were originally believed to be present only on the cytoplasmic surface of the PM. However, they may also be present on intracellular structures. These G-proteins consist of {alpha}-, ß-, and {gamma}-subunits that have molecular weights of 39-46, 37, and 8 kD, respectively (Hepler and Gilman 1992 ). It is the {alpha}-subunit that is a GTPase, i.e., it binds and hydrolyzes GTP and is primarily responsible for the diversity of heterotrimeric G-proteins. The monomeric or small GTP-binding proteins (smgs) differ from the heterotrimeric G-proteins in that they have smaller molecular masses (18-30 kD), consist of a single subunit, have specific GTPase-activating proteins that enhance intrinsic GTPase activity, and may serve different regulatory functions (Hall 1990 ; Takai et al. 1992 ). They belong to the ras superfamily, which is divided into four subfamilies: ras, rho, rab, and arf (Bokoch 1993 ). At least 50 smgs have been identified in mammalian cells (Bokoch 1993 ). The proteins of this superfamily have been shown to be involved in organelle movement (Walworth et al. 1989 ; Balch 1990 ), cell proliferation and differentiation, and transport across membranes (Hall 1990 ; Bokoch 1993 ).

The smg rap has close homology to ras but often differs in cellular and intracellular localization (Bokoch 1993 ). It can be divided into two subgroups, rap1 and rap2, each of which has two subtypes: a and b (Bo-koch 1993 ). These proteins are found in almost all tissues (Bokoch 1993 ). Like other members of the ras superfamily, rap1 has been identified in a variety of cell types, which include human epidermoid carcinoma cells, CHO cells, the Golgi of rat fibroblasts, the zymogen granule, PM and microsomal membrane of rat pancreatic acinar cells, the specific granules and PM in neutrophils, and cytosol, {alpha}-granules, and PM fractions of human platelets (Lapetina et al. 1989 ; Nagata et al. 1989 ; Matsui et al. 1990 ; Beranger et al. 1991 ; Maridonneau-Parini and de Gunzburg 1992 ; Quinn et al. 1992 ; Schneffel et al. 1992 ; Mori et al. 1993 ; Berger et al. 1994 ; Nagata et al. 1995). This indicates that it serves a general function regulated via different effector pathways, depending on the cell type involved (Bokoch 1993 ). For example, it may be involved in controlling cell growth and differentiation (Bokoch 1993 ). Alternatively, the variable localization of rap1 on specific intracellular organelles, such as the SG (Maridonneau-Parini and de Gunzburg 1992 ; Schneffel et al. 1992 ; Berger et al. 1994 ; Nagata et al. 1995) may be responsible for its playing well-defined biological functions that vary with intracellular location and cell type (Bokoch 1993 ). In neutrophils, in which rap1 is located primarily on the specific granules, studies suggest that it plays a crucial role in phagocytosis (Maridonneau-Parini and de Gunzburg 1992 ). In platelets it has been linked to cytoskeletal reorganization (Fischer et al. 1990 ), Ca2+ regulation (Corvazier et al. 1992 ), and to regulation of PLC{gamma} activity (Torti and Lapetina 1992 ).

In this study we identified the smg rap1 on SGM, PM, and CY fractions of the rat parotid gland by radiolabeled GTP-blot overlay analysis, immunoblot analysis, and immunohistochemistry. Rap1 was found to be a tightly bound protein of the SGM, and its location on the cytosolic face of the SG suggests that it may play an important role in exocytosis.


  Materials and Methods
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Materials and Methods
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Materials
Materials were obtained as follows: renografin-60 from ER Squibb (New Brunswick, NJ); N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), diaminobenzidene tetrahydrochloride (DAB), rabbit anti-human {alpha}-amylase antibody, 4',6-diamidino-2-phenylindole (DAPI), and hyaluronidase from Sigma Chemicals (St Louis, MO); crude collagenase CLS2 from Worthington Laboratories (Freehold, NJ); di-thiothreitol (DTT), Tween-20, SDS-PAGE chemicals and low molecular weight protein standards from Bio-Rad (Richmond, CA); SDS-PAGE and Western blot apparatus including mini-cell, precast 12% Tris-glycine minigels, blotting transfer module, and polyvinylidene fluoride (PVDF) filters from Novex (Encinitas, CA); nitrocellulose filters from Schleicher & Schuell, 0.2 µm (Keene, NH); [{alpha}-32P]-GTP from Dupont-New England Nuclear Research Products (Boston, MA); enhanced chemiluminescent (ECL) Western blotting detection system and Hyperfilm MP autoradiography film from Amersham (Arlington Heights, IL); affinity-purified horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG and normal rabbit serum from Jackson ImmunoResearch Laboratories (West Grove, PA); rabbit anti-rap1 affinity-purified polyclonal antibody and its neutralizing peptide from Santa Cruz Biotechnology (Santa Cruz, CA); Vectastain ABC kit, affinity-purified fluorescein-conjugated goat anti-rabbit IgG, affinity-purified Texas red-conjugated goat anti-rabbit IgG, and Vectashield from Vector Laboratories (Burlingame, CA). Recombinant rap1b (rR) was a generous gift from Dr. Thomas H. Fischer, (University of North Carolina). Rabbit antiserum against purified parotid secretory granule membrane was kindly provided by Drs. A. Castle and J.D. Castle (University of Virginia).

Preparation of Rat Parotid Plasma and Secretory Granule Membranes
Briefly, the parotid glands were removed from fasted male Sprague-Dawley rats weighing 100-120 g. The glands were minced and homogenized and a 250 x g supernatant and pellet were obtained as described by Robinovitch et al. 1975 . The pellet was used to prepare the PM fraction following the procedure of Arvan and Castle 1982 , with modification as described by Watson et al. 1992 . Secretory granules were isolated from the supernatant fraction as described by Iversen et al. 1985 . Briefly, the 250 x g supernatant of a homogenate of parotid glands was layered over 30% renografin and centrifuged. Light SGs were obtained in the renografin and heavy or more mature SGs were obtained in the pellet. Preliminary analysis of these fractions indicated no difference in rap1 concentration between these fractions. Hence, the light and heavy SGs were pooled for the studies shown here. The purity of intact SGs was previously described by Watson et al. 1992 . The SGs were lysed overnight and SGM separated from contents as described by Robinovitch et al. 1980 . The SGM was sequentially washed with hypotonic and isotonic buffers to remove loosely adherent proteins.

Preparation of Purified Rough Endoplasmic Reticulum
Endoplasmic reticular membranes (ER) were prepared from whole rat parotid glands as described previously by Watson et al. 1992 , according to the method of Bayerdorffer et al. 1984 for rat pancreas.

Preparation of Rat Parotid Acinar Cells
Parotid acini were prepared as described by Watson et al. 1993 , with modification. Briefly, parotid glands were isolated from male Sprague-Dawley rats (100-120 g), minced in Krebs-Henseleit bicarbonate solution (KHB, pH 7.4) containing 1.13 mM Mg2+, 0.21 mM Ca2+, 30 mM HEPES, 90 U/ml crude collagenase and 1 mg/ml hyaluronidase, and enzymatically digested in a rotary water bath at 37C for 60 min under continuous 5% CO2-95% O2 gassing. The suspension was pipetted up and down, with a 10-ml pipette, 10 times at 20, 40, and 50 min and five times at 60 min. From the 40-min pipetting onwards, a 1-ml pipette tip was attached to the 10 ml pipette. At the end of the digestion period, the acini were washed with KHB buffer (minus enzymes) containing 1% BSA and were subsequently allowed to rest in the same buffer for 30 min at 37C.

Preparation of Cytosol
For the CY preparation, rat parotid acini were prepared as described above, homogenized and centrifuged at 250 x g for 5 min. Cytosol was prepared from the supernatant by centrifugation at 100,000 x g for 1 hr at 4C.

{alpha}-32P]-GTP-blot Overlay Assay
Low molecular mass GTP-binding proteins were detected by the radiolabeled GTP-blot overlay method of Bhullar and Haslam 1987 , with modification as described by Schneffel et al. 1992 and Gromov and Celis 1994 . Briefly, SGM, PM, CY, CO, ER, rR, and molecular weight standards were separated by SDS-12% PAGE and transferred to nitrocellulose filters. The filters were blocked in GTP-binding buffer (0.3% Tween-20, 5 µM MgCl2, 1 mM potassium phosphate, 100 µM ATP, 50 mM Tris-HCl, pH 7.5). The filters were then incubated in the same buffer containing 100 mM DTT and 1 µCi [{alpha}-32P]-GTP/ml, with or without added unlabeled GTP (10 µM) for 60 min at room temperature, and washed repeatedly in cold buffer without DTT. The filters were air-dried and exposed to autoradiographic film with and without intensifying screens for 25 hr to 13 days. The data were analyzed by laser densitometry.

Immunoblot Detection of Rap1
Secretory granule membrane, PM, CY, CO, ER and molecular weight standards were separated by SDS-12% PAGE and transferred to PVDF filters. The filters were incubated with rabbit anti-rap 1 affinity-purified polyclonal antibody (1 µg/ml) or with antibody preincubated with a 20-fold excess of its neutralizing peptide. Antibody binding was detected with the ECL detection system, using HRP-linked donkey anti-rabbit IgG secondary antibody (1:15,000).

Immunohistochemistry
A parotid gland was harvested and placed in Carnoy's fixative (acetic acid 10%, methanol 60%, chloroform 30%) for 3 hr, transferred to decreasing grades of alcohol to replace the choloroform with water, dehydrated to remove the water, which is not miscible with paraffin, and paraffin-embedded. Rap1 was detected on 5-µm tissue sections using rabbit anti-rap1 affinity-purified polyclonal antibody (5 µg/ml) with the avidin-biotin-peroxidase immunoassay using the Vecta-stain ABC kit and the DAB colorimetric detection system.

For widefield microscopy with image deconvolution studies (Agard et al. 1989 ), subsequently referred to as widefield deconvolution microscopy, the 5-µm parotid gland sections were incubated with the rap1 affinity-purified polyclonal antibody (5 µg/ml) and/ or with rabbit antiserum against purified SGM at a concentration of 1:100 (Cameron et al. 1986 ). Indirect immunofluorescence was performed using affinity-purified, fluorescein-conjugated goat anti-rabbit IgG (H+L) and affinity-purified Texas red-conjugated goat anti-rabbit IgG (H+L) as secondary antibodies. Double labeling was done according to the method of Wang and Larsson 1985 . The DNA in the nuclei was stained with DAPI (0.1 µg/ml). Sections were observed on an Olympus fluorescence microscope equipped with epi-illumination with selective filters for excitation at 490 nm (FITC) and 546 nm (TRITC). The data were collected and analyzed using the DeltaVision system (Applied Precision; Mercer Island, WA) for multidimensional microscopy. The system includes an optical sectioning light microscope, a cooled charge-couple device camera, and computer software that can acquire and analyze three-dimensional images of fluorescence-labeled specimens (Agard et al. 1989 ). Optical sections 200 nm thick were obtained and stored on a disk. Improved resolution is obtained by analyzing images with a deconvolution alogorithm to remove out-of-focus information. Widefield deconvolution microscopy is less damaging to the tissue than confocal microscopy because it does not require laser illumination.

Controls for the immunoperoxidase studies included omission of the primary antibody or substitution of the primary antibody with rabbit IgG. For the widefield deconvolution microscopy studies, the controls included substitution of the second primary antibody (against SGM) with buffer or IgG.

Tightly Bound vs Loosely Adherent Protein/Epitope Orientation
To determine whether rap 1 is tightly bound to the SGM or whether it becomes associated with it during isolation procedures, SGMs were treated with 0.1 M sodium carbonate as described by Fujiki et al. 1982 . To determine the epitope orientation of rap 1 found on isolated SGs, i.e., on the cytoplasmic or internal granule surface, isolated SGs were treated with trypsin (100 µg/ml) according to the method of Vidal and Stahl 1993 , before immunoblot analysis.


  Results
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Materials and Methods
Results
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Detection of Monomeric GTP-binding Proteins by [{alpha}-32P]-GTP Overlay Assay
Initial experiments were designed to determine the presence of smg proteins, particularly rap1, in cell fractions isolated from rat parotid gland. When separated by one-dimensional gel electrophoresis and immobilized on a nitrocellulose filter, recombinant rap1, peptides of rat parotid SGM, CY, PM and ER-enriched fractions were found to bind [{alpha}-32P]-GTP (Figure 1A). Four smg proteins with molecular weights of 24.5, 26.5, 27.5, and 29 kD were present on the PM fraction (as determined by laser densitometry). At least three smgs with molecular weights of 24.5, 27, and 28 kD were present on the SGM: the GTP binding did not resolve into discrete bands for finite molecular mass determination of a possible fourth band present between the 24.5- and 27-kD bands. Those membrane-bound smg proteins, having an apparent common molecular mass, were most abundant in the SGM fraction, where the levels were severalfold that found in the PM-enriched fraction. The 26.5- and 29-kD smg signals present in the CY were low compared to those present in the PM. In addition, a ~21-kD smg was also detected in the CY but not in the other subcellular fractions. For those peptides that bind GTP specifically, micromolar concentrations of unlabeled GTP successfully competed for radiolabeled GTP (Figure 1B).



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Figure 1. Autoradiograph of [{alpha}-32P]-GTP binding to blotted proteins of subcellular fractions of the rat paratid gland. Recombinant rap1b (rR) secretory granule membrane (SGM), plasma membrane (PM), cytosol (CY), secretory granule contents (CO), and endoplasmic reticulum membrane (ER) fractions, 7 µg each, were separated by SDS-12% PAGE followed by transfer to nitrocellulose filters. The filters were incubated with [{alpha}-32P]-GTP in GTP-binding buffer without (A) or with (B) 10 µm unlabeled GTP. The nitrocellulose filters were exposed to autoradiographic film, which was analyzed by laser densitometry. Data shown are from autoradiographs exposed for various durations to filters to optimize signals, i.e., to visualize minor bands, for a particular fraction. The duration of exposure for each fraction is as indicated.

Endoplasmic reticulum, found to be the major organelle contaminating the SGM fraction (Arvan and Castle 1982 ), contained three [{alpha}-32P]-GTP-binding proteins, two of which had molecular masses of 26.5 and 29 kD. The third GTP-binding protein had a slightly higher molecular weight than the 26.5-kD signal but could not be resolved for finite molecular weight determination. These signals were in common with those present in the PM, suggesting that these fractions have smgs in common. [{alpha}-32P]-GTP binding to ER fractions, however, was severalfold less than that found in the SGM and PM fractions, based on analysis of similar amounts of resolved proteins. The lower abundance of the 26.5-kD protein in the ER relative to membrane fractions indicates that the SGM population of smg-proteins cannot result solely from ER contamination.

In contrast to Klinz 1994 , we were able to show that rR binds [{alpha}-32P]-GTP on a GTP overlay. This may have been due to a slight difference in the GTP-binding buffer, e.g., the presence of DTT in our buffer. Gromov and Celis 1994 reported that the inclusion of 100 mM DTT in the GTP-binding buffer enhanced [{alpha}-32P]-GTP binding to GTP-binding proteins on a GTP overlay. However, despite the presence of DTT, CY rap1 transferred to a nitrocellulose filter was not detected by GTP. In addition, Leiser et al. 1995 reported, and we also found (Figure 3, reported below), that rap1 was undetectable in the CY fraction with rap1 antibody when a nitrocellulose rather than a PVDF filter was used as the protein-blotting membrane. This may have been due to a very low signal or to absence of (soluble) CY rap 1 on the nitrocellulose filter, which has lower protein binding capacity than PVDF (Leiser et al. 1995 ).



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Figure 2. Immunoblot localization of rap1 in various rat parotid acinar fractions. Secretory granule membrane (SGM), plasma membrane (PM), cytosol (CY), endoplasmic reticulum (ER), and secretory granule contents (CO), 7 µg each, were separated by SDS-PAGE, transferred to PVDF filters, and blotted with (A) rap1 affinity-purified polyclonal antibody (1 µg/ml) or (B) antibody neutralized with a 20-fold excess of its neutralizing peptide. Recombinant rap1b (rR) served as a positive control.



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Figure 3. Correlation of rap 1 identified by immunoblot with the 24.5-kD signal detected by [{alpha}-32P]-GTP in Figure 1. After autoradiographs were obtained, the filter was washed in Tween-20-Tris-buffered saline and probed with rap1 affinity-purified polyclonal antibody (1 µg/ml).

Immunoblot Detection of Rap1 in Subcellular Fractions
The various subcellular fractions prepared were probed on an immunoblot for rap1. Rap1 was detected as an ~24-kD signal and was found to be present in highest concentration in the SGM fraction; rap1 was also detected in the PM, but to a lesser extent (Figure 2A). Comparatively weaker signals were obtained with the ER-enriched fraction, CO and CY. To verify the specificity of this signal, the antibody was incubated with a 20-fold excess (20 µg/ml) of its neutralizing peptide for 1 hr before rap1 detection. In the presence of peptide, the 24-kD rap1 signal was removed in most fractions and markedly reduced in the recombinant rap1 fraction (Figure 2B). The latter would probably have been removed with longer peptide neutralization or a higher concentration of the peptide. The other nonspecific signals were also removed and appeared to be proteins that have immunological epitopes in common with rap 1. The ~55-kD nonspecific signal observed in the SGM, CO, and CY fractions was consistent with the molecular weight for amylase, present in the secretory granule contents. However, this was ruled out because rap1 antibody purified on an amylase column crossreacted with this signal (data not shown). The ~28-kD nonspecific signal also observed in the SGM fraction may be an immunoglobulin breakdown product because the rap1 antibody was found to crossreact with mouse IgG at ~28 kD (data not shown). The peptide with a molecular weight lower than that of rap551 is probably a breakdown product of the latter, since it is also seen in the purified rR lane.

To show that the ~24.5-kD GTP-binding protein identified in the SGM and PM fractions by [{alpha}-32P]-GTP binding analysis (Figure 1A) was, in fact, rap1 identified by immunoblot in Figure 2A, the filter used for the GTP overlay assay was subsequently immunoblotted with the rap1 antibody. Data presented in Figure 3 showed exact correlation between the ~24.5-kD [{alpha}-32P]-GTP-binding protein detected in the PM and SGM (Figure 1A) and the protein labeled with the rap1 antibody.

Immunohistochemistry
Immunohistochemical studies revealed strongest staining with the rap1 antibody in the apical region of the acinar cell where the SGs are located (Figure 4A). This staining was removed when the tissue sections were incubated with antibody that had been preincubated with a 10-fold excess of its neutralizing peptide (Figure 4B). Negative controls, which included omission of the primary antibody or substitution with rabbit IgG, were negative.



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Figure 4. Immunohistochemical detection of rap1 in the rat parotid gland. Parotid gland tissue sections were incubated (A) with rabbit anti-rap1 antibody (5 µg/ml) or (B) with primary antibody preincubated with a tenfold excess of neutralizing peptide for 2 hr at room temperature. Punctate brown staining is most concentrated in the apical regions of the acinar cells (arrows), where the secretory granules are located. Brown DAB reaction product with methyl green counterstain. Bar = 25 µm.

Figure 5. Immunofluorescent detection of rap 1 on the secretory granule membrane with the wide-field deconvolution microscopy (Delta Vision) system. Carnoy's-fixed, paraffin-embedded tissue sections were incubated with rap1 antibody and with rabbit antiserum against SG membrane. Immunofluorescence was performed using Texas red- and fluorescein-conjugated goat anti-rabbit IgG (12 µg/ml). The rap1 (green) and granule membrane (red) antibodies co-localized on the SG membrane (arrows). Rap1 staining is punctate. No staining was observed within the SG contents. The DNA was stained with the fluorescent dye DAPI (blue) to highlight the nucleus. Co-localization of the granule membrane and rap1 antibodies was also observed on the PM (arrowheads). Bar = 2 µm.

To rule out the possibility that labeling of the tissue sections in the apical region of the cell may have been due to the reactivity of the antibody with the nonspecific ~55-kD signal detected by immunoblot in the CO, we utilized widefield deconvolution microscopy, a technique that yields high-resolution images (Agard et al. 1989 ). For these studies, double labeling was done with SGM (red) and rap1 (green) antibodies to verify that rap1 is present on the SGM. Data presented in Figure 5 show that these antibodies co-localized on the SGM (arrows). No localization was observed within the CO. Co-localization of the SGM and rap1 antibodies was also observed on the PM (arrowheads). Staining of the PM with the SGM antibody was previously reported by Cameron et al. 1986 . Because SGs in rat parotid acini vary from about 1 to 4 µm in diameter (Anderson et al. 1990 ), some SGs appear almost half the size of the nucleus if the latter is cut in tangential section. To facilitate orientation, the DNA fluorescent stain DAPI, seen as a blue stain, was used to detect the nucleus.

Negative controls, which included substituting the second primary antibody, i.e., the SGM antibody, with buffer or IgG, were negative for Texas red staining.

Tightly Bound vs Loosely Adherent to the Secretory Granule Membrane
Because proteins may become loosely adherent to an organelle as a function of tissue homogenization and subcellular fraction preparation (Fujiki et al. 1982 ), SGM and PM were treated with 0.1 M sodium carbonate to remove proteins that might be loosely adherent to the membranes (Fujiki et al. 1982 ). Hence, if rap1 is redistributed to the SGM or PM during subcellular fraction preparation, it should not be detected in the pellet (membrane) fraction after treatment with sodium carbonate but should be detected in the supernatant. Treatment with 0.1 M sodium carbonate did not remove rap1 from the SGM-enriched fraction (Figure 6). It did, however, partially remove rap1 from the PM-enriched fraction. This suggests that the SGM-associated rap1 and some of the PM-associated rap1 are tightly bound to the membranes and do not represent artifacts resulting from tissue homogenization procedures.



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Figure 6. Rap1 is tightly bound to the secretory granule membrane. Secretory granule membrane (SGM) and plasma membrane (PM) fractions were treated with 10 mM Tris-HCI (control = 1) or 100 mM sodium carbonate (treated = 2). The supernatant (SN) and pellet (SGM or PM) fractions were separated for each sample. Seven µl (~7 µg of membrane) of each sample was separated by SDS-PAGE and immunoblotted with the rap1 affinity-purified polyclonal antibody (1 µg/ml). Recombinant rap1b (rR, 4 ng) served as a positive control.

Epitope Orientation
In other studies, intact SGs were treated with 100 µg/ml trypsin to determine whether rap1 was present on the cytosolic vs the inner surface of the SG. If rap1 is present on the cytosolic surface, it should be sensitive to protease digestion whereas a protein on the intragranular surface should not. Data presented in Figure 7B suggest that rap1 is present on the cytosolic surface of the SG, because it was sensitive to protease treatment. Amylase, a protein in the CO, was not removed (Figure 7A) thereby indicating that the SGs were intact when treated with trypsin.



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Figure 7. Orientation of rap1 on whole secretory granules. Whole secretory granules were treated with trypsin (100 µg/ml; Lanes 2) or distilled water (control; Lanes 1) for 20 min. The samples were centrifuged and the secretory granule pellets were solubilized in sample buffer. An equivalent amount of each sample was separated by SDS-PAGE an immunoblotted with (B) rap1 affinity-purified polyclonal antibody (1 µg/ml) or (A) rabbit anti-human {alpha}-amylase antibody (0.25 µg/ml).


  Discussion
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

The identification and orientation of smgs on specific cellular organelles suggest their function. For example, rab3A's (smg25A) localization on SGs in endocrine cells suggests a role in exocytosis (Regazzi et al. 1992 ; Darchen et al. 1990 ). Rap1's identification on synaptic vesicles in neurons suggests a similar role (Kim et al. 1990 ). In the present study we identified the smg rap1 associated with rat parotid gland fractions by using complementary methods, including [{alpha}-32P]-GTP-blot overlay, immunoblot analysis, and immunohistochemistry, i.e., immunoperoxidase and widefield deconvolution microscopy. We found, by all of these methods, that rap1 is present on the SGM and PM. Two-dimensional gel electrophoretic analysis of the SGM, PM, and CY fractions may help to ascertain if they do indeed have identical smgs.

Kameyama et al. 1994 have reported rap1 localization on the SGM and on both the apical and basolateral PM fractions of the rat parotid gland. However, in contrast to our results, in which we found that the strongest rap1 signal was associated with the SGM, Kameyama et al. 1994 found that the strongest rap1 signal was associated with the apical and basolateral PM. These differences may be due to differences in the methods of PM preparation, e.g., Kameyama et al. 1994 prepared separate apical and basolateral PM fractions.

The significance of the present findings lies in our ability to provide definitive data showing that rap1 is localized to the SGM. Studies using immunoblot analysis are subject to question, because rap1 could have translocated to the SG from another location. Alternatively or additionally, immunoblot localization of rap 1 on the SGM could have been the consequence of organelle contamination produced during isolation of the granules. Even with stringent investigation of contaminating organelles by enzyme marker analysis, it is difficult to make definitive conclusions about localization. Hence, in our studies, we supported our immunoblot data with immunohistochemical data, i.e., immunoperoxidase and widefield deconvolution microscopy. Although the immunoperoxidase data showed maximal staining in the apical region of acinar cells, where SGs are located, it was compromised by the limited resolution of light microscopy, and therefore was unable to distinguish between the SGM vs the CO. This was considered critical because rap1 crossreacts on immunoblots with a 55-kD signal in the CO. This was addressed by using widefield deconvolution microscopy, which provided a very clear picture of the localization of rap1 on the SGM and PM. Orientation was facilitated with the DNA fluorescent stain, DAPI. We specifically show that rap1 co-localizes with a parotid granule-specific antibody (Cameron et al. 1986 ). No rap 1 was observed in the CO. Interestingly, crossreactive epitopes may be detectable in one technique but undetectable in another, in this case immunoblot vs immunohistochemistry, respectively, due to multivalency of the antigen as well as the antibody (Harlow and Lane 1988 ). Widefield deconvolution microscopy allowed visualization of intracellular organelles and was very beneficial for our localization studies, in that we were able to show that no rap1 staining was observed in the CO, whereas rap1 was present on the SGM.

In other studies, the finding that rap 1 present on the SGM-enriched fraction is a tightly bound protein rather than a loosely adherent contaminant protein is consistent with and supports the immunoperoxidase and widefield deconvolution microscopy studies. The orientation of rap1 on the SG cytoplasmic face is of significance in postulating a function for rap1 because exocytosis involves the fusion of the cytoplasmic face of the SGM with the cytoplasmic face of the PM with subsequent release of CO. Therefore the localization and high concentration of rap1 on the SGM and its cytosolic localization suggest that it may play a role in the regulation of secretion.


  Acknowledgments

Supported by the National Institute of Dental Research of the National Institutes of Health, grants DE 07023 and DE 10733.

Received for publication January 27, 1997; accepted February 6, 1997.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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