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(Circulation Research. 1996;78:343-347.)
© 1996 American Heart Association, Inc.

Articles

Identification of rab12 as a Secretory Granule Associated Small GTP-Binding Protein in Atrial Myocytes

Hiroshi Iida, Luming Wang, Kiyomasa Nishii, Akiko Ookuma, Yosaburo Shibata

From the Department of Anatomy, Faculty of Medicine, Kyushu University, Fukuoka, Japan.

Correspondence to Dr Hiroshi Iida, Department of Anatomy, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan.

	Abstract

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Abstract A subfamily of small GTP-binding proteins, rab, has been shown to be involved in regulation of vesicular traffic in eukaryotic cells. The goal of this study was to identify the rab proteins associated with atrial secretory granules. A [³²P]GTP-overlay assay showed the presence of multiple small GTP-binding proteins on the atrial granules. By biochemical analysis, we have demonstrated that one of the small GTP-binding proteins associated with the atrial granules is a rab12 protein (rab12p), one of the rab proteins that are most closely related to a Sec4 protein of yeast. Association of rab12p with the atrial granules was confirmed by immunogold electron microscopy. Immunoprecipitation followed by immunoblot analysis with anti-rab12 antibody showed that in addition to atria, rab12p was expressed in multiple other organs and cell lines. These results suggest that rab12p may function in vesicular traffic in multiple diverse types of cells.

Key Words: small GTP-binding proteins • atrial granules • rab12 • atrial myocytes • secretion

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent studies have demonstrated the involvement of small GTP-binding proteins, homologues to the proto-oncogene ras, in the regulation of membrane traffic.^{1 2 3} The first evidence that GTPases are essential in membrane traffic came from genetic studies in Saccharomyces cerevisiae, which resulted in the identification of Ypt1p and Sec4p. A mutation in Ypt1 interferes with vesicle transport between the endoplasmic reticulum and the Golgi complex; the Sec4 mutants are characterized by an accumulation of secretory vesicles that have failed to fuse with the plasma membranes.^{4 5} The mammalian homologues of these proteins are called rab proteins and form a large family of at least 30 members.⁶ Several of them have been localized to specific subcellular compartments along exocytic and endocytic pathways, where they appear to confer specificity for transport vesicle targeting to or fusion with their correct acceptor compartment.^{2 3} Although a mammalian homologue of Ypt1p has been identified (rab1p), a mammalian counterpart of Sec4 has not yet been identified. Of many rab proteins, the sequences of rab8, rab10, and rab12 are most closely related to that of Sec4.^{6 7 8} If these rab proteins function as a mammalian counterpart of Sec4p, which is associated with transport vesicles derived from the Golgi complex in yeast, they might be localized on secretory granules, ie, post-Golgi vesicles in endocrine and exocrine cells in mammalian tissues.

ANP, an important regulator of circulating blood volume and of vascular smooth muscle function,⁹ is stored in atrial secretory granules and released from atrial myocytes by various secretagogues. Our preliminary experiments revealed that antibodies against rab8 and rab10 failed to detect any antigens on the nitrocellulose sheets to which proteins of isolated atrial granules were transferred, whereas anti-rab12 antiserum recognized 28-kD protein on the sheets. In the present study, we have performed biochemical and immunocytochemical analyses with affinity-purified antibody against rab12p to investigate whether this protein is associated with the atrial granules. In this way, we have for the first time obtained evidence that one of the small GTP-binding proteins associated with the atrial granules is indeed rab12p.

	Materials and Methods

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Cell Cultures
Primary cultures of atrial myocytes on laminin-coated dishes or coverslips were prepared from the hearts of ether-anesthetized 250- to 300-g Wistar rats as described previously.¹⁰ The cells were used for experiments on day 7 of culture. Cell lines of BHK-21, HeLa, MDCK, and AtT-20 were obtained from the Japanese Cancer Research Resources Bank and cultured in DMEM supplemented with 10% FBS.

Atrial Granule Isolation
Atrial secretory granules of adult Wistar rats were isolated by a Percoll density gradient method described by Thibault et al.¹¹ Briefly, a fraction of a postnuclear supernatant of rat atria was suspended in 20 mL of 53% Percoll solution in 0.25 mol/L sucrose containing 15 mmol/L EDTA and 10 mmol/L Tris-Cl (pH 7.4). After centrifugation at 32 500g for 60 minutes, two bands were observed in the tube: the white band at the bottom, which was a highly purified atrial granule fraction (see Fig 2), and the brown band at the top of the gradient, which was composed of cell debris, mitochondria, lysosomes, myofilaments, and microsomes, as described by Thibault et al. Purified atrial granules were collected as a pellet for subsequent electron microscopy and immunoblot analysis.

View larger version (199K): [in this window] [in a new window]   Figure 2. An electron micrograph of isolated atrial granules. Bar=1 µm.

For subcellular fractionation, fractions (1 mL) of the Percoll gradient were collected from the bottom of the tube with a peristaltic pump, diluted in 0.25 mol/L sucrose containing 15 mmol/L EDTA and 10 mmol/L Tris-Cl (pH 7.4), and centrifuged at 100 000g for 90 minutes. Resultant pellets were dissolved in 70 µL of SDS-PAGE sample buffer, and 20 µL of the samples was analyzed either by SDS-PAGE followed by Coomassie blue staining or by immunoblotting.

Antibodies
The peptide used for raising antibody was derived from the C-terminal hypervariable region (KKMPLDVLRSELSNSILSLQPEPE) of rat rab12p.⁷ The synthesized peptide was coupled to keyhole limpet hemocyanin (Calbiochem). Rabbits were immunized on three occasions at 2-week intervals with 1 mg of peptide mixed with Freund's adjuvant as previously described.¹² Affinity purification of antibodies was carried out over a matrix of the peptide coupled to 2-fluoro-1-methylpyridinium toluene-4-sulfonate–activated Sephadex (Seikagaku-Kogyo) according to the supplier's protocol. The peptide-absorbed antibody was prepared by mixing the antibody and the synthesized peptide as previously described.¹²

RNA Preparation, Probe Generation, and Northern Blot Analysis
Total cellular RNA was isolated by homogenization in guanidinium isothiocyanate and centrifugation in cesium chloride as described by Maniatis et al.¹³ cDNA strands were synthesized from 2 µg of total RNA by using a first-strand synthesis kit (Amersham Corp) with random primers. The reverse-transcribed cDNA was used as the PCR template to synthesize a 413-bp cDNA fragment of rat rab12. The primers used to amplify the fragment of rat rab12 cDNA⁷ were 5'-TACAGATATGGGACACAGC-3' (forward) and 5'-GATCTCAGGCTCTGGTTGTAG-3' (reverse). The PCR-amplified fragment was cloned into a T vector (Amersham) and sequenced using a DNA sequencer (Applied Biosystems). The probe for rab12 detection by Northern blot analysis was generated by digesting the vector with BamHI and HindIII to release the insert from the vector. The probe was purified by extraction after agarose gel electrophoresis. For RNA blots, 18 µg of total RNA was resolved on agarose-formaldehyde gel and transferred to nylon membranes (Amersham). The blots were hybridized in a hybridization buffer (5x SSC, 50% formamide, 2.5x Denhardt's solution, and 100 µg/mL herring sperm DNA) for 18 hours at 42°C with random-primed ³²P-labeled rab12 probe. Membranes were washed at a final stringency of 0.1x SSC/0.1% SDS at 60°C (1x SSC contains 0.15 mol/L NaCl and 0.015 mol/L trisodium citrate [pH 7.0]). Visualization of mRNA hybridized with the probe was performed by a Fuji BAS 2000 image analyzer. RNA quantity and integrity were verified by staining the agarose gels with ethidium bromide.

Immunoprecipitation, Immunoblot Analysis, and GTP-Overlay Assay
Proteins, obtained by solubilizing rat tissues or cultured cells in RIPA buffer (50 mmol/L Tris [pH 7.2], 1 mmol/L EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Nonidet P-40, and protease inhibitors),¹⁰ were centrifuged for 20 minutes at 10 000g. Clarified materials were incubated with anti-rab12 antibody for 12 hours at 4°C with mild agitation. Protein A/agarose (Boehringer Mannheim) was then added, and incubation was continued for 3 hours at room temperature. The resultant protein A/agarose antibody-antigen complexes were washed five times with RIPA buffer and then washed twice with 50 mmol/L Tris-Cl (pH 6.9). After the washing, the antigen was eluted from the beads with 60 µL of SDS-PAGE sample buffer. The elute (20 µL) was subjected to immunoblot analysis or GTP-overlay assay. The protein concentration of the samples was determined with the BCA protein assay kit (Pierce), according to the supplier's protocol.

Proteins of immunoprecipitates or of RIPA-solubilized samples were separated on SDS-PAGE and transferred to nitrocellulose sheets. The sheets were incubated for 1 hour with anti-rab12 antibody diluted 1:1000 with a blocking buffer (PBS containing 5% nonfat milk and 0.1% Tween 20), followed by incubation with protein A conjugated with horseradish peroxidase (BioRad) diluted 1:2000 in the same buffer. Antigen-antibody complexes were visualized using an ECL detection kit (Amersham).

GTP-binding proteins on the nitrocellulose sheets were detected using a [³²P]GTP-overlay assay according to the method by Lapetina and Reep.¹⁴ [³²P]GTP binding was visualized with a Fuji BAS 2000 image analyzer.

Immunoelectron Microscopy
Specimens for immunogold electron microscopy were prepared by the method of Tokuyasu.¹⁵ Rat atria, which were fixed in PBS containing 4% paraformaldehyde and 0.1% glutaraldehyde, were frozen in liquid nitrogen after infiltration with 2.3 mmol/L sucrose. Ultrathin sections were cut on a cryoultramicrotome (Reichert Jung) and stained with anti-rab12 antibody, followed by incubation with 10 nm gold-conjugated goat anti-rabbit IgG (Amersham). For controls, the primary antibody was replaced by preimmune serum. Antibodies were diluted in PBS containing 0.5% BSA and 0.1% gelatin. Immunostained sections were embedded in methyl cellulose after staining with 2% uranyl acetate for examination by a JEOL 2000EX electron microscope.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of rab12 mRNA
Northern blot analysis was carried out to investigate the expression of rab12 mRNA in atria as well as in several other tissues or organs. Fig 1 shows that an 2.4-kb single transcript of rab12 was observed in all tissues examined, although it was detected at a very low level in kidney and lung. The size of the 2.4-kb transcript detected by the rab12 probe is similar to that reported by Olkkonen et al,⁸ which minimizes the possibility of cross hybridization between different rab mRNAs and the cDNA probe used in the present study.

View larger version (72K): [in this window] [in a new window]   Figure 1. Northern blot analysis of rab12 mRNA in various tissues. A, Total RNA (18 µg) from atria (lane 1), ventricle (lane 2), kidney (lane 3), lung (lane 4), and liver (lane 5) was electrophoresed and probed with radiolabeled cDNA fragment of rab12. B, Ethidium bromide–stained 28S ribosomal RNAs are shown, demonstrating that approximately equal amounts of RNA were loaded.

Immunoblot Analysis
Immunoblot analysis was performed to examine the presence of rab12p on the atrial granules. The purity of isolated atrial granules was checked by both electron microscopy (Fig 2) and electrophoresis (Fig 3A, lane 1). On the nitrocellulose sheets to which atrial granule proteins and total proteins of atrial culture homogenates were transferred, antibody against rab12p recognized a single band migrating at 28 kD (Fig 3B). To confirm that the 28-kD protein recognized by the antibody is a GTP-binding protein, the antigen immunoprecipitated from rat atria by the antibody was separated by SDS-PAGE and transferred to nitrocellulose sheets, on which a [³²P]GTP-overlay assay was performed. The result shows that the 28-kD protein immunoprecipitated by the anti-rab12 antibody bound [³²P]GTP, whereas no [³²P]GTP binding was detected in the samples immunoprecipitated by preimmune serum or peptide (antigen)–absorbed anti-rab12 antibody (Fig 3C, lanes 1 to 3). The GTP-overlay assay also showed that the atrial granules contained at least four small GTP-binding proteins with molecular masses of 22 to 28 kD (Fig 3C, lane 4) and that one protein with the highest molecular mass (28 kD) might correspond to rab12p. These results indicate that rab12p, a 28-kD small GTP-binding protein, is expressed in the atrial myocytes and might be a component of the atrial granules.

View larger version (40K): [in this window] [in a new window]   Figure 3. Identification of rab12p immunoreactive antigen in immunoblot analysis and [32P]GTP overlay. A and B, Proteins of isolated atrial granules (lane 1) and of homogenates of cultured atrial myocytes (lane 2) were separated on SDS-PAGE, and fractionated proteins were either stained by Coomassie blue (A) or subjected to immunoblot analysis using anti-rab12 antibody (B). The 18-kD protein in the Coomassie blue–stained gel (panel A, lane 1) is ANP. C, The antigens immunoprecipitated from rat atria by preimmune serum (lane 1), the anti-rab12 antibody (lane 2), and the antigen-absorbed anti-rab12 antibody (lane 3) were resolved in SDS-PAGE and transferred to nitrocellulose membranes on which a [32P]GTP-overlay assay was carried out. In lane 4 of panel C, the atrial granule proteins were resolved on SDS-PAGE and similarly processed for [32P]GTP overlay.

Immunocytochemical Analysis
Immunofluorescence microscopy carried out on frozen sections of rat atria and the cultured atrial myocytes suggested the association of rab12p with the atrial granules (not shown). To obtain more direct evidence that the protein is localized on the granules, immunogold electron microscopy was carried out on cryoultrathin sections of rat atria. The majority of gold particles complexed with anti-rab12 antibody appeared to label the atrial granules, with a tendency of gold particles to be present at the periphery of the granules (Fig 4). Little labeling was observed in the nucleus, mitochondria, myofilaments, or sarcolemma. No specific labeling was detected in controls in which the primary antibody was replaced by preimmune serum (not shown).

View larger version (82K): [in this window] [in a new window]   Figure 4. Immunogold electron microscopy of rab12p. Cryoultrathin sections of rat atria were stained with anti-rab12 antibody, followed by 10-nm gold-conjugated anti-rabbit IgG. Immunogold labeling was detected on the atrial granules; the nucleus (N), mitochondria (M), and sarcolemma (SL) were negative (A and B). Gold particles tend to label the periphery of the atrial granules (B). Bars=0.2 µm.

Identification of rab12p on a Percoll Density Gradient
The results of immunocytochemistry strongly suggested that rab12p is highly concentrated on the atrial granules. To confirm this observation, fractions from a Percoll density gradient were analyzed by immunoblot with anti-rab12 antibody. After centrifugation of a postnuclear supernatant over a Percoll density gradient, the atrial granules were sedimented as a white band at the bottom of the tube and recovered in fractions 2 to 5; other cell organelles that migrated toward the top were recovered in fractions 15 to 18 (Fig 5A), as previously reported.¹¹ High purity of the atrial granules sedimented at the bottom of the tube was proved by electron microscopy (Fig 2). By immunoblotting with the antibody against rab12p, the protein was found to be present in the ANP-containing atrial granule fraction (fraction 3 in Fig 5B) but hardly detectable in fractions 15 to 17, which were composed of diverse cell organelles. The result provides the evidence supporting our conclusion that rab12p is a component of the atrial granules.

View larger version (49K): [in this window] [in a new window]   Figure 5. Identification of rab12p on a Percoll density gradient. A postnuclear supernatant prepared from rat atria was subjected to Percoll gradient centrifugation. The fraction (1 mL) of the gradient was collected from the bottom of the tube and centrifuged, and resultant pellets were dissolved in SDS-PAGE sample buffer. Of 20 fractions, the samples of an odd number were subjected to SDS-PAGE. Fraction 1 indicates the sample from the bottom of the tube. Separated proteins were either stained with Coomassie blue (A) or transferred to nitrocellulose sheets for immunoblot to detect ANP or rab12p (B). The 18-kD protein in fractions 3 and 5 in panel A is ANP concentrated in the purified atrial granules. As shown in panel B, Percoll gradient centrifugation divided ANP into two, the bottom (fractions 3 and 5) and the top (fractions 15 and 17), as previously reported.11 rab12p was detected in fraction 3 of highly purified atrial granules (B).

Expression of rab12p in Various Tissues and Cell Lines
Since our preliminary immunoblot analysis revealed relatively low levels of rab12p expression in many tissues, we examined its expression by immunoprecipitation followed by immunoblot analysis. We first compared rab12p expression between atria and ventricle. The expression level of rab12p in the atria was almost the same as that in the ventricle (Fig 6A). We further examined rab12p expression in many endocrine tissues, as well as in several organs and cell lines (Fig 6B and 6C). The results showed that although the expression levels of rab12p varied between tissues and cells, its expression was detectable in all tissues and cell lines except liver and kidney. Low expression levels of rab12p in liver and kidney are consistent with the results of Northern blot analysis (Fig 1).

View larger version (20K): [in this window] [in a new window]   Figure 6. Expression of rab12p in various tissues and cells. rab12p was immunoprecipitated from 6.5 mg proteins of tissue homogenates (A and B) or from 2.5 mg proteins of cultured cell homogenates (C). The cells, tissues, and organs used for immunoprecipitation are indicated at the top, except for panel A, in which atria and ventricle are numbered 1 and 2, respectively. Precipitated antigens were separated on SDS-PAGE and transferred to nitrocellulose membranes for immunoblot analysis using anti-rab12 antibody. A sample of the atria was included in all blots as the internal standard. The arrow in panel A indicates that immunoglobulin cross-reacted with protein A/horseradish peroxidase.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have shown that multiple small GTP-binding proteins are associated with atrial granules (Fig 3C), a result consistent with the previous report of Wildey and Matyas.¹⁶ This finding is not specific for atrial granules, since multiple small GTP-binding proteins have been reported to be associated with the zymogen granules of the pancreas¹⁷ and the adrenal chromaffin cell granules.¹⁸ Therefore, it is likely that several small GTP-binding proteins may function cooperatively as GTPases for the intracellular traffic of secretory granules in endocrine and exocrine cells. By biochemical and immunocytochemical analysis, we have obtained evidence in the present study supporting the conclusion that one of the small GTP-binding proteins associated with the atrial granules is rab12p.

rab12 cDNA with a complete open reading frame has not previously been obtained.^{7 8} Moreover, little information is available concerning the distribution and the intracellular localization of rab12p, except for one report⁸ in which the protein was reported to be localized by immunofluorescence microscopy in the perinuclear Golgi region of BHK-21 cells. That report, however, lacked the immunoblot analysis necessary for characterization of the anti-rab12 antibody used in the study. To our knowledge, identification of rab12p as a secretory granule–associated GTPase has not previously been reported.

By using immunoprecipitation/immunoblot analysis with specific antibody against rab12p, we have extensively examined the expression of rab12p in multiple tissues and cell lines. The results support the conclusion that rab12p is a ubiquitously expressed small GTP-binding protein. It is noteworthy that in addition to regulated secretory cells (the atrial myocytes and AtT-20), the protein is also expressed in constitutive secretory cell lines (BHK, HeLa, and MDCK) as well as in ventricles from which ANP is constitutively secreted. The observation suggests that the protein may function in vesicular transport in both types of secretory cells. Therefore, it is likely that rab12p is a mammalian counterpart for Sec4p. Identification of other types of small GTP-binding proteins associated with the atrial granules is now under investigation.

	Selected Abbreviations and Acronyms

 

ANP = atrial natriuretic peptide AtT-20 = mouse pituitary tumor cells BHK-21 = hamster kidney fibroblastic cells HeLa = human cervix epithelial cells MDCK = canine kidney epithelial cells PCR = polymerase chain reaction rab12p = rab12 protein

	Acknowledgments

 
This study was supported by grants from the Ministry of Education, Science, and Culture of Japan. We are grateful to Dr Ernest Page for critical review of the manuscript.

Received September 7, 1995; accepted November 15, 1995.

	References

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