Dominant Negative Rab3D Mutants Reduce GTP-bound Endogenous Rab3D in Pancreatic Acini
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m309910200
ISSN1083-351X
AutoresXuequn Chen, Stephen A. Ernst, John A. Williams,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoTwo dominant negative mutants of Rab3D, N135I and T36N were recently reported to inhibit the early phase of regulated amylase secretion from mouse pancreatic acini (Chen, X., Edwards, J. A., Logsdon, C. D., Ernst, S. A., and Williams, J. A. (2002) J. Biol. Chem. 277, 18002–18009). Immunocytochemical studies showed that while the wild-type Rab3D localized to zymogen granules, the two dominant negative mutants did not localize to granules and were primarily in the basolateral regions of the cell. The present study, therefore, evaluated the potential mechanisms by which the dominant negative mutants might act. An affinity precipitation assay based on the property of the Rab3 effector Rim1 to interact only with GTP-bound Rab3D was developed. 78.9 ± 4.5% of total endogenous Rab3D was found in the GTP-bound form. Overexpression of HA-tagged Rab3D, and its Q81L, N135I, and T36N mutants had no effect on the total amount of endogenous Rab3D. However, the dominant negative mutants, T36N and N135I, reduced GTP-bound endogenous Rab3D by 70.0 ± 3.5% and 72.7 ± 1.2%, respectively, while the wild-type Rab3D and Q81L mutant had no effect. Triton X-114 phase separation and cell fractionation studies showed that dominant negative Rab3D mutants did not alter isoprenylation or membrane association of endogenous Rab3D. The dominant negative Rab3D did not affect the amount of endogenous Rab3D on purified zymogen granules as assessed by either Western blotting or immunocytochemistry, but reduced the GTP-bound form by 78.6 ± 3.3%. The two dominant negative Rab3D mutants, therefore, interfere with endogenous Rab3D function by blocking the GDP/GTP exchange but not zymogen granule targeting of endogenous Rab3D. Two dominant negative mutants of Rab3D, N135I and T36N were recently reported to inhibit the early phase of regulated amylase secretion from mouse pancreatic acini (Chen, X., Edwards, J. A., Logsdon, C. D., Ernst, S. A., and Williams, J. A. (2002) J. Biol. Chem. 277, 18002–18009). Immunocytochemical studies showed that while the wild-type Rab3D localized to zymogen granules, the two dominant negative mutants did not localize to granules and were primarily in the basolateral regions of the cell. The present study, therefore, evaluated the potential mechanisms by which the dominant negative mutants might act. An affinity precipitation assay based on the property of the Rab3 effector Rim1 to interact only with GTP-bound Rab3D was developed. 78.9 ± 4.5% of total endogenous Rab3D was found in the GTP-bound form. Overexpression of HA-tagged Rab3D, and its Q81L, N135I, and T36N mutants had no effect on the total amount of endogenous Rab3D. However, the dominant negative mutants, T36N and N135I, reduced GTP-bound endogenous Rab3D by 70.0 ± 3.5% and 72.7 ± 1.2%, respectively, while the wild-type Rab3D and Q81L mutant had no effect. Triton X-114 phase separation and cell fractionation studies showed that dominant negative Rab3D mutants did not alter isoprenylation or membrane association of endogenous Rab3D. The dominant negative Rab3D did not affect the amount of endogenous Rab3D on purified zymogen granules as assessed by either Western blotting or immunocytochemistry, but reduced the GTP-bound form by 78.6 ± 3.3%. The two dominant negative Rab3D mutants, therefore, interfere with endogenous Rab3D function by blocking the GDP/GTP exchange but not zymogen granule targeting of endogenous Rab3D. Small GTPases of the Rab/Ypt family form the largest branch of the Ras-related small G-protein superfamily and are recognized as key protein components involved in vesicular trafficking and membrane fusion in eukaryotic cells (1.Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2091) Google Scholar, 2.Bean A.J. Scheller R.H. Neuron. 1997; 19: 751-754Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Rab proteins act as molecular switches, which cycle between the GDP-bound inactive and GTP-bound active forms. The conversion from the GDP-bound form to the GTP-bound form is stimulated by a Rab GEF 1The abbreviations used are: GEFguanine nucleotide exchange factorHAhemagglutininEGFPenhanced green fluorescent proteinCMVcytomegalovirusRimRab3-interacting moleculeZGzymogen granulesPipes1,4-piperazinediethanesulfonic acidPBSphosphate-buffered salineDTTdithiothreitolGTPγSguanosine 5′-3-O-(thio)triphosphatePNSpostnuclear supernatant fractionsGSTglutathione S-transferase. (guanine nucleotide exchange factor), and the conversion of the GTP-bound form to the GDP-bound form is catalyzed by a Rab GAP (GTPase-activating protein) (3.Segev N. Curr. Opin. Cell Biol. 2001; 13: 500-511Crossref PubMed Scopus (247) Google Scholar). A characteristic of Rab proteins is that a cycle of association with and dissociation from membranes is superimposed onto their GDP/GTP cycle. These two types of cycling are essential for Rab function in vesicle trafficking and fusion. At steady state, a portion of a Rab protein is detected in the cytosol. This pool is maintained in the GDP-bound form through interaction with another important regulator of Rab cycling, Rab GDI (GDP dissociation inhibitor) (1.Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2091) Google Scholar). guanine nucleotide exchange factor hemagglutinin enhanced green fluorescent protein cytomegalovirus Rab3-interacting molecule zymogen granules 1,4-piperazinediethanesulfonic acid phosphate-buffered saline dithiothreitol guanosine 5′-3-O-(thio)triphosphate postnuclear supernatant fractions glutathione S-transferase. A model of Rab GDP/GTP and membrane/cytosol cycling had been developed based on pioneering studies of Rab9 and Rab5 (4.Soldati T. Shapiro A.D. Svejstrup A.B. Pfeffer S.R. Nature. 1994; 369: 76-78Crossref PubMed Scopus (152) Google Scholar, 5.Ullrich O. Horiuchi H. Bucci C. Zerial M. Nature. 1994; 368: 157-160Crossref PubMed Scopus (252) Google Scholar), and modified according to more recent studies (6.Mohrmann K. Gerez L. Oorschot V. Klumperman J. van der Sluijs P. J. Biol. Chem. 2002; 277: 32029-32035Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 7.Ayad N. Hull M. Mellman I. EMBO J. 1997; 16: 4497-4507Crossref PubMed Scopus (48) Google Scholar, 8.Wilson A.L. Erdman R.A. Maltese W.A. J. Biol. Chem. 1996; 271: 10932-10940Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 9.Chou J.H. Jahn R. J. Biol. Chem. 2000; 275: 9433-9440Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 10.Luan P. Balch W.E. Emr S.D. Burd C.G. J. Biol. Chem. 1999; 274: 14806-14817Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In this model, GDI delivers Rab to the target membranes and itself is released into the cytosol. In a consecutive but separate step, the membrane-associated, inactive Rab proteins are converted into their active forms by compartment-specific GEFs (11.Wang W. Sacher M. Ferro-Novick S. J. Cell Biol. 2000; 151: 289-296Crossref PubMed Scopus (166) Google Scholar, 12.Siniossoglou S. Peak-Chew S.Y. Pelham H.R. EMBO J. 2000; 19: 4885-4894Crossref PubMed Scopus (139) Google Scholar, 13.Novick P. Garrett M.D. Nature. 1994; 369: 18-19Crossref PubMed Scopus (17) Google Scholar). The Rab, now in its GTP-bound form, recruits its effector(s) to the vesicle. Following fusion with the acceptor compartment, a GAP stimulates the Rab protein to hydrolyze its bound GTP and the resulting GDP-bound form is recognized and retrieved from acceptor compartment by Rab GDI, possibly facilitated by other factors. Pancreatic acinar cells are the functional unit of digestive enzyme secretion and have long been used as a model to study the packaging and secretion of secretory proteins and its control by neurotransmitters and hormones (14.Palade G. Science. 1975; 189: 347-358Crossref PubMed Scopus (2425) Google Scholar, 15.Jamieson J.D. Palade G.E. J. Cell Biol. 1967; 34: 597-615Crossref PubMed Scopus (372) Google Scholar, 16.Jamieson J.D. Palade G.E. J. Cell Biol. 1971; 50: 135-158Crossref PubMed Scopus (268) Google Scholar). In acinar cells, secretory proteins are transferred by vesicular fusion through a series of compartments to the mature zymogen granule, which upon cellular stimulation fuses with the apical plasma membrane to release its contents into the lumen. The Rab3 proteins are the Rab species associated with synaptic or secretory vesicles in neurons, neuroendocrine, endocrine, and exocrine cells and are thought to play an important role in regulated exocytosis (17.Geppert M. Sudhof T.C. Annu. Rev. Neurosci. 1998; 21: 75-95Crossref PubMed Scopus (224) Google Scholar, 18.Johannes L. Lledo P.M. Roa M. Vincent J.D. Henry J.P. Darchen F. EMBO J. 1994; 13: 2029-2037Crossref PubMed Scopus (189) Google Scholar, 19.Chung S.H. Joberty G. Gelino E.A. Macara I.G. Holz R.W. J. Biol. Chem. 1999; 274: 18113-18120Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 20.Baldini G. Wang G. Weber M. Zweyer M. Bareggi R. Witkin J.W. Martelli A.M. J. Cell Biol. 1998; 140: 305-313Crossref PubMed Scopus (40) Google Scholar, 21.Lledo P.M. Vernier P. Vincent J.D. Mason W.T. Zorec R. Nature. 1993; 364: 540-544Crossref PubMed Scopus (181) Google Scholar). Recent work in our laboratory and others (22.Valentijn J.A. Sengupta D. Gumkowski F.D. Tang L.H. Konieczko E.M. Jamieson J.D. Eur. J. Cell Biol. 1996; 70: 33-41PubMed Google Scholar, 23.Ohnishi H. Ernst S.A. Wys N. McNiven M. Williams J.A. Am. J. Physiol. 1996; 271: G531-G538PubMed Google Scholar) has demonstrated that Rab3D is the only detectable Rab3 isoform in rodent pancreatic acini and is localized on zymogen granules. More recently, we reported (24.Chen X. Edwards J.A. Logsdon C.D. Ernst S.A. Williams J.A. J. Biol. Chem. 2002; 277: 18002-18009Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) that dominant negative mutants of Rab3D, N135I and T36N, introduced by adenoviral vector expression, inhibited regulated amylase secretion by mouse pancreatic acini, while the wild-type and active mutant Q81L had no effect. Localization of adenoviral expressed Rab protein showed that wild-type Rab3D localized to zymogen granules. The two dominant negative mutants did not localize to granules and were primarily in the basolateral cytoplasmic region of the cell. These observations raise the question of how the cytosolic dominant negative Rab3D mutants interfere with the zymogen granules (ZG)-associated endogenous Rab3D and inhibit its function in acinar secretion. To evaluate the potential mechanisms by which the dominant negative mutants might act, it is necessary to directly monitor active, GTP-bound Rab3D in acinar cells. Recently, affinity precipitation assays for detecting cellular GTP bound members of the Rho family have been developed (25.Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1374) Google Scholar, 26.Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 27.Sander E.E. ten Klooster J.P. van Delft S. van der Kammen R.A. Collard J.G. J. Cell Biol. 1999; 147: 1009-1022Crossref PubMed Scopus (741) Google Scholar, 28.Taylor S.J. Shalloway D. Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). This method takes advantage of the fact that the effector proteins interact only with GTP-bound G-protein and that binding of G-protein to the effector protein inhibits the GTPase activity of the G-protein. In the present study, we developed an affinity-precipitation assay to monitor the active state of endogenous Rab3D in pancreatic acinar cells based on the property of the Rab3 effector Rim to interact only with GTP bound Rab3 (29.Sun L. Bittner M.A. Holz R.W. J. Biol. Chem. 2001; 276: 12911-12917Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). A high percentage (∼80%) of endogenous Rab3D was found to be in GTP-bound conformation. In acini infected with adenoviruses expressing either two of the dominant negative Rab3D mutants, N135I and T36N, the GTP-bound endogenous Rab3D was reduced to only 30% of that of control acini. Furthermore, Western blot together with immunocytochemistry demonstrated that the dominant negative Rab3D mutants dramatically reduced the active state, but not the total amount, of endogenous Rab3D on the zymogen granules. Materials—The plasmid encoding GST (glutathione S-transferase)-RIM (Rab3-interacting molecule) (amino acids 1–399) fusion protein was obtained from Dr. Ronald W. Holz (University of Michigan). Anti-Rab3D antisera was a gift from Dr. Mark McNiven (Mayo Clinic, Rochester, MN). Glutathione-Sepharose 4B beads and Percoll were purchased from Amersham Biosciences; Rat monoclonal anti-HA antibody 3F10 from Roche Applied Science (Indianapolis, IN); and collagenase (CLSPA grade) from Worthington Biochemicals (Freehold, NJ). All other reagents were from Sigma. Isolation, Short Term Culture, and Adenoviral Infection of Pancreatic Acini—As previously described (24.Chen X. Edwards J.A. Logsdon C.D. Ernst S.A. Williams J.A. J. Biol. Chem. 2002; 277: 18002-18009Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), pancreatic acini were isolated from male ICR mice by collagenase digestion. Isolated acini from 2–3 pancreas were resuspended in Dulbecco's modified Eagle's medium (DMEM) and divided into 4 or 5 150-mm Petri dishes each containing 30 ml of Dulbecco's modified Eagle's medium enriched with 0.5% fetal bovine serum, 0.02% soybean trypsin inhibitor and antibiotics, and incubated at 37 °C overnight. The HA-tagged wild-type and mutant mouse Rab3D adenoviral constructs were made using AdEASY system as described previously (24.Chen X. Edwards J.A. Logsdon C.D. Ernst S.A. Williams J.A. J. Biol. Chem. 2002; 277: 18002-18009Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The shuttle vector pAdTrack-CMV also encodes EGFP driven by a separate CMV promoter. An adenovirus expressing bacterial β-galactosidase and EGFP, each under the control of a separate CMV promoter was used as the control virus. In the adenoviral infection experiments, either control β-galactosidase or various HA-tagged Rab3D adenoviruses (106 pfu/ml) were added, to the culture medium at the beginning of the incubation. Under this condition, over 95% of acini were infected as indicated by their EGFP expression. Preparation of Zymogen Granules—A previously described method for purifying zymogen granules from rat pancreas was adopted (30.Burnham D.B. Munowitz P. Thorn N. Williams J.A. Biochem. J. 1985; 227: 743-751Crossref PubMed Scopus (28) Google Scholar). In brief, isolated mouse pancreatic acini were homogenized in 3 ml ice-cold homogenization buffer containing 0.3 m sucrose, 25 mm Pipes, 200 μm CaCl2, 200 μm MgCl2, and 2 mm EGTA (pH 6.8) supplemented with 0.02% w/v soybean trypsin inhibitor. Homogenates were centrifuged for 10 min at 300 × g and 4 °C to generate postnuclear supernatant fractions (PNS). The PNS were then centrifuged for 10 min 4 °C at 1,000 × g to generate a crude particulate fraction enriched in secretory granules. The supernatant was removed, and the particulate fraction was resuspended in 1.5 ml of homogenization buffer, and an equal volume of Percoll was added. The mixture was then centrifuged at 100,000 × g for 20 min in a Beckman tabletop ultracentrifuge using a TLA 100.4 rotor. The dense white zymogen granule band near the bottom of the tube was collected and washed once with homogenization buffer. The purity of the preparation was confirmed by measuring the relative specific activity of amylase in each fraction as previously described (30.Burnham D.B. Munowitz P. Thorn N. Williams J.A. Biochem. J. 1985; 227: 743-751Crossref PubMed Scopus (28) Google Scholar). Amylase activity showed a 5–6-fold enrichment over the homogenate. GST Fusion Protein Expression—GST-Rim1 fusion proteins were expressed in Escherichia coli HB101 cells following induction at 37 °C for 2 h with isopropyl-β-d-thiogalactoside (IPTG). Bacterial pellets were lysed by means of a French Press in buffer containing 20% sucrose, 10% glycerol, 50 mm Tris (pH 8.0), 0.2 mm Na2S2O5,2mm MgCl2,2mm DTT, and 1% Triton X-100. GST-Rim was purified by incubation with glutathione-Sepharose 4B beads (Amersham Biosciences) for 1 h at 4 °C. The beads were washed three times with lysis buffer and stored at –20 °C in storage buffer containing 50 mm Hepes (pH 7.4), 150 mm NaCl2,5mm MgCl2, 5 mm DTT, 50% glycerol. Affinity Precipitation of Cellular GTP-bound Rab3D—Isolated pancreatic acini were washed with ice-cold PBS and lysed in buffer containing 50 mm Tris, pH 7.4, 150 mm NaCl, 10 mm MgCl2, 1 mm DTT, 0.2% Triton X-100, 2% glycerol supplemented with 10 μg/ml each of leupeptin and aprotinin, and 1 mm phenylmethylsulfonyl fluoride. Cell lysates were clarified by centrifugation at 20,000 × g at 4 °C for 10 min, and protein concentration was determined using protein assay reagent (Bio-Rad). In most experiments, 1 ml (0.5 mg/ml) of acinar lysate was incubated with 10 μg of GST-Rim attached to glutathione-Sepharose 4B beads for 1 h at 4 °C. Beads were washed three times, and the remaining proteins were eluted with SDS sample buffer, and run on SDS-PAGE. Bound Rab3D proteins were detected by Western blotting using anti-Rab3D and anti-HA antibodies and visualized with ECL reagent and recorded on film. Two Rab3D bands were revealed in each lane: the top corresponding to overexpressed HA-tagged Rab3D and the bottom to endogenous Rab3D. In vitro GDP or GTPγS protein loading was used for negative and positive controls (26.Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 29.Sun L. Bittner M.A. Holz R.W. J. Biol. Chem. 2001; 276: 12911-12917Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). 50 μl of 0.2 m EDTA and 2 μl of 100 mm GTPγS or 10 μl of 100 mm GDP were added to 1 ml of acinar lysate (0.5 mg/ml protein concentration), and the mixtures were incubated at 30 °C for 30 min. The reaction was terminated by adding 60 μl of 1 m MgCl2 and placing the samples on ice. Densitometric analysis of appropriately exposed film was performed and quantified using BioRad Multi-Analyst software. The amount of GST-Rim bound Rab3D was compared among different groups of samples and expressed as percentage of that of the control group. Immunocytochemistry and Confocal Microscopy—Purified zymogen granules prepared from β-galactosidase (control) and Rab3D T36N adenovirus-infected acini were resuspended in a small volume of PBS and transferred onto microscope slides (Fisher Superfrost plus). The granules were allowed to bind to slides for 10 min at room temperature in humid box, and then the PBS solution was replaced with 4% paraformaldehyde in PBS for 1 h at room temperature. After fixation, samples were rinsed with PBS, blocked with 5% normal goat serum in PBS for 30 min at room temperature, and then incubated with rabbit anti-Rab3D antibody diluted 1:200 or rat anti-HA antibody diluted 1:100 in 2% normal goat serum in PBS for 1.5 h at room temperature. After rinsing with PBS, and blocking with 2% normal goat serum in PBS, samples were incubated with Cy3-conjugated donkey anti-rabbit or anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:200 in 2% normal goat serum in PBS. Slides were viewed with a Zeiss LSM 510 confocal microscope and digitized images were processed using Photoshop 6.0 software (Adobe Systems Inc., Mountain View, CA). For quantification of immunofluorescence, digital images from three microscopic fields for each viral construct were chosen at random from each of three independent experiments and analyzed using Metamorph 6.0.4 software (Universal Imaging, Inc., Malvern, PA). After background and any saturated pixels were excluded, the mean pixel intensities from the separate fields of each condition were averaged. Results were expressed as percent of control (β-galactosidase) mean pixel intensity for the three experiments. Subcellular Fractionation and Triton X-114 Phase Separation—Isolated pancreatic acini were resuspended in lysis buffer containing 50 mm Tris, pH 7.4, 150 mm NaCl, 10 mm MgCl2, 1 mm DTT, 2% glycerol supplemented with 10 μg/ml each of leupeptin and aprotinin and 1 mm phenylmethylsulfonyl fluoride. The suspension was sonicated briefly and a PNS prepared by centrifugation for 10 min at 800 × g at 4 °C. High speed supernatants and membrane pellets, designated as cytosolic (C) and membrane (M) were obtained by centrifugation of the PNS at 100,000 × g for 45 min at 4 °C in a Beckman tabletop ultracentrifuge using a TLA 45 rotor. The membrane pellet was dissolved in lysis buffer containing 0.5% Triton X-100 and centrifuged again at 20,000 × g to remove insoluble proteins. To examine Rab3D isoprenylation, a Triton X-114 partitioning method was used as described previously (22.Valentijn J.A. Sengupta D. Gumkowski F.D. Tang L.H. Konieczko E.M. Jamieson J.D. Eur. J. Cell Biol. 1996; 70: 33-41PubMed Google Scholar, 31.Wagner A.C. Wishart M.J. Mulders S.M. Blevins P.M. Andrews P.C. Lowe A.W. Williams J.A. J. Biol. Chem. 1994; 269: 9099-9104Abstract Full Text PDF PubMed Google Scholar). Briefly, isolated acini were resuspended in 1 ml of lysis buffer containing 25 mm Tris, 150 mm NaCl, 1% Triton X-114, 5 mm EDTA, and supplemented with the proteinase inhibitors and sonicated. The acinar lysate was then incubated at 37 °C for 2 min and the resulting turbid solution was centrifuged at 1000 × g for 2 min at room temperature. The upper clear aqueous phase was separated from the bottom turbid detergent phase. The two phases were then both brought to a 1-ml volume with 1% Triton X-114. Both phases were kept on ice until the solutions became clear. The above process was repeated two more times to wash away contaminants. GST-RIM Interaction with GTP-bound Rab3D in Acinar Lysate Can Be Used to Assess the GTP Binding Status of Endogenous Rab3D in Vivo—To determine how the guanine nucleotide state of Rab3D influenced its interaction with Rim, different amounts (250,500 μg) of acinar lysate were treated to load all small G-proteins with GDP or GTPγS as negative or positive controls, respectively, and then incubated with GST-Rim-(1–399) in a "pulldown" protocol. Similar to prior studies with Rab3A (29.Sun L. Bittner M.A. Holz R.W. J. Biol. Chem. 2001; 276: 12911-12917Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), GST-Rim pulled down Rab3D when liganded with GTP but not with GDP (Fig. 1). This demonstrated that GST-RIM associated exclusively with the GTP-bound Rab3D and that the pull-down assay was able to detect the GTP-bound endogenous Rab3D. Moreover, the pull-down signals were proportional to the amount of total lysate. On the basis of these results, we compared the pull-down signal from the same amount of acinar lysate with or without GTPγS loading. Assuming that GTPγS activated all endogenous Rab3D and the signal can be used as 100%, we estimated that about 80% of the endogenous Rab3D was in active, GTP-bound conformation (Fig. 1). In four experiments, quantitative densitometry showed 78.9 ± 4.5% to be GTP-liganded (Table I). In separate subcellular fractionation experiments (Fig. 2A), we found that about 80% of total Rab3D was in the membrane fraction. The similarity of the percentage of GTP-bound and that of membrane-bound Rab3D led us to hypothesize that membrane-bound Rab3D was all GTP-bound while cytosolic Rab3D was GDP-bound. This hypothesis was tested by performing pull-down assays of Rab3D on the membrane and cytosolic fraction separately (Fig. 2, B and C). About 90% of membrane associated Rab3D was found to be GTP-liganded while only 2% of soluble Rab3D was GTP-liganded (Table I).Table IThe amount of total and active Rab3D in different subcellular fractionsTotal Rab3DGTP-bound Rab3D%Cellular lysate10078.9 ± 4.5Cytosol18.8 ± 1.31.7 ± 1.7Membrane81.3 ± 1.390.2 ± 10.4 Open table in a new tab Fig. 2Pools of Rab3D in different GDP/GTP binding status were revealed by subcellular fractionation. To analyze the distribution of total Rab3D, high-speed supernatants and membrane pellets, designated as cytosolic (C) and membrane (M), were subjected to SDS-PAGE and immunoblotted with anti-Rab3D antibody (A). To analyze the GTP-binding status of cytosolic (B) or membrane (C) endogenous Rab3D, cytosolic or solubilized membrane fraction of acinar lysate was incubated with GST-RIM beads and pull-down assay was conducted as described under "Experimental Procedures." In order to enhance the cytosolic pull-down signal, more protein and longer exposure time was used. Results shown are representative of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Dominant Negative Rab3D Mutants Reduce GTP-bound Endogenous Rab3D in Acinar Cells—We have reported that the dominant negative Rab3D mutants, Rab3D N135I and T36N, preferentially inhibited acinar amylase secretion (24.Chen X. Edwards J.A. Logsdon C.D. Ernst S.A. Williams J.A. J. Biol. Chem. 2002; 277: 18002-18009Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To address the mechanism by which dominant negative Rab3D mutants act, we examined the effect of the overexpressed wild-type and mutant Rab3D on the active GTP-bound state of endogenous Rab3D by use of the GST-RIM pull-down assay. The representative Western blot results are shown in Fig. 3. Because of the higher molecular weight introduced by the HA tag, anti-Rab3D antibody revealed two bands in each lane: the top band is overexpressed HA-tagged Rab3D, which may also be visualized by immunoblotting with anti-HA antibody, and the bottom is Rab3D, which does not react with anti-HA. None of the overexpressed Rab3D constructs affected the total amount of endogenous Rab3D (Fig. 3A). When the amount of GTP-bound Rab3D was evaluated using the GST-Rim pull-down, expression of wild-type Rab3D and the Q81L mutant had no effect, but both dominant negative Rab3D mutants, T36N and N135I greatly reduced GTP bound endogenous Rab3D (Fig. 3B). Quantitative data showed that the reduction was 70.0 ± 3.5% and 72.7 ± 1.2% respectively (Fig. 3C). One unexpected difference between T36N and N135I was that N135I interacted with GST-Rim as indicated in the Western blot following pull-down (Fig. 3B). This pull-down of N135I was independent of the presence of GDP or GTPγS and appeared to result from guanine nucleotide-independent interaction of Rab3D N135I and Rim (data not shown). Dominant Negative Rab3D Mutants Did Not Affect Isoprenylation and Membrane Association of Endogenous Rab3D— Early studies (4.Soldati T. Shapiro A.D. Svejstrup A.B. Pfeffer S.R. Nature. 1994; 369: 76-78Crossref PubMed Scopus (152) Google Scholar, 5.Ullrich O. Horiuchi H. Bucci C. Zerial M. Nature. 1994; 368: 157-160Crossref PubMed Scopus (252) Google Scholar) and our current results (Fig. 2) indicate that membrane association and GDP/GTP exchange of Rab proteins are closely coupled. We therefore tested whether or not the reduction of active Rab3D by dominant negative Rab3D mutants was due to the reduction of either isoprenylation or membrane association of endogenous Rab3D. The Triton X-114 partitioning method was used to address this issue since the isoprenylated Rab3D was expected to be in the detergent phase. As shown in Fig. 4A, in control acini, endogenous Rab3D was exclusively in the detergent phase consistent with a complete isoprenylation of Rab3D. In the acini expressing HA-tagged Rab3D constructs, neither the wild type nor the T36N mutant had an effect on the isoprenylation of endogenous Rab3D. However, a significant portion of overexpressed Rab3D from both wild-type and T36N were in the aqueous phase. Similar partitioning of Rab3D N135I mutant was also seen (data not shown). This could be due to a limited capacity for isoprenylation, or more likely the isoprenyl group was not as well protected in the cytosol as on the membrane. In either case, this result demonstrated that dominant negative Rab3D did not interfere with the isoprenylation of endogenous Rab3D. We then examine the effect of dominant negative Rab3D on the membrane association of endogenous Rab3D using a subcellular fractionation method. As shown in Fig. 4B, the amount of membrane associated endogenous Rab3D was the same in control and T36N virus infected acini. On the other hand, T36N itself was largely cytosolic, which is consistent with the basalateral cytosolic localization demonstrated in our previous immunocytochemistry studies (24.Chen X. Edwards J.A. Logsdon C.D. Ernst S.A. Williams J.A. J. Biol. Chem. 2002; 277: 18002-18009Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These data demonstrated that dominant negative Rab3D mutant did not affect membrane association of endogenous Rab3D. Dominant Negative Rab3D Significantly Reduced the Active State but Not the Total Amount of Endogenous Rab3D on Purified ZGs—The precise localization of Rab proteins to the correct organelle or membrane compartment is essential for their functions in regulating vesicular trafficking. Previous studies from our laboratory and others had shown that endogenous Rab3D was highly enriched on zymogen granule membrane in acinar cells (22.Valentijn J.A. Sengupta D. Gumkowski F.D. Tang L.H. Konieczko E.M. Jamieson J.D. Eur. J. Cell Biol. 1996; 70: 33-41PubMed Google Scholar, 32.Ohnishi H. Samuelson L.C. Yule D.I. Ernst S.A. Williams J.A. J. Clin. Investig. 1997; 100: 3044-3052Crossref PubMed Scopus (79) Google Scholar). W
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