Dominant Negative Rab3D Mutants Reduce GTP-bound Endogenous Rab3D in Pancreatic Acini

Abstract

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, 2). 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 1 (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). 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).A model of Rab GDP/GTP and membrane/cytosol cycling had been developed based on pioneering studies of Rab9 and Rab5 (4, 5), and modified according to more recent studies (6 -10). 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 (11-13). The Rab, now in its GTP-bound form, recruits its effector(s) to the vesicle. Following fusion with the acceptor compartment, a GAP stimulates ...

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

“…1). We also showed that VNUT co-localizes with Rab3D, which is a low-molecular weight GTP-binding protein that associates with secretory granules in exocrine glands, e.g., zymogen granules (ZGs) in pancreatic acini [9,21] (Fig. 1a, b).…”

“…Rat acinar cell line AR42J (CRL-1492, ATCC, passages [15][16][17][18][19][20][21][22][23][24][25] was cultured in RPMI media supplemented with 10 % fetal bovine serum (FBS) and penicillin/streptomycin. Cells were cultured in humidified incubators at 37°C, with 5 % CO 2 in air.…”

Male NOD mouse external lacrimal glands exhibit profound changes in the exocytotic pathway early in postnatal development

“…The precise function of rab3D in acinar secretion is still unclear although studies overexpressing active and dominant negative forms in pancreatic acini showed changes in exocytosis (Ohnishi et al, 1997;Chen et al, 2003). Surprisingly, recent generation of a rab3D knockout mouse resulted in no detectable phenotype in release kinetics in the exocrine pancreas or parotid gland (Riedel et al, 2002).…”

“…Although the precise function of rab3D in acinar secretion is still in question, its association with mature SVs is decreased concomitant with apical exocytosis (Wang et al, 2003). Dominant negative forms of rab3D reduce amylase secretion in pancreatic acini (Chen et al, 2003), while overexpression of wild type rab3D can enhance pancreatic acinar exocytosis (Ohnishi et al, 1997), suggesting its direct participation in acinar exocytosis. Actin filaments in lacrimal acini can be detected in association with peripheral membranes, with considerable enrichment beneath the apical membrane due to actin concentration in the dense cortical apical actin array which includes microvilli (da Costa et al, 1998).…”

Sdmg1 is a component of secretory granules in mouse secretory exocrine tissues

“…Pancreatic secretory granules can be purified by Percoll gradient separation of a particulate fraction of pancreatic postnuclear supernantant (Chen et al, 2003). The dense white band of secretory granules was collected in fraction 3 of the Percoll gradient, and microscopic inspection of the Percoll gradient fractions confirmed that large vesicles around 1 m in diameter were enriched and abundant in this fraction ( Fig.…”