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Institut de Biologie Cellulaire et de Morphologie (S.C., T.C., R.R.), University of Lausanne, 1005 Lausanne, Switzerland; and The Physiological Laboratory (L.P.H., R.D.B.), University of Liverpool, Liverpool L69 3BX, United Kingdom
Address all correspondence and requests for reprints to: Dr. Romano Regazzi, Institut de Biologie Cellulaire et de Morphologie, Rue du Bugnon 9, 1005 Lausanne, Switzerland. E-mail: Romano.Regazzi{at}ibcm.unil.ch.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Rab GTPases exert their function by interacting with one or more downstream effectors. Thus, the precise definition of the role of Rab3 and Rab27 in insulin exocytosis will require the identification of their molecular partners. Until recently, Rab effectors were thought to be very selective and to mediate the action of only one GTPase. However, systematic analysis revealed that often Rab effectors can associate with a subset of related GTPases. This is the case of Noc2, a 38-kDa protein abundantly expressed in pancreatic ß-cells and in other endocrine cells (9) that has recently been shown to bind both Rab3 and Rab27 (10). Because of this property, Noc2 would be ideally suited to regulate insulin exocytosis. However, the function of Noc2 in pancreatic ß-cells has not been investigated, and its role in PC12 cells, the only secretory system in which the protein has been studied, is still controversial (9, 11).
Here we determined the subcellular distribution and the function of Noc2 in insulin-secreting cells. We found that a large fraction of the protein is associated with insulin granules and that its recruitment on secretory vesicles is mediated mainly by the interaction with Rab27. In addition, we demonstrate that alteration in the cellular level of Noc2 profoundly impairs ß-cell exocytosis, indicating that the protein is a key element of the machinery controlling insulin secretion.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) but not with other more distantly related members of the Rab family such as Rab13 (not shown). The binding of Noc2 to Rab3 was strictly GTP-dependent (11). In contrast, under our assay conditions, the capacity to associate with Rab27 was not significantly influenced by the nucleotide bound to the GTPase (Fig. 1). The interaction with Rab3 and Rab27 was differentially affected by point mutations within the amino terminus of Noc2. Replacement of valine 58 with alanine (Noc258A) prevented the interaction with Rab3 without interfering with the binding of Rab27 (Fig. 1). Introduction of a glutamic acid at position 65 (Noc265E) and, thus rendering the sequence of Noc2 more similar to the sequence of Rab3-interacting molecule (RIM), a well-known Rab3 effector (12), improved binding to both Rab3-GDP and Rab3-GTP without altering the association with Rab27. In contrast, mutations of cysteine 138 and 141 within the putative Zn2+ finger region to serine (Noc2138S,141S) or replacement of the sequence WFY within the putative Rab-binding motif (13) with alanines (Noc2154A,155A,156A) abolished the binding to both Rab3 and Rab27.
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). To confirm this observation, the organelles of INS-1E cells were separated on a sucrose density gradient (3, 4). As expected, the secretory granule proteins Slp4/Granuphilin-A and -B (7, 8) were enriched in the fractions containing 1.3–1.6 M sucrose (Fig. 3). In contrast, the synaptic-like vesicle marker Synaptophysin was recovered in lighter fractions (0.5–0.8 M sucrose). Noc2 cosedimented with Slp4/Granuphilin isoforms confirming its association with secretory granules (Fig. 3). In agreement with these results, green fluorescence protein (GFP)-tagged wild-type Noc2 transfected in INS-1E cells associated with secretory granules (Fig. 4). Similar results were obtained with the GFP-Noc265E mutant (supplemental Fig. 1
). We then investigated whether the subcellular localization of Noc2 requires the interaction with the two Rab GTPases present on secretory granules. As shown in Fig. 4, the GFP-Noc258A mutant colocalized with secretory granules, indicating that the binding to Rab3 is not essential for proper targeting of the protein to the secretory organelle. In contrast, GFP-Noc2138S,141S and GFP-Noc2154A,155A,156A, which are unable to bind to both Rab3 and Rab27, were homogenously distributed throughout the cell cytoplasm (Fig. 4). To confirm these morphological data, we determined the fraction of each Noc2 mutant present in soluble and membrane-associated forms in INS-1E cells. Endogenous Noc2 was found to be almost exclusively associated with the membrane fraction (data not shown). Wild-type GFP-Noc2 and GFP-Noc258A were predominantly recovered in the membrane fraction (Fig. 5). In contrast, GFP-Noc2154A,155A,156A was almost only detected in the soluble fraction. Similar results were obtained with the GFP-Noc2138S,141S mutant (data not shown).
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). The GFP-Noc258A and GFP-Noc265E mutants displayed the same effect on INS-1E secretion. In contrast, the two mutants (GFP-Noc2138S,141S and GFP-Noc2154A,155A,156A) that are not targeted to insulin granules did not modify the secretory response. Similar results were obtained after overexpression of the corresponding untagged Noc2 constructs (not shown).
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, siRNA-1 did not affect the expression of GFP-Noc2 in INS-1E cells. In contrast, the presence of siRNA-2 decreased dramatically the level of the transfected protein. Similar results were obtained when we analyzed the expression of endogenous Noc2 (Fig. 7A, middle panel). A sizeable reduction of endogenous Noc2 was detectable in homogenates of INS-1E cells transiently transfected with siRNA-2 but not with siRNA-1. Taking in to account that under our experimental conditions only part of the cells (approximately 30–40%) are efficiently transfected, this demonstrates that the silencer can potently diminish the cellular content of Noc2. The effect of siRNA-2 was specific because the expression of other endogenous proteins such as the Rab27 effector MyRIP/Slac2-c was unaffected (Fig. 7A, lower panel). Moreover, siRNA-2 did not modify the coexpression of hGH and had no influence on cell viability (not shown). We then analyzed the effect of Noc2 silencing on insulin exocytosis. Insulin secretion in response to glucose exposure is biphasic, with a first phase peaking after about 5 min, a decrease to almost basal levels and a second phase beginning after 10 min and lasting for hours (17). The early secretory phase is thought to reflect mainly the release of a pool of granules docked or very close to the plasma membrane. The sustained phase necessitates the recruitment of granules from a reserve pool. To determine whether the two secretory phases are affected by the silencing of Noc2, we measured the amount of hGH secreted within the first 10 min (early phase) and between 10 and 45 min (sustained phase). As expected, the secretory response of INS-1E cells transfected with the plasmid encoding siRNA-1 was not significantly different from control cells (Fig. 7B). In contrast, the amount of hGH released by the cells transfected with the plasmid directing the synthesis of siRNA-2 was strongly impaired. The decrease in Noc2 expression caused a reduction in both secretory phases (Fig. 7B). However, the effect was much more pronounced in the sustained phase when secretion was practically abolished. Despite the potent inhibition of hormone release, the distribution of insulin-containing secretory granules in INS-1E cells transfected with siRNA-2 was comparable to that of control cells (data not shown). This indicates that the absence of Noc2 is not affecting the transport of secretory granules from the Golgi apparatus to the cell periphery.
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, left panel) and in vitro translated Noc2 associated with the amino terminal domain of Munc13 (amino acids 3–317) (Fig. 8A, right panel). We then prepared affinity columns containing comparable amounts of either GST, a GST-fusion protein including the Rab-binding domain of RIM, a well-characterized partner of Munc13 (19), or GST-Noc2 (Fig. 8B, left panel). When rat brain extracts were loaded on these affinity columns, similar amounts of Munc13 were retained on the GST-RIM and on the GST-Noc2 (Fig. 8B, right panel).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A previous study on Noc2 concluded that the protein is localized in the cytosol (9). Here we demonstrate that although a minor fraction of endogenous Noc2 and of GFP-tagged Noc2 is indeed cytosolic, in INS-1E cells the bulk of the protein is associated with insulin-containing granules. Mutations that prevent the binding of Rab3 and Rab27 alter the subcellular localization of Noc2 indicating that the protein is recruited on secretory granules by the interaction with Rab GTPases. Selective impairment of Rab3 binding did not affect the distribution of Noc2, suggesting that in ß-cells Rab27 is the privileged partner of the protein. Indeed, Noc2 appears not to be ideally suited to bind Rab3. In fact, we were able to improve the Rab3 binding activity of Noc2 by replacing leucine 65 with glutamic acid. This point mutation renders the sequence of Noc2 more similar to that of RIM, a specific Rab3 partner (12). These findings are in good agreement with a recent study reporting a preferential association of Noc2 with Rab27 (10). However, our experiments do not exclude the possibility of a functional link between Noc2 and Rab3. Indeed, in PC12 cells that express large amounts of Rab3 and lower levels of Rab27, the Noc258A mutant is inactive (11). Thus, in this particular secretory system, the binding to Rab3 is probably functionally relevant. Because Noc2 has the capacity to interact both with Rab3 and Rab27, its preferential partner may differ according to the GTPase that predominates in the cell.
In INS-1E cells, overexpression of Noc2 constructs targeted to secretory granules causes a decrease in stimulated exocytosis. Thus, Noc2 is likely to exert its regulatory function by associating with the secretory organelle. Other Rab27-binding proteins have already been shown to be localized on insulin-containing granules and to regulate pancreatic ß-cell exocytosis (7, 8, 22). Overexpression of Noc2 may lead to a competition with these Rab27 effectors and could alter their regulatory activity. In addition, large amounts of Noc2 may perturb secretion by diminishing the availability of Munc13. Thus, alone overexpression experiments are not appropriate to assess the precise function of Noc2 in insulin exocytosis. To overcome this problem, we took advantage of the possibilities offered by the RNAi process. Specific silencing of the Noc2 gene led to a very profound decrease in the capacity of INS-1E cells to respond to insulin secretagogues. This demonstrates that Noc2 is a key element of the secretory machinery of pancreatic ß-cells that has a positive role in exocytosis. Because Noc2 is abundantly expressed in other endocrine cells (9), the protein is most probably also regulating the release of other hormones. Insulin secretion is a complex process involving a series of highly coordinated steps. Insulin-containing vesicles emerging from the Golgi apparatus are transported to the cell periphery probably through a microtubule-dependent mechanism powered by kinesin (23). The secretory granules are then translocated to and docked at the plasma membrane. In ß-cells, morphologically docked granules represent about one tenth of the total granule content and only a small fraction of these granules is immediately releasable (17). In fact, to become fusion competent morphologically docked granules are believed to undergo an activation process called priming. Short stimulations of insulin release involve exocytosis of immediately releasable granules. In contrast, prolonged stimulations necessitate the recruitment of granules from a reserve pool including undocked granules and docked granules that are not yet primed.
At present, the precise step in the secretory pathway that is controlled by Noc2 remains to be determined. In INS-1E cells lacking Noc2, the secretory granules are transported to the cell periphery, demonstrating the absence of a major defect in the routing of the secretory vesicles from the Golgi apparatus toward the plasma membrane. Silencing of Noc2 led to a very strong decrease in the sustained secretory phase, which was almost abolished. Early secretory phases were also diminished but the effect was less pronounced. This observation would be consistent with impairment in the recruitment of secretory granules from the reserve pool, a process required to compensate for the depletion of the readily realizable granule pool during prolonged stimulation. In agreement with this hypothesis, we found that Noc2 has the capacity to bind Munc13, a well-characterized neuronal protein that plays an essential role in vesicle priming (19). Munc13 has very recently been identified also in pancreatic ß-cells and has been shown to be involved in the regulation of insulin exocytosis (18). Thus, it is possible that at least part of the effect of Noc2 on exocytosis may be mediated through the control of the priming of secretory granules.
In conclusion, we have demonstrated that Noc2 is associated via a Rab27-dependent mechanism to insulin-containing granules and plays an essential role in the secretory process of pancreatic ß-cells. Future experiments will have to determine the precise mode of action of Noc2 and its possible involvement in the pathophysiology of diabetes mellitus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of Noc2 Mutants
The generation of plasmids for mammalian and bacterial expression of untagged wild-type Noc2, Noc258A, and Noc2154A,155A,156A and of their corresponding GST-fusion proteins have been described previously (11). Wild-type GFP-Noc2, GFP-Noc258A and GFP-Noc2154A,155A,156A were produced by subcloning the coding sequence of the untagged proteins in frame with GFP in pcDNA3 (Invitrogen, San Diego, CA). The Noc2138S,141S mutant was generated by site-directed mutagenesis with the QuikChange kit (Stratagene, La Jolla, CA) using the following primers: sense, 5'-CCCCTGTGGCTGAGTAAGATCAGCAGTGAGCAGAG-3'; antisense, 5'-CTCTGCTCACTGCTGATCTTACTCAGCCACAGGGG-3'. To produce the Noc265E mutant we used the primers: sense, 5'-AGAGCAGAGCGGGAGGACATCCTGGAG-3'; antisense, 5'-CTCCAGGATGTCCTCCCGCTCTGCTCT-3'. Mutagenesis was verified by sequence analysis.
Cell Culture and Transfection
The insulin-secreting cell line INS-1E was cultured as previously described (25) in RPMI 1640 medium supplemented with 5% fetal calf serum, 50 IU/ml penicillin, 50 µg/ml streptomycin, 0.1 mM sodium pyruvate, and 0.001% ß-mercaptoethanol. Transient transfection experiments were performed using the Effectene transfection kit (QIAGEN, Valencia, CA) with a DNA/Effectene ratio of 1/25.
Immunocytochemistry
Untransfected cells or cells expressing the Noc2 constructs were seeded on glass coverslips coated with 20 µg/ml laminin and 2 mg/ml poly-L-lysine. After 2 d, the cells were fixed in 4% paraformaldehyde and incubated for 2 h with the first antibody diluted in PBS (pH 7.5), 0.1% goat serum, 0.3% Triton X-100, and 20 mg/ml BSA. The coverslips were rinsed with PBS, incubated for 30 min with the secondary antibody diluted in the same buffer, and mounted for confocal microscopy (Leica, model TCS NT, Lasertechnik, Heidelberg, Germany).
Sucrose Gradient
Insulin-containing secretory granules were separated as described (3, 4). Briefly, INS-1E cells were homogenized in 5 mM HEPES (pH 7.4), 1 mM EGTA, 10 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.25 M sucrose. Cell debris and nuclei were eliminated by centrifuging the homogenate for 10 min at 3000 x g. The supernatant obtained was loaded onto a sucrose density gradient (0.45–2 M) and centrifuged for 18 h at 110,000 x g. The fractions were collected from the top of the gradient.
Subcellular Fractionation
INS-1E cells were transiently transfected with the GFP-Noc2 constructs. After 3 d the cells were disrupted by brief sonication at 4 C in 10 mM Tris-HCl (pH 7.5), 5 mM KCl, 2 mM MgCl2, 5 µg/ml leupeptin and 5 µg/ml aprotinin. The homogenate was centrifuged at 100,000 x g for 1 h. The supernatant was collected and used as cytosolic fraction. The pellet was resuspended in the same buffer and centrifuged again at 100,000 x g for 1 h. The pellet yielded by the second centrifugation was used as crude membrane fraction.
Interaction of Noc2 with Rab GTPases
INS-1E cells transiently transfected with wild-type GFP-Rab3a or wild-type GFP-Rab27a were lysed in buffer A (20 mM Tris-HCl, 200 mM NaCl, 10% glycerol, 1% Triton X-100, 5 µg/ml leupeptin, and 5 µg/ml aprotinin). The homogenates were exposed to 100 µM GDP or GTP
S for 15 min at 30 C and then incubated for 1 h at 4 C with 2 µg of GST-Noc2 fusion proteins immobilized on glutathione-agarose beads (Sigma, St. Louis, MO). At the end of the incubation the beads were washed in buffer A and the proteins remaining attached to the beads analyzed by Western blotting.
Interaction of Noc2 with Munc13
Bacterially expressed GST, GST-Noc2, and GST-Munc13–1 (amino acids 3–317) were immobilized on glutathione-agarose beads and resuspended in buffer B [20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM dithiothreitol, 5% glycerol, 0.05% Tween-20, and 1 mg/ml BSA]. The beads were incubated for 1 h at 4 C with 35S-labeled Munc13.1 or Noc2 produced by in vitro translation (Promega, Madison, WI). After several washes in buffer B, the proteins associated with the affinity columns were separated by SDS-PAGE and visualized by autoradiography. For the experiment presented in Fig. 8B, a rat brain fragment was homogenized in 320 mM sucrose in the presence of a cocktail of protease inhibitors (Roche, Rotkreuz, Switzerland). The homogenate was centrifuged for 10 min at 2000 x g and the supernatant incubated for 1 h at 4 C with GST, GST-RIM1 (amino acids 11–398) or GST-Noc2 immobilized on glutathione-agarose beads. At the end of the incubation the beads were rinsed in buffer A. The proteins remaining attached to the beads were analyzed by Western blotting with an antibody directed against Munc13.
Preparation of Vectors for Noc2 Silencing
Two mammalian expression vectors (siRNA-1 and siRNA-2) directing the synthesis of small interfering RNAs targeted against Noc2 were prepared according to the method of Brummelkamp et al. (24). cDNA fragments encoding 19-nucleotide sequences derived from rat Noc2 and separated from its reverse 19-nucleotide complement by a short spacer were synthesized by MWG Biotech Co. (Ebersberg, Germany). For the siRNA-1 construct we used the following oligonucleotides: sense, 5'-GATCCCCGTACATCTTGCCCCTGAAATTCAAGAGATTTCAGGGGCAAGATGTACTTTTTGGAAA-3'; antisense, 5'-AGCTTTTCCAAAAAGTACATCTTGCCCCTGAAATCTCTTGAATTTCAGGGGCAAGATGTACGGG-3'. For siRNA-2: sense, 5'-GATCCCCTATGACAAGCCTAGAGGGGTTCAAGAGACCCCTCTAGGCTTGTCATATTTTTGGAAA-3'; antisense, 5'-AGCTTTTCCAAAAATATGACAAGCCTAGAGGGGTCTCTTGAACCCCTCTAGGCTTGTCATAGGG-3'. The cDNA fragments were annealed and cloned in front of the H1-RNA promoter in the pSUPER vector (24). The specificity of each sequence was verified by basic local alignment search tool (BLAST) search against GenBank.
The capacity of each siRNA plasmid to reduce the expression of exogenously expressed Noc2 was first determined by transiently cotransfecting them in INS-1E cells with GFP-Noc2. The expression level of GFP-Noc2 obtained after 3 d was determined by Western blotting using an antibody against GFP. The impact of the silencers on the expression of endogenous Noc2 and Slac2c/MyRIP was estimated by analyzing by Western blotting the amount of each protein remaining after 3 d in INS-1E cells transiently transfected with the siRNA plasmids. Cell viability was estimated by transiently cotransfecting the silencers with a plasmid encoding GFP. The total cell number and the number of GFP-positive cells present in the wells 3 d after transfection was the same in control cells and in cells expressing the silencers.
Secretion Experiments
INS-1E cells were transiently cotransfected with a plasmid encoding hGH and with plasmids encoding either the silencers or the Noc2 expression constructs. Three days later, the cells were preincubated for 30 min in 20 mM HEPES (pH 7.4), 128 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 2.7 mM CaCl2. The medium was then removed and the cells incubated for the indicated periods at 37 C either in the same buffer (basal) or in a buffer containing 20 mM HEPES (pH 7.4), 53 mM NaCl, 80 mM KCl, 1 mM MgCl2, 2.7 mM CaCl2, 20 mM glucose, 1 µM forskolin, and 1 mM 3-isobutyl-1-methylxanthine (IBMX) (stimulated). Exocytosis from transfected cells was assessed by measuring by ELISA the amount of hGH released in the medium during the incubation period (Roche).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: GFP, Green fluorescence protein; GST, glutathione-S-transferase; hGH, human GH; IBMX, 3-isobutyl-1-methylxanthine; RIM, Rab3-interacting molecule; RNAi, RNA interference; siRNA, small interfering RNA.
Received for publication July 31, 2003. Accepted for publication October 21, 2003.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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