© 2016. Published by The Company of Biologists Ltd.
Rab2a and Rab27a cooperatively regulate transition from granule maturation to exocytosis through the dual effector Noc2
Kohichi Matsunaga1, Masato Taoka3, Toshiaki Isobe3 and Tetsuro Izumi1,2*
1
Laboratory of Molecular Endocrinology and Metabolism, Department of Molecular
Medicine, Institute for Molecular and Cellular Regulation, and 2Research Program for Signal Transduction, Division of Endocrinology, Metabolism and Signal Research, Gunma University Initiative for Advanced Research, Gunma University, Maebashi 371-8512, Japan 3
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University,
Hachioji, Tokyo 192-0397, Japan
*Correspondence to Tetsuro Izumi (
[email protected])
Pancreatic beta cells, Diabetes Summary statement: Although regulated exocytosis comprises several sequential steps, the mechanisms coordinating each step are poorly understood. The present findings suggest that Noc2 regulates the transition between Rab2a- and Rab27a-mediated exocytosis.
JCS Advance Online Article. Posted on 7 December 2016
Journal of Cell Science • Advance article
Key words: Small GTPase, Secretory granules, Regulated exocytosis, Insulin,
Abstract
Exocytosis of secretory granules entails budding from the trans-Golgi network, sorting and maturation of cargo proteins, and trafficking and fusion to the plasma membrane. Rab27a regulates the late steps in this process, such as granule recruitment to the fusion site, whereas Rab2a functions in the early steps, such as granule biogenesis and maturation. Here, we demonstrate that these two small GTPases simultaneously bind to Noc2 in GTP-dependent manners, although Rab2a binds only after Rab27a has bound. In pancreatic beta cells, the ternary Rab2a-Noc2-Rab27a complex specifically localizes on perinuclear immature granules, whereas the binary Noc2-Rab27a complex localizes on peripheral mature granules. In contrast to the wild type, Noc2 mutants defective in binding to Rab2a or Rab27a fail to promote glucose-stimulated insulin secretion. Although knockdown of any component of the ternary complex markedly inhibits insulin secretion, only that of Rab2a or Noc2, and not that of Rab27a, impairs cargo processing from proinsulin to insulin. These results suggest that the dual effector, Noc2,
granule exocytosis.
Journal of Cell Science • Advance article
regulates transition from Rab2a-mediated granule biogenesis to Rab27a-mediated
Introduction
Regulated secretion is a main pathway in the delivery of a cell’s bioactive molecules to the extracellular environment. The pathway comprises coordinated sequential steps, such as secretory vesicle biogenesis, maturation, trafficking, and fusion with the plasma membrane. Although the molecular machinery for these individual processes has been characterized, the precise mechanisms connecting each process remain poorly understood. Previous studies have shown that the small GTPase, Rab27, regulates the late steps of this pathway through its multiple effector proteins (Izumi et al., 2003; Fukuda, 2006). For example, in pancreatic beta cells, Rab27a localizes on insulin granules (Yi et al., 2002) and forms a complex with its effectors, such as granuphilin/Slp4 (Wang et al., 1999; Gomi et al., 2005), exophilin7/JFC1/Slp1 (Wang et al., 2013), exophilin8/MyRIP/Slac2c (Waselle et al., 2003; Mizuno et al., 2011), and Noc2 (Kotake et al., 1997; Cheviet et al., 2004), and it regulates a specific step of insulin granule trafficking and exocytosis. Namely, granuphilin and exophilin7 controls
whereas exophilin8 reserves granules in the cortical actin network for subsequent release. However, it remains unknown at which step or by what mechanism Noc2 functions, despite the finding that Noc2 knockout mice exhibit impaired insulin secretion when under acute stress (Matsumoto et al., 2004). This is partly because Noc2 is a relatively small protein compared with other Rab27a effectors, and it appears to lack functional domains other than the Rab27-binding domain. In the present study, we
Journal of Cell Science • Advance article
exocytosis of granules docked and undocked to the plasma membrane, respectively,
show that Noc2 binds to another GTPase, Rab2a, in addition to Rab27a. The ternary Rab2a-Noc2-Rab27a complex specifically localizes on immature granules, and interference in the complex formation inhibits cargo processing and granule exocytosis. We present evidence that this novel complex regulates a transitional step from granule
Journal of Cell Science • Advance article
maturation to exocytosis in the regulated secretory pathway.
Results
Rab2a binds to Rab27a via Noc2 in a GTP-dependent manner To identify Rab27a-interacting proteins more comprehensively, we stably expressed Rab27a in the beta-cell line, MIN6, with the MEF-tag (myc-TEV-FLAG) that consists of myc and FLAG epitope tags connected by a spacer sequence containing a TEV protease cleavage site, and then performed tandem affinity purification (Ichimura et al., 2005; Matsunaga et al., 2009). The protein bands specific to the Rab27a immunoprecipitate were then analyzed by a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) system (Fig. S1A; Table S1). In addition to the known Rab27-interacting proteins, such as granuphilin, exophilin9/Slp5 (Kuroda et al., 2002), and
Noc2,
two
Rab
GTPases,
Rab2
and
Rab18,
were
also
identified.
Coimmunoprecipitation experiments showed that Rab2a, but not Rab18a, interacts with Rab27a in MIN6 cells (Fig. S1C). Because the Rab2a-immunoprecipitate also contained Noc2, but not granuphilin (Fig. S1C,D), we performed similar tandem purification in
protein, as is Rab27a (Fig. S1B; Table S2). Furthermore, Noc2 formed an endogenous complex with Rab2a in both of the beta-cell lines, mouse MIN6 and rat INS1 832/13 (Fig. 1A,B). Noc2 specifically bound to the Rab2a Q65L mutant mimicking the GTP-bound form, but not with the S20N mutant mimicking the GDP-bound form (Fig. 1C). As previously reported (Fukuda et al., 2004), Noc2 interacted with the corresponding Rab27a Q78L mutant, but not with the T23N mutant (Fig. S1E).
Journal of Cell Science • Advance article
MIN6 cells stably expressing MEF-Noc2, and found Rab2a to be a Noc2-interacting
Although Noc2 also forms a complex with Rab3a (Fig. 1B), as previously reported (Haynes et al., 2001; Cheviet et al., 2004), no Rab3a was present in either the Rab2a- or Rab27a-immunoprecipitate (Fig. 1C; Fig. S1E). Furthermore, neither the established Rab2a effector, ICA69 (Buffa et al., 2008), nor its interacting protein, PICK1 (Cao et al., 2007), were found in the Noc2- or Rab27a-immunoprecipitate, in contrast to the Rab2a-immunoprecipitate (Fig. 1B,C; Fig. S1E). When Noc2 was downregulated by specific short-hairpin RNA (shRNA), the interaction between Rab2a and Rab27a disappeared (Fig. 1D). These findings indicate that the two Rab proteins interact through Noc2, and that Noc2 simultaneously binds to Rab2a and Rab27a. Furthermore, the ternary Rab2a-Noc2-Rab27a complex appears to exist separately from either the Rab2a-ICA69-PICK1 complex or the Noc2-Rab3a complex.
Rab2a binds to Noc2 only in the presence of Rab27a We next investigated the formation of the ternary complex. Coexpression of Rab2a and Noc2 in HEK293A cells did not lead to a complex formation between the two proteins,
mutation known to dramatically reduce the Rab27a-binding activity (Fukuda et al., 2004) simultaneously lost its Rab2a-binding activity in MIN6 cells (Fig. 2B). Furthermore, in contrast to the wild-type beta cell-lines (Fig. 1), the Rab2a-Noc2 complex was absent in Rab27a-null beta cells derived from ashen mice (Wilson et al., 2000); however, the complex was present after the introduction of wild-type Rab27a into the cells (Fig. 2C). These findings indicate that Rab2a interacts with Noc2 only in
Journal of Cell Science • Advance article
although additional expression of Rab27a did (Fig. 2A). Further, the Noc2 E51A/I55A
the presence of Rab27a, and probably after Rab27a binds to Noc2. To substantiate this conclusion, we simultaneously expressed different amounts of Rab2a and Rab27a in HEK293A cells expressing One-STrEP-Flag (OSF)-tagged Noc2. Noc2 and the binding proteins were pulled down using Strep-Tactin beads and were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue (CBB) staining (Fig. 2D). There were no specific protein bands other than Rab2a, Rab27a, and Noc2. Furthermore, the amounts of Rab2a bound with Noc2 were proportional to those of Rab27a bound with Noc2. These findings eliminate the possibilities that other proteins are involved in the ternary complex formation and that Rab2a and Rab27a competitively interact with Noc2.
Rab2a and Rab27a bind to Noc2 through distinct N-terminal regions We next determined the Rab2a-interacting domain of Noc2. A series of Noc2 deletion mutants were expressed as bait in MIN6 cells (Fig. 3A). Rab27 effectors, including Noc2, possess a highly conserved N-terminal Rab27-biding domain, named RBD
were unable to bind to either Rab27a or Rab2a (Fig. 3B,C). However, residues further towards the N-terminal of the RBD were specifically required for the binding to Rab2a (Fig. 3C,D). In fact, the minimum deletion mutant Δ(11-20) showed a marked decrease in binding activity to Rab2a, but not to Rab27a (Fig. 3E). These findings indicate that the two Rab proteins interact with Noc2 through different N-terminal regions.
Journal of Cell Science • Advance article
(Izumi et al., 2003). Indeed, the Noc2 mutants devoid of its RBD (41-158 residues)
Site-directed mutagenesis analysis in the N-terminus region of Noc2 revealed that mutation of threonine 38, but not that of glutamine 12, specifically eliminated the binding activity to Rab2a (Fig. 3E,F). We explored the possibility that phosphorylation at this threonine residue might be involved in the binding to Rab2a, because endogenous Noc2 proteins in beta cells were detected as multiple bands in gels (Figs. 1D, 2C). However, the phosphomimetic mutants, T38D and T38E, failed to interact with Rab2a (Fig. 3F). Furthermore, although in vitro phosphatase treatment in the Noc2-immunoprecipitate did increase the gel mobility of Noc2 protein, it failed to influence the interaction with either Rab2a or Rab27a (Fig. S2), suggesting that phosphorylation of Noc2 plays no role in the formation of the ternary complex.
The Rab2a-Noc2-Rab27a complex localizes on immature proinsulin granules We next investigated the intracellular localization of Rab27a, Rab2a, and Noc2 in INS1 832/13 cells. Because available antibodies to Rab2a and Noc2 are not durable for
an N-terminal MEF tag. Although Rab27a and Noc2 colocalized with insulin-positive puncta in the overall cytoplasm, Rab2a did not, and was instead restricted to the perinuclear region (Fig. 4A). However, MEF-Rab2a almost completely colocalized with proinsulin-positive puncta in the perinuclear region, although MEF-Rab27a and MEF-Noc2 also colocalized there (Fig. 4B). The insulin- and proinsulin-puncta did not colocalize, indicating that the anti-insulin and anti-proinsulin antibodies hardly
Journal of Cell Science • Advance article
immunostaining, we examined the localization of exogenously expressed proteins with
crossreacted to the other protein under our experimental condition (Fig. S3A). Furthermore, none of the proteins colocalized with the endosome marker, EEA1, the trans-Golgi network (TGN) marker, TGN38, or the endoplasmic reticulum marker, PDI (Fig. S3B). These findings suggest that the Rab2a-Noc2-Rab27a ternary complex specifically locates on immature granules, whereas the Noc2-Rab27a binary complex locates on mature granules. In contrast to the wild-type Noc2, the E51A/I55A mutant defective in binding to Rab27a neither localized on insulin-positive mature nor proinsulin-positive immature granules (Fig. 5). However, the Noc2 mutants specifically defective in binding to Rab2a, Δ(11-20) or T38A, still localized to both mature and immature granules. These results indicate that the interaction with Rab27a, but not that with Rab2a, determines the localization of Noc2 on secretory granules.
Rab2a-Noc2-Rab27a complex formation is essential for insulin granule exocytosis To investigate the functional roles of the Rab2a-Noc2-Rab27a complex, we expressed
cells, and examined the effects on insulin secretion (Fig. 6A). The cells expressing exogenous wild-type Noc2 similar to the endogenous level showed a 1.5-fold higher glucose-stimulated insulin secretion compared with control cells (Fig. 6B). By contrast, none of the Noc2 mutants, Δ(11-20), T38A, or E51A/I55A, showed such enhancement. The insulin content was not affected by expression of either wild-type or mutant Noc2 (Fig. 6C). These results suggest that the ternary complex formation is instrumental for
Journal of Cell Science • Advance article
wild-type Noc2 or its mutants defective in binding to these Rab proteins in INS1 832/13
evoked granule exocytosis. We next investigated the effects of downregulation of each component of the complex by adenovirus-mediated shRNA expression (Fig. 7A). Surprisingly, Rab2a knockdown simultaneously downregulated Noc2, despite the presence of Rab27a. This is not an off-targeting effect, because independent knockdown using double stranded small interfering RNA (siRNA) against a different sequence of Rab2a also led to markedly decreased expression of Noc2 (Fig. S4A). Because Noc2 stably associates with Rab27a on peripheral mature granules without Rab2a (Fig. 4A), and because Noc2 mutants defective in Rab2a binding still locate on perinuclear immature granules (Fig. 5B), the instability of Noc2 in the absence of Rab2a requires more than the loss of the interaction between the two proteins. Rather, the impairment of granule biogenesis by Rab2a-depletion appears to extinguish the membrane-association site for nascent Noc2 to be stabilized. Consistent with this idea, knockdown of ICA69, which is thought to function with Rab2a in the early stage, such as in granule biogenesis and maturation (Sumakovic et al., 2009; Cao et al., 2013), dislodges Noc2 from immature granules (Fig.
Depletion of any component of the complex markedly inhibited glucose-stimulated insulin secretion (Fig. 7B; Fig. S4B). Because insulin secretion could be impaired by inhibition of insulin or granule synthesis, we also measured total insulin content in the cells (Fig. 7C; S4C). There were notable differences among the cells:
Rab27a-knockdown
markedly
increased
insulin
content,
whereas
Rab2a-knockdown markedly decreased it. Although the effect of ICA69-depletion was
Journal of Cell Science • Advance article
S4D).
similar to that of Rab2a-knockdown, Noc2-depletion caused an intermediate effect between those of the Rab27a- and Rab2a-depletions. Although normalization of insulin secretion by its content confirmed the inhibition of exocytosis in all the downregulated cells (Fig. 7D), Rab27a-depletion appeared to primarily affect granule exocytosis because of the increased insulin content, whereas Rab2a-depletion likely impaired granule biogenesis and/or maturation because of the decreased insulin content. The intermediate phenotypes of Noc2-depleted cells suggest that Noc2 plays a regulatory role in the transition between Rab2a- and Rab27a-mediated processes. Because the Rab2a-Noc2-Rab27a complex should specifically localize on immature proinsulin granules (Fig. 4), we also examined proinsulin secretion and content (Fig. 7E-G). Knockdown of Rab2a, ICA69, or Noc2, but not that of Rab27a, significantly increased the amount of proinsulin secreted in the media and the relative ratio between proinsulin and insulin levels in the cells. These results suggest that Noc2, as well as Rab2a and
Journal of Cell Science • Advance article
ICA69, is involved in granule maturation and cargo processing.
Discussion
The present findings indicate that Rab2a and Rab27a bind together through Noc2 protein. The mode of the complex formation is very unique. Although some Rab effector proteins are known to interact with different Rab proteins, such as RUFY1 (Yamamoto et al., 2010), Nischarin (Kuij et al., 2013), and golgin family proteins (Gillingham and Munro, 2016), they do so separately or sequentially. There is no instance in which the same effector molecule simultaneously binds two different Rab proteins. Although the Rab2a-binding region locates towards the N-terminus further from the Rab27-binding domain, the GTP-bound Rab2a cannot bind to the Noc2 mutant that is incapable of binding to Rab27a, yet can bind to wild-type Noc2 in the presence of Rab27a. These findings indicate that the ternary complex forms after the formation of the binary Noc2-Rab27a complex. The interaction between Rab2a and Rab27a is intriguing because they have been shown to be involved in early and late stages of secretory granule exocytosis,
orthologue of Rab2) or in RIC-19 (the orthologue of ICA69) is unable to prevent cargos of secretory granules from inappropriately entering endosomal compartments during granule maturation (Edwards et al., 2009; Sumakovic et al., 2009). Rab2a forms a complex with ICA69 and PICK1, both of which contain a BAR domain known to bind to lipid membranes and to initiate vesicle formation (Peter et al., 2004). Furthermore, mice deficient in these proteins exhibit defects in granule biogenesis and/or maturation
Journal of Cell Science • Advance article
respectively. In Caenorhabditis elegans (C. elegans), a mutation in UNC-108 (the
(Cao et al., 2013; Holst et al., 2013). On the other hand, Rab27a associates with secretory granules (Yi et al., 2002) and regulates the late steps of granule exocytosis, such as recruitment and/or docking to fusion sites, through its multiple effector proteins (Izumi, 2007, 2011). We consistently found that knockdown of Rab2a or ICA69 inhibits insulin synthesis and processing, whereas that of Rab27a primarily affects insulin secretion. The intermediate phenotype caused by knockdown of Noc2 suggests that Noc2 plays a regulatory role in connecting the early and late exocytic pathways by forming a complex with both Rab2a and Rab27a. Consistent with this model, Rab2a forms a complex with Noc2 and Rab27a, separately from that with ICA69 and PICK1. Although Noc2 and Rab27a locate on both perinuclear immature and peripheral mature granules, Rab2a is restricted to immature granules. ICA69 and PICK1 are also known to localize on immature granules and/or granule budding site from the TGN (Spitzenberger et al., 2003; Cao et al., 2013; Holst et al., 2013). Interestingly, knockdown of ICA69 dislodges Noc2 from immature granules, and that of Rab2a can eliminate Noc2 expression, which suggests that the two
ICA69 knockdowns likely reflect the presence of other Rab2 effectors. It has recently been reported that the two other Rab2 effectors, RUND-1 and CCCP-1, function in granule biogenesis and maturation in C. elegans (Ailion et al., 2014). RUND-1 binds RIC-19 (ICA69 ortholog), whereas CCCP-1 does not bind it and forms a different complex with Rab2. Therefore, Rab2 seems to function in parallel pathways through its multiple effectors, and its absence likely induces more profound effects on granule
Journal of Cell Science • Advance article
proteins function earlier than does Noc2. The differential effects between Rab2a and
biogenesis than that of one of its effectors, ICA69. In fact, Rab2a-knockdown has a severer effect on insulin content in cells compared with ICA69-knockdown. Furthermore, Rab2a-knockdown decreases the amount of proinsulin in cells, as was found in Rab2 (UNC-108)-mutated C. elegans that fails to prevent granule cargos from inappropriately entering endosomal compartments and exhibits their losses due to the increased degradation (Edwards et al., 2009; Sumakovic et al., 2009). By contrast, ICA69-knockdown increases it, as was found in ICA69-knockout beta cells where the exit of proinsulin from TGN is blocked (Cao et al., 2013). Despite these differences, the absence of Rab2a or ICA69 appears to prevent granules from being generated or becoming mature enough for nascent Noc2 to associate with them. Taken together, these findings suggest that Rab2a may first regulate granule budding and/or proper cargo sorting at the TGN, through an interaction with ICA69 and other effectors, and then regulates granule maturation and cargo processing by forming a complex with Noc2 bound to Rab27a on immature granules. Further research is required to identify the mechanism and timing of Rab2a dissociation from mature granules, although we
In contrast to the wild type, Noc2 mutants defective in binding to Rab2a or Rab27a fail to enhance glucose-stimulated insulin secretion, indicating that the ternary complex formation is pivotal for evoked granule exocytosis. The phenotype of Noc2 knockout mice, with impaired insulin secretion under condition of acute stress (Matsumoto et al., 2004), may represent imbalance between granule maturation and exocytosis. The complex may also play a role in the pathogenesis of human type 2
Journal of Cell Science • Advance article
suspect that the process is unlikely to involve the phosphorylation of Noc2.
diabetes, because these patients exhibit a disproportionate level of circulating proinsulin (Røder et al., 1998). However, the complex is not specific to β cells, because it was found by similar tandem purification and LC-MS/MS analyses performed in pancreatic alpha and melanocyte cell-lines (our unpublished observations), both of which employ a Rab27a-regulated exocytic system (Bahadoran et al., 2001; Hume et al., 2001; Yu et al., 2007). Therefore, the present ternary complex may play a universally conserved role in
Journal of Cell Science • Advance article
secretory cells.
Materials and methods
Cell culture All cells were cultured in a humidified incubator with 95% air and 5% CO 2 at 37 C, MIN6 cells (Miyazaki et al., 1990) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 15% fetal bovine serum (FBS) supplemented with 1 mM L-glutamine and 50 M 2-mercapthoethanol. MIN6 cells stably expressing MEF-tagged Rab27a, Rab2a, or Noc2 were cultured in a medium containing 0.5
g ml-1 of
puromycin (Invivogen). INS1 832/13 cells (Hohmeier et al., 2000) were cultured in RPMI1640 containing 10% FBS supplemented with 1 mM L-glutamine, 1 mM HEPES, 1 mM sodium pyruvate, and 50 M 2-mercapthoethanol. HEK293A cells (Invitrogen) were cultured in DMEM containing 10% FBS supplemented with 1 mM L-glutamine. Rab27a-null β-cell lines were established from Rab27a-mutated ashen mice (Wilson et al., 2000), by a method similar to that by which granuphilin-null β-cell lines have been established (Mizuno et al., 2016), and will be described in detail elsewhere. All animal
Care and Experimentation Committee, Gunma University.
Antibodies Rabbit polyclonal antibodies against Rab27a and granuphilin were described previously (Yi et al., 2002). Guinea pig anti-porcine insulin serum was a gift from H. Kobayashi (Gunma University). Mouse anti-myc 9E10 monoclonal antibody was purified from the
Journal of Cell Science • Advance article
experiments were performed in accordance with the rules and regulations of the Animal
ascites fluid of a hybridoma-injected mouse. The following commercially purchased antibodies were also used: rabbit polyclonal antibodies toward FLAG (F7425, Sigma-Aldrich), hemagglutinin (HA; 561, MBL), green fluorescent protein (GFP; 598, MBL), Rab27a/b (18975, IBL), Noc2 (15297-1-AP, Proteintech), Rab2a (15420-1-AP, Proteintech), ICA69 (ab81500, Abcam), and PICK1 (ab3420, Abcam); and mouse monoclonal antibodies toward Rab3 (610379, BD Biosciences), EEA1 (610457, BD Biosciences),
TGN38
(610849,
BD
Biosciences),
PDI
(MA3-018,
Affinity
BioReagents), α-tubulin (T5168, Sigma-Aldrich), and proinsulin (clone 3A1; ab8301, Abcam).
DNA construction Mouse Rab27a wild-type and mutant cDNAs were described previously (Yi et al., 2002). Mouse Rab2a and Noc2 cDNAs were reverse transcribed from RNA of MIN6 cells. Point and deletion mutants of Rab2a and Noc2 were generated using a standard polymerase chain reaction-based mutagenesis strategy, and were verified by DNA
(Saitoh et al., 2003), where an MEF-tag or FLAG-tag sequence from a pcDNA3-MEF vector (Ichimura et al., 2005) had been incorporated. They were also subcloned into the pCAG vector with or without an OSF tag (Morita et al., 2007). Sub-confluent HEK293A cells were transfected with the plasmids using Lipofectamine 2000 reagent (Invitrogen). For generation of recombinant adenoviruses, the cDNAs of Rab27a, Rab2a, and Noc2 in pENTR-3C-MEF/FLAG were transferred into a pAd/CMV vector
Journal of Cell Science • Advance article
sequencing. These cDNAs were subcloned into pENTR-3C (Invitrogen) or pMRX
(Invitrogen) by LR Clonase recombination (Invitrogen). Adenoviral production and infection were performed according to the manufacturer’s protocol.
Immunoprecipitation and immunoblotting Cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (w/v) glycerol, 100 mM NaF, 10 mM ethylene glycol tetraacetic acid, 1 mM Na3VO4, 1% Triton X-100, 5
M ZnCl2, 1 mM phenylmethylsulfonyl fluoride, and
complete protease inhibitor cocktail (Roche). The lysates were cleared by centrifugation at 15,000 rpm for 15 min at 4 C. The supernatants were subjected to immunoprecipitation by primary antibody and Protein G-Sepharose 4FF (GE Healthcare Bioscience), anti-FLAG resin (A2220; Sigma-Aldrich), or Strep-Tactin beads (GE Healthcare Bioscience). After being washed five times with TBS (50 mM Tris-HCl, pH 7.5; 150 mM NaCl) containing 0.1% Triton X-100, the immunoprecipitates were subjected to SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with TBST (TBS plus 0.1% Tween-20) containing 0.5%
antibody diluted in Can Get Signal solution I (TOYOBO). It was then washed three times with TBST, was incubated for 1 h at room temperature with a 5,000× dilution of horseradish peroxidase-conjugated secondary antibody (GE Healthcare Bioscience) in TBST containing 0.5% nonfat dry milk, and was washed five times. Immunoreactive signals were then detected using ECL prime and an LAS-4000 chemiluminescence detection system (GE Healthcare Bioscience).
Journal of Cell Science • Advance article
nonfat dry milk, and then incubated overnight at room temperature with primary
MEF tag-based protein purification and mass spectrometry The purification procedure was similar to that reported previously (Ichimura et al., 2005; Matsunaga et al., 2009), with minor modifications. Briefly, ~2 × 108 cells were lysed in 15 ml of lysis buffer. The interacting proteins were immunoprecipitated with anti-myc
antibody,
cleaved
by
TEV
protease
(12575015;
Invitrogen),
reimmunoprecipitated with anti-FLAG antibody, and eluted by FLAG peptides. The final eluate was separated by SDS-PAGE and visualized by Oriole fluorescent gel staining (BioRad). Specific bands were excised and digested in gels with trypsin, and the resulting peptide mixtures were analyzed by LC-MS/MS. All MS/MS spectra were searched against the RefSeq protein sequence database at the National Center for Biotechnology Information using Mascot software (Matrix Science).
Immunofluorescence and microscopy INS1 832/13 cells cultured on coverslips were fixed with 3% paraformaldehyde in
in PBS for 30 min. The cells were then treated with 50 mM NH4Cl-PBS for 10 min at room temperature and blocked with PBS containing 1% bovine serum albumin (BSA) for 15 min. The coverslips were incubated with primary antibody (1:100 or 1:200 dilution) overnight, washed three times with PBS, and incubated with Alexa Fluor 488or 568-conjugated secondary antibody (Invitrogen; 1:500 dilution) for 60 min. Samples were washed five times and mounted using SlowFade Gold (Invitrogen). The
Journal of Cell Science • Advance article
phosphate buffered saline (PBS) for 30 min and permeabilized with 0.1% Triton X-100
microscopic images were obtained by a Fluoview FV1000 (Olympus) confocal laser scanning microscope equipped with a 100× oil immersion objective lens (1.40 NA), or by an A1 (Nikon) confocal laser scanning microscope equipped with a 100× oil immersion objective lens (1.49 NA) and NIS elements. The images were adjusted using Adobe Photoshop CS4 software (Adobe Systems).
shRNA-mediated RNA interference The oligonucleotide sequences used for shRNA interference were as follows: 298-316 bp of rat Rab27a (5’-GACCTGACAAACGAGCAAA-3’), 11-29 bp of rat Noc2 (5’-CCATCTTCAGCAGTGGAAA-3’), (5’-GCTTATTGCTACAGTTTAC-3’), (5’-GGAAGATGAACATGTCGTT-3’),
61-83
bp
of
rat
Rab2a,
129-147
bp
of
rat
ICA69
and
(5’-GCGATCACATGATCTACTT-3’), respectively,
647-665
bp
of
GFP
followed by a 9-nucleotide
non-complementary spacer (TTCAAGAGA) and the reverse complement of the initial 19-nucleotide sequence. These dsDNA oligos were cloned into the pENTR/U6 vector
recombination. Sub-confluent INS1 832/13 cells in 35-mm dishes were infected with adenovirus, and were transferred to 60-mm dishes 48 h later. The cells were infected again with the virus 24 h later, and were transferred to 12-well dishes after an additional 24 h. Experiments were performed 24 h after the transfer.
Journal of Cell Science • Advance article
(Invitrogen) and transferred into a pAd/PL vector (Invitrogen) by LR Clonase
siRNA-mediated RNA interference On-Target plus SMARTpool siRNA against rat Noc2 (catalog no. 171123) and Rab2a (catalog no. 65158), as well as control On-Target plus non-targeting pool siRNA, were purchased from GE Dharmacon. INS1 832/13 cells plated at a density of 2.5 × 106 in a 6-well dish were grown for 24 h. Suspended cells after trypsinization were transfected twice with siRNAs using Lipofectamine RNAiMAX reagent (Invitrogen), according to the manufacturer’s instructions. The second transfection was performed 48 h later, and the cells were analyzed 48 h thereafter.
Insulin and proinsulin secretion assay INS1 832/13 cells plated on 6- or 12-well plates were cultured in the RPMI medium for 24 h. The cells were incubated in modified Krebs-Ringer bicarbonate buffer [KRB; 120 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2, 15 mM HEPES (pH 7.4), 0.1% BSA, 2 mM glucose] for 2 h followed by the same buffer or a buffer
immunoassay kit (PerkinElmer), as described previously (Wang et al., 2013). Proinsulin was measured by a proinsulin ELISA assay kit (Shibayagi).
Statistical analysis Statistical significance was determined using a two-tailed unpaired t-test.
Journal of Cell Science • Advance article
containing 25 mM glucose for 2 h. Insulin was measured by an AlphaLISA
Acknowledgement We are grateful to Drs. Shoji Yamaoka (Tokyo Medical and Dental University), Eiji Morita (Hirosaki University), Toshio Kitamura (The University of Tokyo), and Christopher Newgard (Duke University) for supplying pMRX-puro vector, pCAG-OSF vector, PLAT-E cells, and INS1 832/13 cells, respectively. We thank Drs. Takuji Fujita and Horoshi Gomi for generation and characterization of the beta-cell lines from ashen mice. We also thank S. Shigoka for her assistance in preparing the manuscript.
Competing Interests The authors declare no competing or financial interests.
Author contributions K.M. designed and performed experiments and wrote the article. M.T. and T.Is. contributed to the mass analysis. T.Iz. designed experiments and wrote the article.
This work was supported by grants in aid for scientific research from JSPS KAKENHI Grant Numbers JP20113005, JP24390068, and JP16K/5211 to T.Iz., and JP23790354 and JP25860208 to K.M. It was also supported by grants from Novo Nordic Insulin Study Award (to T.Iz.), and from The Uehara Memorial Foundation, Takeda Science Foundation, The Tokyo Biochemical Research Foundation, and The NOVARTIS Foundation (Japan) for the promotion of Science (To K.M.).
Journal of Cell Science • Advance article
Funding
References
Ailion, M., Hannemann, M., Dalton, S., Pappas, A., Watanabe, S., Hegermann, J., Liu, Q., Han, H.F., Gu, M., Goulding, M.Q., et al. (2014). Two Rab2 interactors regulate dense-core vesicle maturation. Neuron 82, 167-180. Bahadoran, P., Aberdam, E., Mantoux, F., Busca, R., Bille, K., Yalman, N., de Saint-Basile, G., Casaroli-Marano, R., Ortonne, J.-P., and Ballotti, R. (2001). Rab27a: a key to melanosome transport in human melanocytes. J. Cell Biol. 152, 843-850. Buffa, L., Fuchs, E., Pietropaolo, M., Barr, F., and Solimena, M. (2008). ICA69 is a novel Rab2 effector regulating ER-Golgi trafficking in insulinoma cells. Eur. J. Cell Biol. 87, 197-209. Cao, M., Mao, Z., Kam, C., Xiao, N., Cao, X., Shen, C., Cheng, K. K., Xu, A., Lee, K.-M., Jiang, L., et al. (2013). PICK1 and ICA69 control insulin granule trafficking
Cao, M., Xu, J., Shen, C., Kam, C., Huganir, R. L., and Xia, J. (2007). PICK1-ICA69 heteromeric BAR domain complex regulates synaptic targeting and surface expression of AMPA receptors. J. Neurosci. 27, 12945-12956. Cheviet, S., Coppola, T., Haynes, L. P., Burgoyne, R. D., and Regazzi, R. (2004). The Rab-binding protein Noc2 is associated with insulin-containing secretory granules and is essential for pancreatic β-cell exocytosis. Mol. Endocrinol. 18, 117-126.
Journal of Cell Science • Advance article
and their deficiencies lead to impaired glucose tolerance. PLoS Biol. 11, e1001541.
Edwards, S. L., Charlie, N. K., Richmond, J. E., Hegermann, J., Eimer, S., and Miller, K. G. (2009). Impaired dense core vesicle maturation in Caenorhabditis elegans mutants lacking Rab2. J. Cell Biol. 186, 881-895. Fukuda, M. (2006). Rab27 and its effectors in secretory granule exocytosis: a novel docking machinery composed of a Rab27.effector complex. Biochem. Soc. Trans. 34, 691-695. Fukuda, M., Kanno, E., and Yamamoto, A. (2004). Rabphilin and Noc2 are recruited to dense-core vesicles through specific interaction with Rab27A in PC12 cells. J. Biol. Chem. 279, 13065-13075. Gillingham, A. K., and Munro, S. (2016). Finding the Golgi: golgin coiled-coil proteins show the way. Trends Cell Biol. 26, 399-408. Gomi, H., Mizutani, S., Kasai, K., Itohara, S., and Izumi, T. (2005). Granuphilin molecularly docks insulin granules to the fusion machinery. J. Cell Biol. 171, 99-109. Haynes, L. P., Evans, G. J., Morgan, A., and Burgoyne, R. D. (2001). A direct inhibitory role for the Rab3-specific effector, Noc2, in Ca2+-regulated exocytosis in
Hohmeier, H. E., Mulder, H., Chen, G., Henkel-Rieger, R., Prentki, M., and Newgard, C. B. (2000). Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion. Diabetes 49, 424-430. Holst, B., Madsen, K.L., Jansen, A. M., Jin, C., Rickhag, M., Lund, V. K., Jensen, M., Bhatia, V., Sørensen, G., Madsen, A. N., et al. (2013). PICK1 deficiency impairs
Journal of Cell Science • Advance article
neuroendocrine cells. J. Biol. Chem. 276, 9726-9732.
secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance. PLoS Biol. 11, e1001542. Hume, A. N., Collinson, L. M., Rapak, A., Gomes, A. Q., Hopkins, C. R., and Seabra, M. C. (2001). Rab27a regulates the peripheral distribution of melanosomes in melanocytes. J. Cell Biol. 152, 795-808. Ichimura, T., Yamamura, H., Sasamoto, K., Tominaga, Y., Taoka, M., Kakiuchi, K., Shinkawa, T., Takahashi, N., Shimada, S., and Isobe, T. (2005). 14-3-3 proteins modulate the expression of epithelial Na+ channels by phosphorylation-dependent interaction with Nedd4-2 ubiquitin ligase. J. Biol. Chem. 280, 13187-13194. Izumi, T. (2007). Physiological roles of Rab27 effectors in regulated exocytosis. Endocr. J. 54, 649-657. Izumi, T. (2011). Heterogeneous modes of insulin granule exocytosis: molecular determinants. Front. Biosci. (Landmark Ed) 16, 360-367. Izumi, T., Gomi, H., Kasai, K., Mizutani, S., and Torii, S. (2003). The roles of Rab27 and its effectors in the regulated secretory pathways. Cell Struct. Funct. 28, 465-474.
Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells. J. Biol. Chem. 272, 29407-29410. Kuij, C., Pilli, M., Alahari, S.K., Janssen, H., Khoo, P. S., Ervin, K. E., Calero, M., Jonnalagadda, S., Scheller, R. H., Neefjes, J., et al. (2013). Rac and Rab GTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria. EMBO J. 32, 713-727.
Journal of Cell Science • Advance article
Kotake, K., Ozaki, N., Mizuta, M., Sekiya, S., Inagaki, N., and Seino, S. (1997).
Kuroda, T.S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002). Synaptotagmin-like protein 5: a novel Rab27A effector with C-terminal tandem C2 domains. Biochem. Biophys. Res. Commun. 293, 899-906. Matsumoto, M., Miki, T., Shibasaki, T., Kawaguchi, M., Shinozaki, H., Nio, J., Saraya, A., Koseki, H., Miyazaki, M., Iwanaga, T., et al. (2004). Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells. Proc. Natl. Acad. Sci. U. S. A. 101, 8313-8318. Matsunaga, K., Saitoh, T., Tabata, K., Omori, H., Satoh, T., Kurotori, N., Maejima, I., Shirahama-Noda, K., Ichimura, T., Isobe, T., et al. (2009). Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nat. Cell Biol. 11, 385-396. Miyazaki, J., Araki, K., Yamato, E., Ikegami, H., Asano, T., Shibasaki, Y., Oka, Y., and Yamamura, K. (1990). Establishment of a pancreatic β cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127, 126-132.
mediates functional granule docking to the plasma membrane. Sci. Rep. 6, 23909. Mizuno, K., Ramalho, J. S., and Izumi, T. (2011). Exophilin8 transiently clusters insulin granules at the actin-rich cell cortex prior to exocytosis. Mol. Biol. Cell 22, 1716-1726. Morita, E., Sandrin, V., Alam, S.L., Eckert, D.M., Gygi, S.P., and Sundquist, W.I. (2007). Identification of human MVB12 proteins as ESCRT-I subunits that function in
Journal of Cell Science • Advance article
Mizuno, K., Fujita, T., Gomi, H., and Izumi, T. (2016). Granuphilin exclusively
HIV budding. Cell Host Microbe 2, 41-53. Peter, B. J., Kent, H. M., Mills, I. G., Vallis, Y., Butler, P. J. G., Evans, P. R., and McMahon, H. T. (2004). BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495-499. Røder, M. E., Porte, D., Jr., Schwartz, R. S., and Kahn, S. E. (1998). Disproportionately elevated proinsulin levels reflect the degree of impaired B cell secretory capacity in patients with noninsulin-dependent diabetes mellitus. J. Clin. Endocrinol. Metab. 83, 604-608. Saitoh, T., Nakayama, M., Nakano, H., Yagita, H., Yamamoto, N., and Yamaoka, S. (2003). TWEAK induces NF-κB2 p100 processing and long lasting NF-κB activation. J. Biol. Chem. 278, 36005-36012. Spitzenberger, F., Pietropaolo, S., Verkade, P., Habermann, B., Lacas-Gervais, S., Mziaut, H., Pietropaolo, M., and Solimena, M. (2003). Islet cell autoantigen of 69 kDa is an arfaptin-related protein associated with the Golgi complex of insulinoma INS-1 cells. J. Biol. Chem. 278, 26166-26173.
C., Schoofs, L., Richmond, J., and Eimer, S. (2009). UNC-108/RAB-2 and its effector RIC-19 are involved in dense core vesicle maturation in Caenorhabditis elegans. J. Cell Biol. 186, 897-914. Wang, H., Ishizaki, R., Xu, J., Kasai, K., Kobayashi, E., Gomi, H., and Izumi, T. (2013). The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma membrane. Mol. Biol. Cell 24, 319-330.
Journal of Cell Science • Advance article
Sumakovic, M., Hegermann, J., Luo, L., Husson, S. J., Schwarze, K., Olendrowitz,
Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999). Novel rabphilin-3-like protein associates with insulin-containing granules in pancreatic beta cells. J. Biol. Chem. 274, 28542-28548. Waselle, L., Coppola, T., Fukuda, M., Iezzi, M., El-Amraoui, A., Petit, C., and Regazzi, R. (2003). Involvement of the Rab27 binding protein Slac2c/MyRIP in insulin exocytosis. Mol. Biol. Cell 14, 4103-4113. Wilson, S. M., Yip, R., Swing, D. A., O'Sullivan, T. N., Zhang, Y., Novak, E. K., Swank, R. T., Russell, L. B., Copeland, N. G., and Jenkins, N. A. (2000). A mutation in Rab27a causes the vesicle transport defects observed in ashen mice. Proc. Natl. Acad. Sci. U. S. A. 97, 7933-7938. Yamamoto, H., Koga, H., Katoh, Y., Takahashi, S., Nakayama, K., and Shin, H.-W. (2010). Functional cross-talk between Rab14 and Rab4 through a dual effector, RUFY1/Rabip4. Mol. Biol. Cell 21, 2746-2755. Yi, Z., Yokota, H., Torii, S., Aoki, T., Hosaka, M., Zhao, S., Takata, K., Takeuchi, T., and Izumi, T. (2002). The Rab27a/granuphilin complex regulates the exocytosis of
Yu, M., Kasai, K., Nagashima, K., Torii, S., Yokota-Hashimoto, H., Okamoto, K., Takeuchi, T., Gomi, H., and Izumi, T. (2007). Exophilin4/Slp2-a targets glucagon granules to the plasma membrane through unique Ca2+-inhibitory phospholipid-binding activity of the C2A domain. Mol. Biol. Cell 18, 688-696.
Journal of Cell Science • Advance article
insulin-containing dense-core granules. Mol. Cell. Biol. 22, 1858-1867.
Journal of Cell Science • Advance article
Figures
Figure 1. Rab27a interacts with Rab2a through Noc2 (A) MIN6 cell lysates were incubated with control IgG or anti-Noc2 antibody. The immunoprecipitates, as well as an aliquot of the original lysates, were subjected to immunoblot detection with anti-Rab2a antibody. (B) INS1 832/13 cell lysates were analyzed as in (A) with the indicated antibodies. (C) MIN6 cells were infected with adenoviruses expressing control LacZ, MEF-Rab2a wild type (WT), or its mutants Q65L or S20N. The cells were lysed 48 h after the infection, and the immunoprecipitates with anti-FLAG antibody-conjugated beads were immunoblotted with the indicated antibodies. (D) MIN6 cells stably expressing MEF-Rab2a were infected with adenoviruses encoding shRNA against control GFP or Noc2. The immunoprecipitants by anti-FLAG antibody were immunoblotted with the indicated
Journal of Cell Science • Advance article
antibodies.
Figure 2. Rab2a interacts with the Noc2-Rab27a binary complex (A) HEK293A cells were transfected with plasmids expressing GFP-Noc2, HA-Rab27a, and/or FLAG-Rab2a. (B) MIN6 cells were infected with adenoviruses encoding
Beta-cell lines from ashen mice, and those stably expressing MEF-Rab27a, were established. Cell lysates (A-C) were analyzed by immunoprecipitation with anti-FLAG antibody followed by immunoblotting with the indicated antibodies. (D) HEK293A cells were transfected with the indicated amount (0.2, 1, or 5 μg) of plasmids expressing Rab27a, Rab2a, and OSF-tagged Noc2. Noc2 and the binding proteins were pulled down using Strep-Tactin beads, and were subjected to SDS-PAGE and CBB staining.
Journal of Cell Science • Advance article
MEF-tagged Noc2 wild type (WT), or its mutants E51A, I55A, or E51A/I55A. (C)
The band shown by an asterisk below Rab27a is a nonspecific protein. Note that the expression levels of Noc2 were decreased in the absence of either Rab2a or Rab27a in
Journal of Cell Science • Advance article
HEK293A cells.
(A) Diagrams of the domain structure and deletion mutant construction of Noc2. (B-F) MIN6 (B-E) or INS1 823/13 cells (F) were infected with adenoviruses expressing MEFor FLAG-tagged Noc2 wild type or its mutants. The immunoprecipitates with anti-FLAG antibody were by immunoblotted with anti-FLAG, anti-Rab27a or anti-Rab2a antibodies.
Journal of Cell Science • Advance article
Figure 3. The N-terminal region of Noc2 is required for binding to Rab2a
Journal of Cell Science • Advance article
Figure 4. Localization of Rab27a, Rab2a, and Noc2 on mature or immature granules INS1 832/13 cells were infected with adenoviruses expressing MEF-tagged Rab27a, Noc2, or Rab2a. Cells were fixed and coimmunostained with anti-FLAG and either
Journal of Cell Science • Advance article
anti-insulin (A) or anti-proinsulin (B) antibodies. Bar, 10 m.
Journal of Cell Science • Advance article
Figure 5. Localization of Noc2 mutants defective in binding to Rab27a and/or Rab2a INS1 832/13 cells were infected with adenoviruses expressing the MEF-Noc2 mutants Δ(11-20), T38A, or E51A/I55A. Cells were fixed and coimmunostained with
Journal of Cell Science • Advance article
anti-FLAG and either anti-insulin (A) or anti-proinsulin antibodies (B). Bar, 10 m.
Figure 6. Effects of overexpression of Noc2 and its mutants on insulin secretion INS1 832/13 cells were infected with adenoviruses expressing control GFP, FLAG-tagged Noc2 wild type, or its mutants defective in binding to Rab27a and/or to Rab2a. (A) The expression levels of endogenous and exogenous Noc2, as well as that of α-tubulin, were examined by immunoblotting. (B,C) The cells were preincubated in low-glucose (2.8 mM) KRB buffer for 2 h, and were then incubated in the same low-glucose buffer or the high-glucose (25 mM) buffer for 2 h. Insulin secreted in the media (B) and that left in the cells (C) were measured. Data are expressed as the mean
Journal of Cell Science • Advance article
±SD (n = 3). *P<0.05.
Journal of Cell Science • Advance article
Figure 7. Effects of depletion of Rab27a, Noc2, Rab2a, or ICA69 on insulin secretion and processing INS1 832/13 cells were infected with adenoviruses harboring shRNAs targeting Rab27a, Rab2a, Noc2, ICA69, or control GFP. (A) Protein expression levels were examined by immunoblotting with the indicated antibodies. The band shown by an asterisk in the panel of Rab27a is a nonspecific protein. (B-G) The cells treated with the shRNAs were incubated in low-glucose (2.8 mM) KRB buffer for 2 h, and were then incubated in either low-glucose or high-glucose (25 mM) buffer for 2 h. The amount of insulin (B) or proinsulin (E) secreted in the media and that of insulin (C) or proinsulin (F) left in the cells were measured. The ratios of insulin secreted in the media to insulin content left in the cells (D) and those of proinsulin to insulin content in the cells (G) were also shown.
Journal of Cell Science • Advance article
Data are expressed as the mean ± SD (n = 3). *P<0.05.
1 2 3 5 4 6 8 9 10 11 12 13 14 15 Noc2 FLAGRab27a 7 16 17 18 19 Rab2a
25 20
D
100 75
MEF-Rab27a Rab27a
MEF-Rab2a Rab2a
25 20 50
Noc2
1 2
37
3
100
4
75
5
Granuphilin A
Granuphilin B
E
MIN6 cells Input
anti-FLAG IP
La cZ W T Q 78 T2 L 3 N N 13 La 3I c W Z T Q 78 T2 L 3 N N 13 3I
6
50
MEF-Rab18 MEF-Rab2a
MIN6 cells Input anti-FLAG IP
25 MEF-Noc2
Empty vector
250 150
Granuphilin A Granuphilin B
50
20 MIN6 cells
KDa
GFP
100 75
KDa
B
MEF-Rab2a
20 150
KDa FLAGNoc2 11 Noc2
37
20
Rab2a
50
37 75
25
7 Rab27a 50
20
8 9 Rab2a 10
25
Noc2 ICA69
Rab3
MIN6 cells
Figure S1. Analysis of the Rab2a-Noc2-Rab27a complex (A,B) MIN6 cells, or those stably expressing MEF-Rab27a (A) or MEF-Noc2 (B), were lysed and subjected to MEF tag-based purification. Purified proteins bound to bait proteins were detected by SDS-PAGE followed by Oriole fluorescent gel staining. (C,D) MIN6 cells were infected with adenoviruses expressing control GFP, MEF-Rab2a, MEF-Rab18 (C), or MEF-Rab27a (D). The cells were lysed 48 h after infection and the immunoprecipitates by anti-FLAG antibody were immunoblotted with the indicated antibodies. (E) MIN6 cells were infected with adenoviruses expressing control LacZ; MEF-Rab27a wild type (WT); or its mutants, Q78L, T23N, or N133I. The cells were lysed 48 h after infection, and the immunoprecipitates by anti-FLAG antibodywere immunoblotted with the indicated antibodies.
Journal of Cell Science • Supplementary information
37
Rab27a
25
MEF-Rab2a
50
37
MEF-Rab27a
75
25
GFP
100
MEF-Rab2a
150
MEF-Rab2a MEF-Rab18
MEF-Rab27a
250
MEF-Rab18
KDa 37
GFP
MIN6 cells Input anti-FLAG IP
GFP
KDa
C
MEF-Rab27a
A
Empty vector
J. Cell Sci. 130: doi:10.1242/jcs.195479: Supplementary information
J. Cell Sci. 130: doi:10.1242/jcs.195479: Supplementary information
50
Control IP
KDa
Input
MIN6 cells anti-FLAG IP
- +
CIAP
Noc2
37
25
20
FLAGNoc2
Rab27a
Rab2a
Figure S2. Effects of Noc2 dephosphorylation on the complex formation MIN6 cells were infected with adenoviruses expressing control GFP or MEF-Noc2. The immunoprecipitates by anti-FLAG antibody were incubated with or without calf intestine alkaline phosphatase (CIAP; CAP-101, TOY OBO) for 1 h at 37 , and were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies.
Journal of Cell Science • Supplementary information
50
J. Cell Sci. 130: doi:10.1242/jcs.195479: Supplementary information
A
INS1 832/13 cells anti-Insulin
B
anti-Proinsulin
anti-Insulin/anti-Proinsulin
INS1 832/13 cells anti-FLAG
anti-EEA1/anti-FLAG
anti-PDI
anti-FLAG
anti-PDI/anti-FLAG
anti-TGN38
anti-FLAG
anti-TGN38/anti-FLAG
Figure S3. Intracellular localization of Rab27a, Rab2a, and Noc2 (A) INS1 832/13 cells were fixed and coimmunostained with anti-insulin and anti-proinsulin antibodies. (B) INS1 832/13 cells were infected with adenoviruses expressing MEF-tagged Rab27a, Noc2, or Rab2a. Cells were fixed and coimmunostained with anti-FLAG and either anti-EEA1, anti-TGN38, or anti-PDI antibodies. Bars, 10 m.
Journal of Cell Science • Supplementary information
MEF-Noc2
MEF-Rab2a
MEF-Rab27a
MEF-Noc2
MEF-Rab2a
MEF-Rab27a
anti-EEA1
J. Cell Sci. 130: doi:10.1242/jcs.195479: Supplementary information
shRab2a α-Tubulin
50
*
*
0.8 0.6 0.4
2.0 1.5 1.0 0.5
0.2
si Rab2a
si Noc2
0
si Control
si Rab2a
si Noc2
si Control
D
*
2.5
1.0
0
C
Total insulin (relative ratio to control)
Noc2 37
25 mM *
1.2
Rab2a
20
Glucose 2.8 mM
Insulin secretion (relative ratio to control)
shNoc2
KDa
B
INS1 832/13 cells shGFP
A
INS1 832/13 cells anti-FLAG
anti-Proinsulin / anti-FLAG
Figure S4. Effects of Noc2-, Rab2a-, Rab27a-, or ICA69-depletion (A-C) INS1 832/13 cells were transfected with siRNAs against GFP, Noc2, or Rab2a. (A) The protein expression levels were analyzed by immunoblotting with the indicated antibodies. (B,C) The siRNA-treated cells were preincubated in low-glucose (2.8 mM) KRB buffer for 2 h, and were then incubated in either the low-glucose or high-glucose (25 mM) buffer for 2 h. Insulin secreted in the media (B) and that left in the cells (C) were measured. Data are expressed as the mean ± SD (n = 3). *P<0.05. (D) INS1 832/13 cells expressing MEF-Noc2 were infected with adenoviruses harboring shRNAs targeting control GFP, Rab27a, Rab2a, or ICA69, as in Figure 7A. The cells were coimmunostained with anti-proinsulin and anti-FLAG antibodies. Note that Rab2a-depletion simultaneously caused Noc2-depletion, as shown in Figure 7A. Bar, 10 μm.
Journal of Cell Science • Supplementary information
sh ICA69 MEF-Noc2
sh Rab2a MEF-Noc2
sh Rab27a MEF-Noc2
sh Control MEF-Noc2
anti-Proinsulin
J. Cell Sci. 130: doi:10.1242/jcs.195479: Supplementary information
Supplementary Tables
Table S1. Identification of Rab27a-interacting proteins by LC-MS/MS Protein bands specific to MEF-Rab27a isolated by MEF-tag purification (Fig. S1A) were identified by LC-MS/MS and MASCOT software. For the protein band for MEF-Rab27a bait, 1:10 volume of the peptide mixtures was analyzed.
Table S2. Identification of Noc2-interacting proteins by LC-MS/MS Protein bands specific to MEF-Noc2 isolated by MEF-tag purification (Fig. S1B) were identified by LC-MS/MS and MASCOT software. For the protein band for MEF-Noc2 bait, 1:10 volume of the peptide mixtures was analyzed.
Click here to Download Table S2
Journal of Cell Science • Supplementary information
Click here to Download Table S1
1 2 3 5 4 6 8 9 10 11 12 13 14 15 Noc2 FLAGRab27a 7 16 17 18 19 Rab2a
25 20
D
100 75
MEF-Rab27a Rab27a MEF-Rab2a Rab2a
25 20 50
Noc2
1 2
37
3
100
4
75
5
Granuphilin A Granuphilin B
E
MIN6 cells Input
anti-FLAG IP
La cZ W T Q 78 T2 L 3 N N 13 La 3I c W Z T Q 78 T2 L 3 N N 13 3I
6
50
MEF-Rab18 MEF-Rab2a
MIN6 cells Input anti-FLAG IP
25 MEF-Noc2
Empty vector
250 150
Granuphilin A Granuphilin B
50
20 MIN6 cells
KDa
GFP
100 75
KDa
B
MEF-Rab2a
20 150
KDa FLAGNoc2 11 Noc2
37
20
Rab2a
50 37 75
25
7 Rab27a 50
20
8 9 Rab2a 10
25
Noc2 ICA69
Rab3
MIN6 cells
Figure S1. Analysis of the Rab2a-Noc2-Rab27a complex (A,B) MIN6 cells, or those stably expressing MEF-Rab27a (A) or MEF-Noc2 (B), were lysed and subjected to MEF tag-based purification. Purified proteins bound to bait proteins were detected by SDS-PAGE followed by Oriole fluorescent gel staining. (C,D) MIN6 cells were infected with adenoviruses expressing control GFP, MEF-Rab2a, MEF-Rab18 (C), or MEF-Rab27a (D). The cells were lysed 48 h after infection and the immunoprecipitates by anti-FLAG antibody were immunoblotted with the indicated antibodies. (E) MIN6 cells were infected with adenoviruses expressing control LacZ; MEF-Rab27a wild type (WT); or its mutants, Q78L, T23N, or N133I. The cells were lysed 48 h after infection, and the immunoprecipitates by anti-FLAG antibodywere immunoblotted with the indicated antibodies.
Journal of Cell Science • Supplementary information
37
Rab27a
25
MEF-Rab2a
50
37
MEF-Rab27a
75
25
GFP
100
MEF-Rab2a
150
MEF-Rab2a MEF-Rab18
MEF-Rab27a
250
MEF-Rab18
KDa 37
GFP
MIN6 cells Input anti-FLAG IP
GFP
KDa
C
MEF-Rab27a
A
Empty vector
J. Cell Sci. 130: doi:10.1242/jcs.195479: Supplementary information
Journal of Cell Science • Supplementary information
J. Cell Sci. 130: doi:10.1242/jcs.195479: Supplementary information
Journal of Cell Science • Supplementary information
J. Cell Sci. 130: doi:10.1242/jcs.195479: Supplementary information
Journal of Cell Science • Supplementary information
J. Cell Sci. 130: doi:10.1242/jcs.195479: Supplementary information
J. Cell Sci. 130: doi:10.1242/jcs.195479: Supplementary information
Supplementary Tables
Table S1. Identification of Rab27a-interacting proteins by LC-MS/MS Protein bands specific to MEF-Rab27a isolated by MEF-tag purification (Fig. S1A) were identified by LC-MS/MS and MASCOT software. For the protein band for MEF-Rab27a bait, 1:10 volume of the peptide mixtures was analyzed.
Table S2. Identification of Noc2-interacting proteins by LC-MS/MS Protein bands specific to MEF-Noc2 isolated by MEF-tag purification (Fig. S1B) were identified by LC-MS/MS and MASCOT software. For the protein band for MEF-Noc2 bait, 1:10 volume of the peptide mixtures was analyzed.
Click here to Download Table S2
Journal of Cell Science • Supplementary information
Click here to Download Table S1
