Rab3 Proteins Involved in Vesicle Biogenesis and Priming in Embryonic Mouse Chromaffin Cells

The four Rab3 paralogs A–D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) (rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD^?/?) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD^?/? animals large dense‐core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD^?/? cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short‐term (4–6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state.     

The Slp homology domain of synaptotagmin-like proteins 1-4 and Slac2 functions as a novel Rab27A binding domain.

rab27A, which encodes a small GTP-binding protein, was recently identified as a gene in which mutations caused human hemophagocytic syndrome (Griscelli syndrome) and ashen mice, which exhibit defects in melanosome transport as well as in regulated granule exocytosis in cytotoxic T lymphocytes. However, little is known about the molecular mechanism of Rab27A-dependent membrane trafficking or the specific effector molecules of Rab27A. In this study, we discovered that the Slp (synaptotagmin-like protein) homology domain (SHD) of Slp1--3 and Slac2-a/b specifically and directly binds the GTP-bound form of Rab27A both in vitro and in intact cells but not of the other Rabs tested (Rab1, Rab2, Rab3A, Rab4, Rab5A, Rab6A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab28, and Rab37). Immunocytochemical analysis revealed that Slp2 (or Slp1) colocalized with Rab27A in the melanosomes of melanoma cells. Slp2 and Rab27A were distributed to the periphery of the cells (especially at the dendritic tips) in the wild-type melanoma cells, whereas they accumulated in the perinuclear region in the melanosome transport-defective cells (S91/Cloudman). These results strongly indicated that the SHD of Slp1--3 and Slac2 functions as an in vivo Rab27A binding domain.     

Carboxyl-methylation of Rab3D in the rat pancreatic acinar tumor cell line AR42J.

Rab3D is a small GTPase implicated in regulated exocytosis, and is a marker of secretory granules in exocrine cells. We have previously shown that rab3D undergoes reversible carboxyl-methylation in adult rat pancreatic acinar cells, and that carboxyl-methylation of rab3D is developmentally regulated concomitantly with the maturation of the regulated secretory apparatus in rat pancreas. We also observed that dexamethasone treatment of the rat pancreatic acinar tumor cell line, AR42J, led to a significant increase in the size of the unmethylated pool of a rab3-like protein. The current study was designed to further characterize this rab3-like protein. Here we show that AR42J cells express rab3D, and that the protein focuses on 2D gels as two spots with pI values of 4.9 and 5.0. Treatment of AR42J cells with N-acetyl-S-geranylgeranyl-l-cysteine, an inhibitor of carboxyl-methylation, led to a decrease in the basic form of rab3D and a proportional increase in the acidic form. In contrast, N-acetyl-S-farnesyl-l-cysteine, which inhibits carboxyl-methylation of farnesylated proteins, had no effect. Lovastatin, an inhibitor of geranylgeranylation, also induced an accumulation of the acidic form of rab3D. Taken together, these data indicate that rab3D can undergo reversible carboxyl-methylation in AR42J cells by a geranylgeranyl-specific methyltransferase. The 2D gel and immunoblotting analyses indicated that dexamethasone treatment of AR42J cells led to an increase in the proportion of the unmethylated form of rab3D concurrent to inducing a regulated secretory pathway, similar to the rab3D profile change in developing rat pancreas. Our data, along with previous studies done on developing rat pancreas, indicate that the tumor cell line AR42J represents a good model system for studying the regulated secretory pathway, and that carboxyl-methylation of rab3D may play a role in the acquisition of stimulus-secretion coupling.     

八肋游仆虫Rab家族新成员Eo-rab-1N基因的克隆与序列分析

Rab蛋白家族属于小分子GTP结合蛋白家族Ras超家族中最大的亚家族,主要在囊泡运输中起作用。本实验运用PCR、RT-PCR等技术,从八肋游仆虫中克隆到一种新的rab基因。序列分析结果表明：在大核中,该基因全长884bp,除去两端的端粒与非编码区,该基因在大核中由723bp组成。从小核中克隆相应的基因片段,此基因片段序列与大核中序列一致,表明该基因在小核中无内部删除序列的存在。通过RT-PCR,从mRNA获得的该基因的开放读框为663bp,表明该基因在转录过程中有内含子的删除。大核基因序列和cDNA序列比较,发现60bp的内含子序列位于大核基因的153~212bp之间,并符合一类内含子GU-AG剪切规则。在遗传密码使用上,该基因内部含有2个TGA,在游仆虫中编码半胱氨酸。同时首次发现,八肋游仆虫基因使用TAG作为终止密码子。NCBI上序列比对表明该基因翻译的蛋白与其它物种Rab1蛋白的同源性达49%~52%,因此我们将它命名为Eo-rab-1N,GenBank登录号为DQ105562。Eo-rab-1N与其他物种的Rab1蛋白构建进化树,发现该蛋白的进化与物种的进化保持一致,表明该基因在细胞中具有重要功能。     

Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2

We evaluated the contribution ofrab5a and rab11a to trafficking and signaling of protease-activatedreceptor 2 (PAR2), a receptor for trypsin and tryptase. Agonistsstimulated internalization of PAR2 into early endosomes containingrab5a. Dominant negative rab5aS34N disrupted early endosomes andinhibited agonist-stimulated endocytosis of PAR2. Internalized PAR2 wassorted to lysosomes, and rab5a remained in early endosomes. Rab5apromoted and rab5aS34N impeded resensitization of trypsin-inducedcalcium mobilization. Rab11a was detected in the Golgi apparatus withPAR2, and PAR2 agonists stimulated redistribution of rab11a intovesicles containing PAR2 that migrated to the cell surface. Dominantnegative rab11aS25N was mostly confined to the Golgi apparatus.Although expression of rab11aS25N caused retention of PAR2 in the Golgiapparatus, it did not abolish trafficking of PAR2 to the cell surface.However, expression of wild-type rab11a accelerated both recovery ofPAR2 at the cell surface and resensitization of PAR2 signaling. Thus rab5a is required for PAR2 endocytosis and resensitization, whereas rab11a contributes to trafficking of PAR2 from the Golgi apparatus tothe plasma membrane.

     

rab5a基因在肿瘤转移中的作用研究

为研究rab5a基因在肿瘤转移机制中的作用,将该基因稳定转染至低转移肺腺癌细胞系AGZY83-a中,采用Superarray肿瘤转移相关基因微芯片分析rab5a对肿瘤转移相关基因的表达影响,共获得了5个差异表达基因,rab5a基因促进s100a4的表达,同时抑制了nm23a、rac1、cst3、col4a2等基因的表达,并分别在RNA及蛋白水平进行验证,确认rab5a基因影响了肿瘤转移的多个途径,促进了肿瘤细胞转移能力增强。     

Rab3a is involved in transport of synaptic vesicles to the active zone in mouse brain nerve terminals

The rab family of GTP-binding proteins regulates membrane transport between intracellular compartments. The major rab protein in brain, rab3A, associates with synaptic vesicles. However, rab3A was shown to regulate the fusion probability of synaptic vesicles, rather than their transport and docking. We tested whether rab3A has a transport function by analyzing synaptic vesicle distribution and exocytosis in rab3A null-mutant mice. Rab3A deletion did not affect the number of vesicles and their distribution in resting nerve terminals. The secretion response upon a single depolarization was also unaffected. In normal mice, a depolarization pulse in the presence of Ca(2+) induces an accumulation of vesicles close to and docked at the active zone (recruitment). Rab3A deletion completely abolished this activity-dependent recruitment, without affecting the total number of vesicles. Concomitantly, the secretion response in the rab3A-deficient terminals recovered slowly and incompletely after exhaustive stimulation, and the replenishment of docked vesicles after exhaustive stimulation was also impaired in the absence of rab3A. These data indicate that rab3A has a function upstream of vesicle fusion in the activity-dependent transport of synaptic vesicles to and their docking at the active zone.     

Melanophilin, the product of the leaden locus, is required for targeting of myosin-Va to melanosomes

The formation of complex subcellular organelles requires the coordinated targeting of multiple components. Melanosome biogenesis in mouse melanocytes is an excellent model system for studying the coordinated function of multiple gene products in intracellular trafficking. To begin to order events in melanosome biogenesis and distribution, we employed the classical coat-color mutants ashen , dilute , and leaden , which affect melanosome distribution, but not melanin synthesis. The loci have been renamed Rab27a , Myo5a , and Mlph for their gene products. While each of the three loci has been shown to be required for melanosome distribution, the point(s) at which each acts is unknown. We have utilized primary melanocytes to examine the interdependencies between rab27a, myosin-Va, and melanophilin. The localization of rab27a to melanosomes did not require the function of either myosin-Va or melanophilin, but leaden function was required for the association of myosin-Va with melanosomes. In leaden melanocytes permeabilized before fixation, myosin-Va immunoreactivity was greatly attenuated, suggesting that myosin-Va is free in the cytoplasm. Finally, we have complemented both the leaden and ashen phenotypes by cell fusion and observed redistribution of mature melanosomes in the absence of both protein and melanin synthesis. Together, our data suggest a model for the initial assembly of the machinery required for melanosome distribution.     

Inhibition of Endosome Fusion by Wortmannin Persists in the Presence of Activated rab5

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5^Q79L or of a xanthosine triphosphate-binding mutant, rab5^D136N, in the presence of the nonhydrolysable analogue XTPγS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPγS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5^Q79L, beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPγS.     

Expression and cellular localization of rab28 mRNA and Rab28 protein during maize embryogenesis

The maize abscisic acid (ABA) responsive gene rab 28, has been shown to be ABA-inducible in embryos and vegetative tissues. A polyclonal antiserum was raised against Rab28 protein. Using immunoblotting and immunoprecipitation, the antiserum specifically recognized a protein of about 30 kDa and pl 6 which is in close agreement with the molecular weight and pl predicted by the deduced amino acid sequence. The rab 28 gene product accumulated during late embryogenesis. In vegetative tissues, dehydration stress induced rab 28 gene expression both in the light and in the dark. The spatial and temporal pattern of rab 28 mRNA expression during embryogenesis was investigated by in situ hybridization using digoxigenin-labelled rab 28 probes, and the immunochemical localization of Rab28 protein using anti-Rab28 antibodies. Expression of rab 28 mRNA is restricted to provascular tissues in young embryos, and at later stages of development the most prevalent accumulation occurred in meristem and in the vascular elements of the plumule, root and scutellum. Using immunoelectron microscopy the Rab28 protein has been located in the nucleolus of different cell types. In light of these results the stress regulation of rab 28 and a likely role for this protein during late embryogenesis are discussed.     

Rabaptin-5alpha/rabaptin-4 serves as a linker between rab4 and gamma(1)-adaptin in membrane recycling from endosomes

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-adaptin regulate membrane recycling.     

Association of Rab3A with synaptic vesicles at late stages of the secretory pathway

Rab3A is a small GTP-binding protein highly concentrated on synaptic vesicles. Like other small GTP-binding proteins it is thought to cycle between a soluble and a membrane-associated state. To determine at which stage of the life cycle of synaptic vesicles rab3A is associated with their membranes, the localization of the protein in neurons and neuroendocrine cells at different developmental and functional stages was investigated. In all cases, rab3A was colocalized with synaptic vesicle markers at the cell periphery, but was absent from the Golgi area, suggesting that rab3A associates with vesicles distally to the Golgi complex and dissociates from vesicle membranes before they recycle to this region. Immunofluorescence experiments carried out on frog motor end plates demonstrated that massive exocytosis of synaptic vesicles is accompanied by a translocation of rab3A to the cell surface. The selective localization of rab3A on synaptic vesicles at stages preceding their fusion with the plasmalemma suggests that the protein is part of a regulatory machinery that is assembled onto the vesicles in preparation for exocytosis.     

Distinct Rab binding specificity of Rim1, Rim2, rabphilin,and Noc2. Identification of a critical determinant of Rab3A/Rab27A recognition by Rim2

Rabphilin, Rim, and Noc2 have generally been believed to be the Rab3 isoform (Rab3A/B/C/D)-specific effectors that regulate secretory vesicle exocytosis in neurons and in some endocrine cells. The results of recent genetic analysis of rabphilin knock-out animals, however, strongly refute this notion, because there are no obvious genetic interactions between Rab3 and rabphilin in nematoda (Staunton, J., Ganetzky, B., and Nonet, M. L. (2001) J. Neurosci. 21, 9255-9264), suggesting that Rab3 is not a major ligand of rabphilin in vivo. In this study, I tested the interaction of rabphilin, Rim1, Rim2, and Noc2 with 42 different Rab proteins by cotransfection assay and found differences in rabphilin, Rim1, Rim2, and Noc2 binding to several Rab proteins that belong to the Rab functional group III (Rab3A/B/C/D, Rab26, Rab27A/B, and Rab37) and/or VIII (Rab8A and Rab10). Rim1 interacts with Rab3A/B/C/D, Rab10, Rab26, and Rab37; Rim2 interacts with Rab3A/B/C/D and Rab8A; and rabphilin and Noc2 interact with Rab3A/B/C/D, Rab8A, and Rab27A/B. By contrast, the synaptotagmin-like protein homology domain of Slp homologue lacking C2 domains-a (Slac2-a)/melanophilin specifically recognizes Rab27A/B but not other Rabs. I also found that alternative splicing events in the first alpha-helical region (alpha(1)) of the Rab binding domain of Rim1 alter the Rab binding specificity of Rim1. Site-directed mutagenesis and chimeric analyses of Rim2 and Slac2-a indicate that the acidic cluster (Glu-50, Glu-51, and Glu-52) in the alpha(1) region of the Rab binding domain of Rim2, which is not conserved in the synaptotagmin-like pro tein homology domain of Slac2-a, is a critical determinant of Rab3A recognition. Based on these results, I propose that Rim, rabphilin, and Noc2 function differently in concert with functional group III and/or VIII Rab proteins, including Rab3 isoforms.     

Rab9 functions in transport between late endosomes and the trans Golgi network.

Rab proteins represent a large family of ras-like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion. We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9. The majority of rab9 appears to be located on the surface of late endosomes. Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate. In vitro-prenylated rab9 protein, but not C-terminally truncated rab9, stimulated the transport of mannose 6-phosphate receptors from late endosomes to the trans Golgi network in a cell-free system that reconstitutes this transport step. Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP. These results strongly suggest that rab9 functions in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Moreover, our results confirm the observation that a given organelle may bear multiple rab proteins with different biological functions.     

Adiponectin and leptin are secreted through distinct trafficking pathways in adipocytes

Adiponectin and leptin are two adipokines secreted by white adipose tissue that regulate insulin sensitivity. Previously we reported that adiponectin but not leptin release depends on GGA-coated vesicle formation, suggesting that leptin and adiponectin may follow different secretory routes. Here we have examined the intracellular trafficking pathways that lead to the secretion of these two hormones. While adiponectin and leptin displayed distinct localization in the steady-state, treatment of adipocytes with brefeldin A inhibited both adiponectin and leptin secretion to a similar level, indicating a common requirement for class III ADP-ribosylating factors and an intact Golgi apparatus. Adiponectin secretion was significantly reduced by endosomal inactivation in both 3T3L1 and rat isolated adipocytes, whereas this treatment had no effect on leptin secretion. Importantly, endosomal inactivation completely abolished the insulin stimulatory effect on adiponectin release in rat adipocytes. Confocal microscopy studies revealed colocalization of adiponectin with endogenous rab11 a marker for the recycling endosome, and with expressed rab5-GFP mutant (rab5Q75L) a marker for the early endosome compartment. Colocalization of adiponectin and rab5Q75L was increased in endosome inactivated cells. Consistent with these findings adiponectin secretion was reduced in cells expressing mutants of Rab11 and Rab5 proteins. In contrast, expression of an inactive (kinase dead) mutant of Protein Kinase D1 moderately but significantly inhibited leptin secretion without altering adiponectin secretion. Taken together, these results suggest that leptin and adiponectin secretion involve distinct intracellular compartments and that endosomal compartments are required for adiponectin but not for leptin secretion.