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Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells
are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone
(Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were
analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D
and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated
in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared
to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to
basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression
of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release
or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may
regulate amylase content in these cells. 相似文献
3.
Collins PR Stack CM O'Neill SM Doyle S Ryan T Brennan GP Mousley A Stewart M Maule AG Dalton JP Donnelly S 《The Journal of biological chemistry》2004,279(17):17038-17046
The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated. Only the fully processed and active mature enzyme can be detected in medium in which adult F. hepatica are cultured. However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut. These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a conserved GXNXFXD motif in the propeptide generates an active 30-kDa intermediate form. Further activation of the enzyme was initiated by decreasing the pH to 5.0 and involved the progressive processing of the 37 and 30-kDa forms to other intermediates and finally to a fully mature 24.5 kDa cathepsin L with an additional 1 or 2 amino acids. An active site mutant procathepsin L, constructed by replacing the Cys(26) with Gly(26), failed to autoprocess. However, [Gly(26)]procathepsin L was processed by exogenous wild-type cathepsin L to a mature enzyme plus 10 amino acids attached to the N terminus. This exogenous processing occurred without the formation of a 30-kDa intermediate form. The results indicate that activation of procathepsin L1 by removal of the propeptide can occur by different pathways, and that this takes place within the parasite gut where the protease functions in food digestion and from where it is liberated as an active enzyme for additional extracorporeal roles. 相似文献
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Fukuda M Kanno E Saegusa C Ogata Y Kuroda TS 《The Journal of biological chemistry》2002,277(42):39673-39678
Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a was originally identified as a protein specifically associated with insulin-containing vesicles in pancreatic beta-cells (Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999) J. Biol. Chem. 274, 28542-28548). Previously, we showed that the N-terminal Slp homology domain of Slp4-a interacts with the GTP-bound form of Rab3A, Rab8, and Rab27A both in vitro and in intact cells (Kuroda, T. S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 9212-9218). How Slp4-a.Rab complex controls regulated secretion, and which Rab isoforms dominantly interact with Slp4-a in vivo, however, have remained unknown. In this study, we showed by immunocytochemistry and subcellular fractionation that three Rabs, Rab3A, Rab8, and Rab27A, and Slp4-a are endogenously expressed in neuroendocrine PC12 cells and localized on dense-core vesicles, and we discovered that the Slp4-a.Rab8 and Slp4-a.Rab27A complexes, but not Slp4-a.Rab3A complexes, are formed on dense-core vesicles in PC12 cells, although the majority of Rab8 is present in the cell body and is free of Slp4-a. We further showed that expression of Rab27A, but not of Rab8, promotes high KCl-dependent secretion of neuropeptide Y (NPY) in PC12 cells, whereas expression of Slp4-a, but not of an Slp4-a mutant incapable of Rab27A binding, inhibits NPY secretion in PC12 cells. In contrast, expression of Slp3-a, but not of Slp3-b lacking an N-terminal Rab27A-binding domain, promotes NPY secretion. These findings suggest that the Slp family controls regulated dense-core vesicle exocytosis via binding to Rab27A. 相似文献
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Fukuda M 《The Journal of biological chemistry》2003,278(17):15390-15396
Slp4-a (synaptotagmin-like protein 4-a)/granuphilin-a is specifically localized on dense-core vesicles in PC12 cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A via the N-terminal Slp homology domain (SHD) (Fukuda, M., Kanno, E., Saegusa, C., Ogata, Y., and Kuroda, T. S. (2002) J. Biol. Chem. 277, 39673-39678). However, the mechanism of the inhibition by Slp4-a has never been elucidated at the molecular level and is still a matter of controversy. In this study, I discovered an unexpected biochemical property of Slp4-a, that Slp4-a, but not other Rab27 effectors reported thus far, is capable of interacting with both Rab27A(T23N), a dominant negative form that mimics the GDP-bound form, and Rab27A(Q78L), a dominant active form that mimics the GTP-bound form, whereas Slp4-a specifically recognizes the GTP-bound form of Rab3A and Rab8A and does not recognize their GDP-bound form. I show by deletion and mutation analyses that the TGDWFY sequence in SHD2 is essential for Rab27A(T23N) binding, whereas SHD1 is involved in Rab27A(Q78L) binding. I further show by immunoprecipitation and cotransfection assays that Munc18-1, but not syntaxin IA, directly interacts with the C-terminal domain of Slp4-a in a Rab27A-independent manner. Expression of Slp4-a mutants that lack Rab27A(T23N) binding activity (i.e. specific binding to Rab27A(Q78L)) completely reverses the inhibitory effect of the wild-type Slp4-a on high KCl-dependent neuropeptide Y secretion in PC12 cells. The results strongly indicate that interaction of Slp4-a with the GDP-bound form of Rab27A, not with syntaxin IA or Munc18-1, is the primary reason that Slp4-a expression inhibits dense core vesicle exocytosis in PC12 cells. 相似文献
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Cohen-Solal KA Sood R Marin Y Crespo-Carbone SM Sinsimer D Martino JJ Robbins C Makalowska I Trent J Chen S 《Biochimica et biophysica acta》2003,1651(1-2):68-75
Rab proteins, a subfamily of the ras superfamily, are low molecular weight GTPases involved in the regulation of intracellular vesicular transport. Cloning of human RAB32 was recently described. Presently, we report the cloning and characterization of the mouse homologue of Rab32. We show that murine Rab32 exhibits a ubiquitous expression pattern, with tissue-specific variation in expression level. Three cell types with highly specialized organelles, melanocytes, platelets and mast cells, exhibit relatively high level of Rab32. We show that in murine amelanotic in vitro transformed melanocytes as well as in human amelanotic metastatic melanoma cell lines, the expression of Rab32 is markedly reduced or absent, in parallel with the loss of expression of two key enzymes for the production of melanin, tyrosinase and Tyrp1. Therefore, in both mouse and human systems, the expression of Rab32 correlates with the expression of genes involved in pigment production. However, in melanoma samples, amelanotic due to a mutation in the tyrosinase gene, the expression of Rab32 remains at levels comparable to those observed in pigmented melanoma samples. Finally, we observed co-localization of Rab32 and the melanosomal proteins, Tyrp1 and Dct, indicating an association of Rab32 with melanosomes. Based on these data, we propose the inclusion of Rab32 to the so-called melanocyte/platelet family of Rab proteins. 相似文献
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Ishikura S Bilan PJ Klip A 《Biochemical and biophysical research communications》2007,353(4):1074-1079
Insulin causes translocation of glucose transporter GLUT4 to the membrane of muscle and fat cells, a process requiring Akt activation. The Rab GTPase-activating protein (Rab-GAP) AS160 is inhibited upon phosphorylation by insulin-activated Akt, thereby allowing GLUT4 translocation. Although several Rab proteins are detected on GLUT4 vesicles, the target Rabs of AS160 involved in the GLUT4 translocation have not been identified. We test whether Rabs 8A, 10, and 14 (in vitro targets of AS160) rescue the inhibition of GLUT4 translocation caused by 'constitutively active' 4P-AS160 in L6 muscle cells. Coexpression of GFP-tagged Rabs 8A or Rab14 with 4P-AS160 prevented the inhibition of GLUT4 translocation imposed by 4P-AS160. GFP-tagged, constitutively active Rab8A also elicited this rescue. In contrast, neither wild-type nor constitutively active GFP-tagged Rab10 restored GLUT4 translocation. These results suggest that Rab8A and possibly Rab14 may be targets of AS160 leading to GLUT4 translocation in L6 muscle cells. 相似文献
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Yang M Chen T Han C Li N Wan T Cao X 《Biochemical and biophysical research communications》2004,318(3):792-799
Rab7 is a small Rab GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Here we report the cloning and characterization of a novel Rab7-like GTPase, which shares highest homology with Rab7 and thus is designated as Rab7b. Northern blot analysis shows that Rab7b mRNA is expressed in human heart, placenta, lung, skeletal muscle, and peripheral blood leukocyte. RT-PCR or Western blot analysis of Rab7b expression shows that Rab7b is selectively expressed in monocytes, monocyte-derived immature dendritic cells (DCs), and promyeloid or monocytic leukemia cell lines. In the peripheral blood, Rab7b is specifically detected in CD14(+) cells, but not in CD4(+), CD8(+), CD19(+) or CD56(+) cells. When immature DCs are matured with lipopolysaccharide (LPS), Rab7b expression is gradually downregulated, while Rab7b is upregulated when monocytes are activated by LPS treatments. In acute promyelocytic leukemia (APL) HL-60 and NB4 cell lines, Rab7b expression is upregulated after phorbol myristate acetate (PMA)-induced monocytic differentiation. By immunofluorescence confocal microscopy, we demonstrate that Rab7b is associated with lysosomal organelles. Our data suggest that Rab7b is a lysosome-localized monocytic cell-specific small GTPase, and is involved in PMA-induced APL cell differentiation and possibly in regulation of monocyte functions. 相似文献
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Faust F Gomez-Lazaro M Borta H Agricola B Schrader M 《Traffic (Copenhagen, Denmark)》2008,9(6):964-979
Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas, which allow digestive enzyme storage and regulated apical secretion. To understand the function of these important organelles, we are conducting studies to identify and characterize ZG membrane proteins. Small guanosine triphosphatases (GTPases) of the Rab family are key protein components involved in vesicular/granular trafficking and membrane fusion in eukaryotic cells. In this study, we show by morphological studies that Rab8 (Rab8A) localizes to ZGs in acinar cells of the pancreas. We find that Rab8 is present on isolated ZGs from rat pancreas and in the ZG membrane fraction obtained after granule subfractionation. To address a putative role of Rab8 in granule biogenesis, we conducted RNA interference experiments to 'knock down' the expression of Rab8 in pancreatic AR42J cells. Silencing of Rab8 (but not of Rab3) resulted in a decrease in the number of ZGs and in an accumulation of granule marker proteins within the Golgi complex. By contrast, the trafficking of lysosomal and plasma membrane proteins was not affected. These data provide first evidence for a role of Rab8 early on in ZG formation at the Golgi complex and thus, apical trafficking of digestive enzymes in acinar cells of the pancreas. 相似文献
11.
Rab3A is a small G-protein of the Rab family that is involved in the late steps of exocytosis. Here, we studied the role of Rab3A and its relationship with Munc13-1 and Munc18-1 during vesicle priming. Phorbol 12-myristate 13-acetate (PMA) is known to enhance the percentage of fusion-competent vesicles and this is mediated by protein kinase C (PKC)-independent Munc13-1 activation and PKC-dependent dissociation of Munc18-1 from syntaxin 1a. Our results show that the effects of PMA varied in cells overexpressing Rab3A or mutants of Rab3A and in cells with Rab3A knockdown. When Munc13-1 was overexpressed in Rab3A knockdown cells, secretion was completely inhibited. In cells overexpressing a Rab-interacting molecule (RIM)-binding deficient Munc13-1 mutant, 128-Munc13-1, the effects of Rab3A on PMA-induced secretion was abolished. The effect of PMA, which disappeared in cells overexpressing GTP-Rab3A (Q81L), could be reversed by co-expressing Munc18-1 but not its mutant R39C, which is unable to bind to syntaxin 1a. In cells overexpressing Munc18-1, manipulation of Rab3A activity had no effect on secretion. Finally, Munc18-1 enhanced the dissociation of Rab3A, and such enhancement correlated with exocytosis. In summary, our results support the hypothesis that the Rab3A cycle is coupled with the activation of Munc13-1 via RIM, which accounts for the regulation of secretion by Rab3A. Munc18-1 acts downstream of Munc13-1/RIM/Rab3A and interacts with syntaxin 1a allowing vesicle priming. Furthermore, Munc18-1 promotes Rab3A dissociation from vesicles, which then results in fusion. 相似文献
12.
Interaction of human breast fibroblasts with collagen I increases secretion of procathepsin B 总被引:6,自引:0,他引:6
Koblinski JE Dosescu J Sameni M Moin K Clark K Sloane BF 《The Journal of biological chemistry》2002,277(35):32220-32227
Interactions of stromal and tumor cells with the extracellular matrix may regulate expression of proteases including the lysosomal proteases cathepsins B and D. In the present study, we determined whether the expression of these two proteases in human breast fibroblasts was modulated by interactions with the extracellular matrix component, collagen I. Breast fibroblasts were isolated from non-malignant breast tissue as well as from tissue surrounding malignant human breast tumors. Growth of these fibroblasts on collagen I gels affected cell morphology, but not the intracellular localization of vesicles staining for cathepsin B or D. Cathepsins B and D levels (mRNA or intracellular protein) were not affected in fibroblasts growing on collagen I gels or plastic, nor was cathepsin D secreted from these cells. In contrast, protein expression and secretion of cathepsin B, primarily procathepsin B, was induced by growth on collagen I gels. The induced secretion appeared to be mediated by integrins binding to collagen I, as inhibitory antibodies against alpha(1), alpha(2), and beta(1) integrin subunits prevented procathepsin B secretion from fibroblasts grown on collagen. In addition, procathepsin B secretion was induced when cells were plated on beta(1) integrin antibodies. To our knowledge, this is the first examination of cathepsin B and D expression and localization in human breast fibroblasts and their regulation by a matrix protein. Secretion of the cysteine protease procathepsin B from breast fibroblasts may have physiological and pathological consequences, as proteases are required for normal development and for lactation of the mammary gland, yet can also initiate and accelerate the progression of breast cancer. 相似文献
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Urwyler S Nyfeler Y Ragaz C Lee H Mueller LN Aebersold R Hilbi H 《Traffic (Copenhagen, Denmark)》2009,10(1):76-87
Legionella pneumophila , the causative agent of Legionnaires' disease, replicates in macrophages and amoebae within ' Legionella -containing vacuoles' (LCVs), which communicate with the early secretory pathway and the endoplasmic reticulum. Formation of LCVs requires the bacterial Icm/Dot type IV secretion system. The Icm/Dot-translocated effector protein SidC selectively anchors to LCVs by binding the host lipid phosphatidylinositol-4-phosphate (PtdIns(4) P ). Here, we describe a novel and simple approach to purify intact vacuoles formed by L. pneumophila within Dictyostelium discoideum by using magnetic immunoseparation with an antibody against SidC, followed by density gradient centrifugation. To monitor LCV purification by fluorescence microscopy, we used Dictyostelium producing the LCV marker calnexin-GFP and L. pneumophila labeled with the red fluorescent protein DsRed. A proteome analysis of purified LCVs by liquid chromatography coupled to tandem mass spectrometry revealed 566 host proteins, including known LCV components, such as the small GTPases Arf1, Rab1 and Rab7. Rab8, an endosomal regulator of the late secretory pathway originating from the trans Golgi network, and the endosomal GTPase Rab14 were identified as novel LCV components, which were found to be present on vacuoles harboring wild-type but not Icm/Dot-deficient L. pneumophila . Thus, LCVs also communicate with the late secretory and endosomal pathways. Depletion of Rab8 or Arf1 by RNA interference reduced the amount of SidC on LCVs, indicating that the GTPases promote the recruitment of Legionella effectors by regulating the level of PtdIns(4) P . 相似文献
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Evidence of Rab3A expression, regulation of vesicle trafficking, and cellular secretion in response to heregulin in mammary epithelial cells
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Vadlamudi RK Wang RA Talukder AH Adam L Johnson R Kumar R 《Molecular and cellular biology》2000,20(23):9092-9101
Heregulin beta1 (HRG), a combinatorial ligand for human growth factor receptors 3 and 4, is a regulatory polypeptide that promotes the differentiation of mammary epithelial cells into secretory lobuloalveoli. Emerging evidence suggests that the processes of secretory pathways, such as biogenesis and trafficking of vesicles in neurons and adipose cells, are regulated by the Rab family of low-molecular-weight GTPases. In this study, we identified Rab3A as a gene product induced by HRG. Full-length Rab3A was cloned from a mammary gland cDNA library. We demonstrated that HRG stimulation of human breast cancer cells and normal breast epithelial cells induces the expression of Rab3A protein and mRNA in a cycloheximide-independent manner. HRG-mediated induction of Rab3A expression was blocked by an inhibitor of phosphatidylinositol 3-kinase but not by inhibitors of mitogen-activated protein kinases p38(MAPK) and p42/44(MAPK). Human breast epithelial cells also express other components of regulated vesicular traffic, such as rabphilin 3A, Doc2, and syntaxin. Rab3A was predominantly localized in the cytosol, and HRG stimulation of the epithelial cells also raised the level of membrane-bound Rab3A. HRG treatment induced a profound alteration in the cell morphology in which cells displayed neuron-like membrane extensions that contained Rab3A-coated, vesicle-like structures. In addition, HRG also promoted the secretion of cellular proteins from the mammary epithelial cells. The ability of HRG to modify exocytosis was verified by using a growth hormone transient-transfection system. Analysis of mouse mammary gland development revealed the expression of Rab3A in mammary epithelial cells. Furthermore, expression of the HRG transgene in Harderian tumors in mice also enhanced the expression of Rab3A. These observations provide new evidence of the existence of a Rab3A pathway in mammary epithelial cells and suggest that it may play a role in vesicle trafficking and secretion of proteins from epithelial cells in response to stimulation by the HRG expressed within the mammary mesenchyma. 相似文献
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Slavin I García IA Monetta P Martinez H Romero N Alvarez C 《European journal of cell biology》2011,90(4):301-311
In eukaryotic cells, proteins destined for secretion are translocated into the endoplasmic reticulum (ER) and packaged into so-called COPII-coated vesicles. In the ER exit sites (ERES), COPII has the capacity of deforming the lipid bilayer, where it modulates the selective sorting and concentration of cargo proteins. In this study, we analyze the involvement of Rab1b in COPII dynamics and function by expressing either the Rab1b negative-mutant (Rab1N121I) or the Rab1b GTP restricted mutant (Rab1Q67L), or performing short interference RNA-based knockdown. We show that Rab1b interacts with the COPII components Sec23, Sec24 and Sec31 and that Rab1b inhibition changes the COPII phenotype. FRAP assays reveal that Rab1b modulates COPII association/dissociation kinetics at the ERES interface. Furthermore, Rab1b inhibition delays cargo sorting at the ER exit sites. We postulate that Rab1b is a key regulatory component of COPII dynamics and function. 相似文献
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Rabphilin and Noc2 were originally described as Rab3A effector proteins involved in the regulation of secretory vesicle exocytosis, however, recently both proteins have been shown to bind Rab27A in vitro in preference to Rab3A (Fukuda, M. (2003) J. Biol. Chem. 278, 15373-15380), suggesting that Rab3A is not their major ligand in vivo. In the present study we showed by means of deletion and mutation analyses that rabphilin and Noc2 are recruited to dense-core vesicles through specific interaction with Rab27A, not with Rab3A, in PC12 cells. Rab3A binding-defective mutants of rabphilin(E50A) and Noc2(E51A) were still localized in the distal portion of the neurites (where dense-core vesicles had accumulated) in nerve growth factor-differentiated PC12 cells, the same as the wild-type proteins, whereas Rab27A binding-defective mutants of rabphilin(E50A/I54A) and Noc2(E51A/I55A) were present throughout the cytosol. We further showed that expression of the wild-type or the E50A mutant of rabphilin-RBD, but not the E50A/I54A mutant of rabphilin-RBD, significantly inhibited high KCl-dependent neuropeptide Y secretion by PC12 cells. We also found that rabphilin and its binding partner, Rab27 have been highly conserved during evolution (from nematoda to humans) and that Caenorhabditis elegans and Drosophila rabphilin (ce/dm-rabphilin) specifically interact with ce/dm-Rab27, but not with ce/dm-Rab3 or ce/dm-Rab8, suggesting that rabphilin functions as a Rab27 effector across phylogeny. Based on these findings, we propose that the N-terminal Rab binding domain of rabphilin and Noc2 be referred to as "RBD27 (Rab binding domain for Rab27)", the same as the synaptotagmin-like protein homology domain (SHD) of Slac2-a/melanophilin. 相似文献
19.
Rab small GTPases are crucial regulators of the membrane traffic that maintains organelle identity and morphology. Several Rab isoforms are present in the Golgi, and it has been suggested that they regulate the compacted morphology of the Golgi in mammalian cells. However, the functional relationships among the Golgi-resident Rabs, e.g. whether they are functionally redundant or different, are poorly understood. In this study, we used specific siRNAs to perform genome-wide screening for human Rabs that are involved in Golgi morphology in HeLa-S3 cells. The results showed that knockdown of any one of the six Rab isoforms (Rab1A/1B/2A/2B/6B/8A) induced fragmentation of the Golgi in HeLa-S3 cells and that its phenotype was rescued by re-expression of their respective siRNA-resistant construct. We then performed systematic knockdown-rescue experiments in relation to each of the six Rabs. Interestingly, with the exception of the Rab8A knockdown, the Golgi fragmentation phenotype induced by knockdown of a single Rab isoform, e.g. Rab2B, was efficiently rescued by re-expression of its siRNA-resistant Rab alone, not by any of the other five Rabs, e.g. Rab2A, which is highly homologous to Rab2B, indicating that these Rab isoforms non-redundantly regulate Golgi morphology possibly through interaction with isoform-specific effector molecules. In addition, we identified Golgi-associated Rab2B interactor-like 4 (GARI-L4) as a novel Golgi-resident Rab2B-specific binding protein whose knockdown also induced fragmentation of the Golgi. Our findings suggest that the compacted Golgi morphology of mammalian cells is finely tuned by multiple sets of Rab (or Rab-effector complexes) that for the most part function independently. 相似文献
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Shin YC Yoon JH Jang TH Kim SY Do Heo W So I Jeon JH Park HH 《Biochemical and biophysical research communications》2012,424(2):269-273
The Ras small G protein-superfamily is a family of GTP hydrolases whose activity is regulated by GTP/GDP binding states. Rab6A, a member of the Ras superfamily, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, biosynthesis, secretion, cell differentiation and cell growth. Rab6A exists in two isoforms, termed RabA and Rab6A′. Substitution of Gln72 to Leu72 (Q72L) at Rab6 family blocks GTP hydrolysis activity and this mutation usually causes the Rab6 protein to be constitutively in an active form. Here, we report the crystal structure of the human Rab6A′(Q72L) mutant form at 1.9 Å resolution. Unexpectedly, we found that Rab6A′(Q72L) possesses GDP/Mg2+ in the GTP binding pockets, which is formed by a flexible switch I and switch II. Large conformational changes were also detected in the switch I and switch II regions. Our structure revealed that the non-hydrolysable, constitutively active form of Rab6A′ can accommodate GDP/Mg2+ in the open conformation. 相似文献