Sec15 Is an Effector for the Rab11 GTPase in Mammalian Cells

Rab/Ypt GTPases play key roles in the regulation of vesicular trafficking. They perform most of their functions in a GTP-bound form by interacting with specific downstream effectors. The exocyst is a complex of eight polypeptides involved in constitutive secretion and functions as an effector for multiple Ras-related small GTPases, including the Rab protein Sec4p in yeast. In this study, we have examined the localization and function of the Sec15 exocyst subunit in mammalian cells. Overexpressed Sec15 associated with clusters of tubular/vesicular elements that were concentrated in the perinuclear region. The tubular/vesicular clusters were dispersed throughout the cytoplasm upon treatment with the microtubule-depolymerizing agent nocodazole and were accessible to endocytosed transferrin, but not exocytic cargo (vesicular stomatitis virus glycoprotein). Consistent with these observations, Sec15 colocalized selectively with the recycling endosome marker Rab11 and exhibited a GTP-dependent interaction with the Rab11 GTPase, but not with Rab4, Rab6, or Rab7. These findings provide the first evidence that the exocyst functions as a Rab effector complex in mammalian cells. Rab/Ypt GTPases play key roles in the regulation of vesicular trafficking. They perform most of their functions in a GTP-bound form by interacting with specific downstream effectors. The exocyst is a complex of eight polypeptides involved in constitutive secretion and functions as an effector for multiple Ras-related small GTPases, including the Rab protein Sec4p in yeast. In this study, we have examined the localization and function of the Sec15 exocyst subunit in mammalian cells. Overexpressed Sec15 associated with clusters of tubular/vesicular elements that were concentrated in the perinuclear region. The tubular/vesicular clusters were dispersed throughout the cytoplasm upon treatment with the microtubule-depolymerizing agent nocodazole and were accessible to endocytosed transferrin, but not exocytic cargo (vesicular stomatitis virus glycoprotein). Consistent with these observations, Sec15 colocalized selectively with the recycling endosome marker Rab11 and exhibited a GTP-dependent interaction with the Rab11 GTPase, but not with Rab4, Rab6, or Rab7. These findings provide the first evidence that the exocyst functions as a Rab effector complex in mammalian cells. Rab/Ypt proteins are members of the Ras superfamily of small GTP-binding proteins that play key roles in the regulation of intracellular vesicular trafficking (1Schimmoller F. Simon I. Pfeffer S.R. J. Biol. Chem. 1998; 273: 22161-22164Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 2Novick P. Zerial M. Curr. Opin. Cell Biol. 1997; 9: 496-504Crossref PubMed Scopus (666) Google Scholar). The Rab proteins encompass a large family of related GTPases that function predominantly in distinct trafficking pathways. The Rab functional cycle is coupled to GTP binding and hydrolysis, which are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins, respectively. In the GTP-bound state, Rab proteins are recruited to membranes where they associate with specific downstream effector molecules that direct vesicle targeting and docking to the appropriate acceptor compartment. It is emerging that GTP-bound Rab proteins regulate a diverse array of effector molecules, including factors that promote vesicle formation, motility, docking, and fusion (3Segev N. Curr. Opin. Cell Biol. 2001; 13: 500-511Crossref PubMed Scopus (247) Google Scholar). GTPase-activating protein-catalyzed GTP hydrolysis results in the dissociation of effector complexes and extraction of GDP-bound Rab from membranes. Rab11 is a ubiquitously expressed Rab protein that is involved in the endosomal recycling pathway in mammalian cells. It colocalizes with the transferrin receptor (TfnR) 1The abbreviations used are: TfnR, transferrin receptor; RE, recycling endosome; Tfn, transferrin; TGN, trans-Golgi network; GST, glutathione S-transferase; GFP, green fluorescent protein; hTfnR, human transferrin receptor; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; GTPγS, guanosine 5′-O-(3-thiotriphosphate); GDPβS, guanosine 5′-O-(2-thiodiphosphate); HRP, horseradish peroxidase; VSVG, vesicular stomatitis virus glycoprotein. on pericentriolar recycling endosomes (REs) and is involved in recycling of transferrin (Tfn) to the plasma membrane (4Ren M. Xu G. Zeng J. De Lemos-Chiarandini C. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6187-6192Crossref PubMed Scopus (397) Google Scholar, 5Ullrich O. Reinsch S. Urbe S. Zerial M. Parton R.G. J. Cell Biol. 1996; 135: 913-924Crossref PubMed Scopus (1084) Google Scholar). Rab11 has also been implicated in apical recycling and transcytosis in Madin-Darby canine kidney cells (6Wang X. Kumar R. Navarre J. Casanova J.E. Goldenring J.R. J. Biol. Chem. 2000; 275: 29138-29146Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) and trans-Golgi network (TGN) to plasma membrane trafficking via REs in baby hamster kidney cells (7Chen W. Feng Y. Chen D. Wandinger-Ness A. Mol. Biol. Cell. 1998; 9: 3241-3257Crossref PubMed Scopus (320) Google Scholar). Several Rab11 effectors have been described that are involved in recycling, including Rab11-binding protein/rabphilin-11, pp75/Rip11, myosin Vb, Rab11-FIP1, Rab11-FIP3/Eferin, Rab11-FIP4, and Rabcoupling protein (8Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 9Wallace D.M. Lindsay A.J. Hendrick A.G. McCaffrey M.W. Biochem. Biophys. Res. Commun. 2002; 292: 909-915Crossref PubMed Scopus (67) Google Scholar). The exocyst complex (also known as the Sec6/8 complex), one of the more extensively studied Rab effectors, is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) in Saccharomyces cerevisiae and mammalian cells (10TerBush D.R. Maurice T. Roth D. Novick P. EMBO J. 1996; 15: 6483-6494Crossref PubMed Scopus (681) Google Scholar, 11Kee Y. Yoo J.S. Hazuka C.D. Peterson K.E. Hsu S.C. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14438-14443Crossref PubMed Scopus (152) Google Scholar). It is required for constitutive secretion as well as polarized exocytosis (12Hsu S.C. TerBush D. Abraham M. Guo W. Int. Rev. Cytol. 2004; 233: 243-265Crossref PubMed Scopus (209) Google Scholar). In yeast, the Sec15p subunit interacts specifically with GTP-bound Sec4p (13Guo W. Roth D. Walch-Solimena C. Novick P. EMBO J. 1999; 18: 1071-1080Crossref PubMed Scopus (502) Google Scholar), a Rab protein involved in secretion. The association of activated Sec4p with Sec15p on secretory vesicles is believed to regulate interactions between exocyst subunits, leading to vesicle docking with the plasma membrane at sites defined by Sec3p (13Guo W. Roth D. Walch-Solimena C. Novick P. EMBO J. 1999; 18: 1071-1080Crossref PubMed Scopus (502) Google Scholar). The molecular basis for exocyst function in vesicle targeting in higher organisms has remained obscure. Reagents—An anti-Sec15 polyclonal antibody was prepared by immunization of rabbits with a C-terminal peptide (KDTSKKNNIFAQFRKNDRDRQKC) conjugated to diphtheria toxin. The antibody was affinity-purified from the antiserum using the same peptide immobilized on Amino Link Plus coupling gel (Pierce). Mouse anti-Xpress monoclonal antibody and LipofectAMINE transfection reagent were purchased from Invitrogen. Rabbit anti-glutathione S-transferase (GST) and anti-Rab11 polyclonal antibodies were from Zymed Laboratories Inc. (South San Francisco, CA). Anti-green fluorescent protein (GFP) monoclonal antibody, Alexa Fluor-conjugated secondary antibodies, and Texas Red-labeled Tfn were purchased from Molecular Probes, Inc. (Eugene, OR). Anti-Sec6 monoclonal antibody (clone 9H5) was from Stressgen Biotech Corp. (Victoria, Canada). All other reagents were from Sigma unless indicated otherwise. Construction of Expression Plasmids—The clones encoding Sec10 and Sec15 were obtained from Dr. R. H. Scheller (Stanford University, Stanford, CA), and human TfnR (hTfnR) was from Drs. M. Silverman and G. Banker (Oregon Health Sciences University, Portland, OR). Complementary DNAs encoding wild-type Sec15 (amino acids 2–822), Sec10 (amino acids 2–707), and TfnR were amplified by PCR and subcloned into the pCR-Blunt-TOPO shuttle vector (Invitrogen). The cDNA clones encoding wild-type Rab4a, Rab6a, Rab7, and Rab11a were generated by PCR from a 3T3-L1 adipocyte cDNA library and subcloned into the pPCR-Script-Amp shuttle vector (Stratagene, La Jolla, CA). The Rab11 mutants Rab11(S25N) and Rab11(Q70L) were generated using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. For construction of expression vectors, the cDNAs were inserted in-frame into the following plasmids: pcDNA4-HisMax-C (Invitrogen) for Xpress-tagged proteins, pEGFP-C (Clontech) for GFP fusion proteins, pEBG (14Tanaka M. Gupta R. Mayer B.J. Mol. Cell. Biol. 1995; 15: 6829-6837Crossref PubMed Scopus (221) Google Scholar) for mammalian expression of GST fusion proteins, and pET28a (Novagen, Madison, WI) for bacterial expression of histidine-tagged Rab11 (His6-Rab11). Cell Culture, Transient Transfections, and Fluorescence Microscopy— COS-7, Chinese hamster ovary (CHO), and 293 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium with and and of cells was using the LipofectAMINE reagent according to the manufacturer's instructions. For and 293 were by of cells in was to a in and at and For the cells were and to for at 37 The cells were with phosphate-buffered with for at and in at °C for with PBS, the cells were with and in for of antibodies was for at in at the following anti-Xpress antibody, and anti-Rab11 antibody, The cells were with and with secondary antibodies at in for at The were with and were obtained using a and cells were with expression encoding and and the cells were with Dulbecco's modified Eagle's medium and for by at 37 °C in Dulbecco's modified Eagle's medium with The cells were with Texas Red-labeled Tfn for at 37 with on and with for The cells were on to and as described in binding cells were with expression as of cells were in of and were with or for at and with GST fusion proteins were by with of glutathione for at For in binding proteins were with or in binding and for at 37 from cells were with glutathione for at °C to the GST fusion The were with binding and for an in the same with and with or binding between Sec15 and was with and in The was at for and the was by with glutathione for or GST was with or in binding for at 37 °C and with the same The were with and with immobilized GST proteins for at The were with and and proteins were by in gel were to and were using anti-Xpress or affinity-purified anti-Sec15 antibodies, by horseradish secondary The were using and by gel was as described J. H. R. Cell. Full Text PDF PubMed Scopus Google Scholar). The cDNAs encoding Sec15 and the Rab proteins were into the and respectively. cells were with the and medium and The cells were medium as and to expression of the fusion The binding were in using a cells were with expression encoding and cells were with medium for an at 37 the medium was with human Tfn conjugated to for at 37 The was as described W. J. Cell Biol. 1996; PubMed Scopus Google Scholar). the the cells were on with PBS, and using and in for at The cells were with with from the and by were prepared using a modified J. C. 2001; PubMed Scopus Google Scholar). of the was using a monoclonal antibody Molecular Probes, or an of a monoclonal antibody by at a of of Overexpressed the localization of Sec15, we generated an affinity-purified polyclonal antibody a Sec15 C-terminal In of the antibody were required to a and in the was throughout the cytoplasm with evidence of the perinuclear S. Hsu S.C. PubMed Scopus Google Scholar). on these was that the anti-Sec15 peptide antibody is not for an we used to the localization of Sec15 with an or a fusion protein in cells that the expression the of Sec15 polypeptides of the appropriate molecular not of or in cells in the of of that were concentrated in the perinuclear and In intracellular was with the of a of the of was upon transfection for of to at localization was in and 293 where of were in the perinuclear and The of were not in cells in with a GFP vector It that the localization of Sec15 is to from and not at expression Sec15 has been to to the perinuclear in cells and to to the and upon S. Hsu S.C. PubMed Scopus Google Scholar). we examined the of the microtubule-depolymerizing agent nocodazole on the localization of in cells The perinuclear localization of endosomal and is by to nocodazole treatment for the of dispersed throughout the in cells with their perinuclear localization was These findings that the localization of Sec15 from association with a or vesicle and not from of the The that the their the cytoplasm also that their not from a direct association of Sec15 with have that Sec10 with Sec15 in and mammalian cells (13Guo W. Roth D. Walch-Solimena C. Novick P. EMBO J. 1999; 18: 1071-1080Crossref PubMed Scopus (502) Google Scholar, C. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). we examined Sec10 was recruited to the fluorescent upon of Sec15 In this cells were with expression encoding or in with expressed exhibited with C. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). In with exhibited a from the to the that the to with Sec10 in mammalian cells and is a functional In not with the that Sec10 recruited to the of other exocyst exocyst subunits have been implicated in the to plasma membrane of the secretory was to the perinuclear of upon of Sec15 elements of the or the of vesicular stomatitis virus to GFP J. 1997; PubMed Scopus Google Scholar) was as an exocytic is and in the at the and is to the and to the plasma membrane following at the the cells are from the to is from the and in cells and were at °C for and at °C for to in the and examined by The fusion protein used for these has been to exocytic trafficking that are from of in cells J. 1997; PubMed Scopus Google Scholar). from to °C for a of was in of the that were concentrated in the perinuclear in cells and that was between the of and these The that was was most a of of Sec15 and in the perinuclear on membranes the by were distinct from the of defined by In of the localization of Sec15p in A. Novick J. Cell Biol. PubMed Scopus Google Scholar), we that the a of exocytic cells that been at °C for were to °C to the of vesicular and of to the plasma were to between and following to °C for a of the was in with to the plasma membrane not The of between and Sec15 to the by the exocyst subunit are accessible to The Tfn recycling pathway has been studied extensively in mammalian cells. binding to TfnR at the Tfn is and from where is to the plasma membrane via the compartment. the are accessible to Tfn, we cells with expression encoding and have that functions in a that is from the receptor in cells J. Cell Biol. PubMed Scopus Google Scholar). was in which the cells were in the of Tfn for at 37 °C and with Tfn for The localization of and endocytosed Tfn was examined by trafficking was following the the endocytosed Tfn dispersed that were of and was with The of Tfn that of Sec15 not at the we of Tfn with in the perinuclear and at and was a of the perinuclear that were for Sec15 were also predominantly These that Sec15 is associated with an endosomal that is accessible to Overexpressed Sec15 with of the of the of Tfn in the are with the that the exocyst subunit with elements of the O. Reinsch S. Urbe S. Zerial M. Parton R.G. J. Cell Biol. 1996; 135: 913-924Crossref PubMed Scopus (1084) Google Scholar, J. Curr. Opin. Cell Biol. 1995; PubMed Scopus Google Scholar). In of we to the of between and the endosome and that the by or were in to the of for Sec15, their were predominantly distinct from the exocyst subunit The of with is with the that Sec15 not with the that were at of and that not the between Sec15 and Tfn, was cells were with expression encoding and the cells were with for at 37 and endosomal were with W. J. Cell Biol. 1996; PubMed Scopus Google Scholar). prepared from the cells were with an monoclonal antibody, by a secondary reagent that clusters of tubular/vesicular that were and for the exocyst subunit was to these with the of a of The tubular/vesicular clusters not to by a that they are distinct from endosomal were not on the tubular/vesicular clusters the antibody was for an that the of was The between Sec15 and Tfn to of the exocyst subunit endosomal were to perform a recycling to Sec15 transfection a was in which cells and were with Texas Red-labeled Tfn for at 37 °C and with for at 37 of Tfn in cells was by trafficking was following the Tfn was throughout the cytoplasm in to In cells the Tfn exhibited a dispersed in this was a higher of perinuclear which is to have from of a of the protein in the tubular/vesicular the the intracellular Tfn was to recycling and from the cells. In in cells the Tfn colocalized with the exocyst subunit and exhibited a higher of intracellular in the cells at the same These that Tfn in the with a on recycling the Sec15 an for the association of Sec15 with the we examined of with a of well Rab proteins For this cells were with expression encoding and Xpress-tagged Rab4a, Rab6a, Rab7, or for endosomes P. M. A. A. I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), the A. A. J. PubMed Scopus Google Scholar), P. Parton R.G. Zerial M. Cell. Full Text PDF PubMed Scopus Google Scholar), and REs O. Reinsch S. Urbe S. Zerial M. Parton R.G. J. Cell Biol. 1996; 135: 913-924Crossref PubMed Scopus (1084) Google Scholar), respectively. between Rab4a, Rab6a, or and Sec15 was was that their were predominantly distinct In Rab11a exhibited a with the tubular/vesicular The localization are of in in which Sec4p is recruited to exocytic vesicles upon of Sec15p (13Guo W. Roth D. Walch-Solimena C. Novick P. EMBO J. 1999; 18: 1071-1080Crossref PubMed Scopus (502) Google Scholar, A. Novick J. Cell Biol. PubMed Scopus Google Scholar). to that Sec15 an effector for Rab11 in mammalian cells. this a interaction was in which cells were with a vector encoding Sec15 with a vector encoding wild-type Rab4a, Rab6a, Rab7, the or the of cells that Rab proteins were expressed at not The of the interaction was using a cells Sec15 and wild-type Rab11 exhibited a higher Sec15 and the other Rab the of the interaction was with the Rab11(S25N) and with Rab11(Q70L) to the wild-type with the that Sec15 functions as an effector for used to the interaction between Sec15 and an in binding was in which cells were with expression encoding and wild-type Rab11a or to from the cells were in the of or GDPβS, and the GST fusion proteins were affinity-purified on glutathione The of GST proteins and on the was by cells were with and GST or the exocyst subunit was not in the following with and the GST were for a was in the In in which was for GDPβS, the of was with a GTP-dependent In an in binding was using histidine-tagged Rab proteins and In this was from cells using glutathione expressed or was with or and with immobilized and the were was on the was with GTPγS, but not and In the protein was not in the following with or and the interaction between the proteins, we Rab11 with Sec15 In this immobilized was with or and with a The were and proteins were by using affinity-purified anti-Sec15 Sec15 was not on the the fusion protein was for GST in the of Sec15 on the was the was with with and the GTP-dependent interaction between Rab11 and Sec15 was using multiple and is with the that the exocyst functions as a Rab11 effector complex in mammalian cells. In this study, we have into the localization and function of The association of Sec15 with elements of the is with that in cells D.M. Zeng W. M. S. J. Cell Biol. 2000; PubMed Scopus Google Scholar) and cells Hsu S.C. J. 2001; PubMed Google Scholar), exocyst subunits are associated with an perinuclear that to distinct from the The localization of Sec15 is also with the that at distinct from the to the plasma membrane in that are on the exocyst subunit Sec6, one of which via endosomes R. J. Cell Biol. 2002; PubMed Scopus Google Scholar, A. J. Cell Biol. 1995; PubMed Scopus Google Scholar). the localization of Sec15 in mammalian cells is a has that colocalizes with in a of the in kidney cells C. J.R. J. Cell Biol. 2001; PubMed Scopus Google Scholar). It is and Sec15 to to distinct intracellular we is that Sec15 results in an that not the localization of the the that Sec10 is recruited specifically to the tubular/vesicular clusters and that Sec15 the a GTP-dependent interaction with Rab11 that the Sec15 fusion proteins are in S. the specific of Sec15p in secretion from of exocytic is with the of the protein in the secretory pathway on P. C. R. Cell. Full Text PDF PubMed Scopus Google Scholar). are a of for the that Sec15 not to the Sec15 function of other subunits of the In this is in yeast, Sec15p and have been to in a in to their in the exocyst (13Guo W. Roth D. Walch-Solimena C. Novick P. EMBO J. 1999; 18: 1071-1080Crossref PubMed Scopus (502) Google Scholar). has been that Sec10 to GFP not with or and with an perinuclear in polarized Madin-Darby canine kidney cells C. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). the of exocyst subunits in plasma trafficking In of this have that Sec10 is not involved in polarized trafficking or but is for secretion N. 2002; PubMed Scopus Google Scholar). Sec15 to a that not to a of in mammalian cells. a family of proteins that function in distinct trafficking a human protein has been described that with the human of Sec15 and is a of a of the exocyst complex that interacts with the small A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). are the by at the that the protein associated with clusters of tubular/vesicular that is to the tubular/vesicular of the in cells S.R. Cell. Full Text PDF PubMed Scopus Google Scholar). the localization of Sec15 distinct from the of the pericentriolar which is as a concentrated of in mammalian cells O. Reinsch S. Urbe S. Zerial M. Parton R.G. J. Cell Biol. 1996; 135: 913-924Crossref PubMed Scopus (1084) Google Scholar). from an of Sec15 on the of the compartment. the tubular/vesicular clusters in the recycling pathway that upon Sec15 the tubular/vesicular clusters are of the of secretory vesicles upon of Sec15p in S. cerevisiae (13Guo W. Roth D. Walch-Solimena C. Novick P. EMBO J. 1999; 18: 1071-1080Crossref PubMed Scopus (502) Google Scholar, A. Novick J. Cell Biol. PubMed Scopus Google Scholar). The of the secretory vesicle clusters is believed to a association between Sec15p and the vesicles is on the function of the Rab Sec4p and nucleotide exchange have the first that Sec15 functions as an effector for a Rab in mammalian cells. have a of Rab11 effectors, pp75/Rip11, Rab11-FIP1, Rab11-FIP3/Eferin, Rab11-FIP4, and protein (8Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 9Wallace D.M. Lindsay A.J. Hendrick A.G. McCaffrey M.W. Biochem. Biophys. Res. Commun. 2002; 292: 909-915Crossref PubMed Scopus (67) Google Scholar), that a Rab11-binding The of Sec15 not a is that Sec15 and to as for myosin and the protein C.M. Goldenring J.R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (221) Google Scholar). In to the interaction with we were to an interaction between Sec15 and Rab4a, Rab6a, or Rab7. endosomal trafficking and is involved in the pathway from to endosomes as well as recycling from endosomes to the plasma membrane M.W. A. G. S. M. F. C. 2001; PubMed Scopus Google Scholar, P. M. P. P. I. Cell. Full Text PDF PubMed Scopus Google Scholar). The that Sec15 not with that not function as an effector for Rab proteins involved in plasma trafficking pathways. have indicated that the exocyst is an effector complex for at small GTPases in and mammalian including and K.E. Curr. Biol. 2002; Full Text Full Text PDF PubMed Google Scholar, P. Guo W. Cell Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The that Sec15 is an effector for Rab11 the of small GTPases that with the exocyst to and are members of the family of GTPases that regulate predominantly their on the the exocyst is emerging as a in exocytic that interacts with multiple Ras-related small including that regulate vesicle trafficking as well as the these interactions are a key for Drs. R. H. Scheller and J. and M. Silverman and G. Banker (hTfnR) for the cDNA clones used in this

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.